The MgaSpn Global Transcriptional Regulator Mediates the Biosynthesis of Capsular Polysaccharides and Affects Virulence via the Uracil Synthesis Pathway in Streptococcus pneumoniae

The MgaSpn Global Transcriptional Regulator Mediates the Biosynthesis of Capsular Polysaccharides and Affects Virulence via the Uracil Synthesis Pathway in Streptococcus pneumoniae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The MgaSpn Global Transcriptional Regulator Mediates the Biosynthesis of Capsular Polysaccharides and Affects Virulence via the Uracil Synthesis Pathway in Streptococcus pneumoniae Xinlin Guo, shuhui wang, Ye Tao, Xuemei Zhang, Weicai Suo, Yapeng Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4618066/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Background Uracil metabolism is an important step in the growth and metabolism of Streptococcus pneumoniae , and pyrimidine nucleotides play an important role in the expression and production of S. pneumoniae capsules. Mga Spn ( spd_1587 ),as a transcriptional ragulator of host environment adaptation, regulates the biosynthesis of the capsules and phosphorylcholine. However, the underlying regulation mechanism between uracil metabolism and biosynthesis of capsules remains incompletely understood. Here, we first described the relationship between uracil metabolism and capsule expression via the pyrR gene( spd_1134 ) in S. pneumoniae . Results Electrophoretic mobility-shift assays (EMSAs) and DNase I footprinting assays showed a direct interaction between Mga Spn and the pyrR promoter (P pyrR ) at two specific binding sites. MgaSpn negatively regulated capsule production through pyrR as confirmed by complementing pyrR expression in D39Δ mgaSpn Δ pyrR . Virulence experiments showed that the Mga Spn - pyrR interaction was necessary for both pneumococcal colonization and invasive infection. Conclusions For the first time, the present study demonstrated that the de novo synthesis gene pyrR of S. pneumoniae is regulated by the Mga Spn transcriptional regulator.Taken together,these results provide an insight into the regulation of capsule production mediated by uracil metabolism and its important roles in pneumococcal pathogenesis. streptococcus pneumoniae capsular polysaccharides MgaSpn pyrR uracil Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Background Streptococcus pneumoniae is a clinically common gram-positive pathogen that generally colonizes the human nasopharynx without symptoms, but it can also invade different ecological niches of the host and cause various diseases, such as acute otitis media, bronchitis, pneumonia, and meningitis 1 – 3 . Bacterial metabolic networks and virulence share an intimate relationship. Sensing nutrient availability drives the regulation and production of virulence factors in a multitude of bacterial pathogens 4 . S. pneumoniae capsule metabolism is significant for its pathogenicity; however, the specific regulatory mechanism is not fully elucidated. Uracil plays an important role in metabolism and virulence of S. pneumoniae . Uridine triphosphate is usually synthesized by the pyrimidine biosynthesis salvage pathway, and it is also endogenously synthesized through de novo synthesis pathway via pyrimidine biosynthesis genes 5 (such as pyrR , pyrF , carA , carB ). De novo biosynthesis of pyrimidine begins with a pyrimidine ring assembled with aspartate, bicarbonate, and glutamine. The pyrimidine ring reacts with ribosyl pyrophosphate (PRPP) to form orotidine 5-monophosphate (OMP). Subsequently, OMP decarboxylates to form uridine monophosphoric acid (UMP), which is converted into other essential pyrimidine nucleotides. The pyrimidine de novo synthesis pathway consists of six enzymatic steps, and the genes encoding these enzymes ( carA , carB , pyrB , pyrC , pyrD , pyrE , and pyrF ) are widely conserved in bacteria 6 – 8 . CarAB ( spd_1132 , spd_1131 )encodes two subunits of carbamyl phosphate synthetase in Escherichia coli , and this enzyme catalyzes the formation of carbamyl phosphate, an intermediate in the pyrimidine nucleotide and arginine biosynthetic pathways 9 . It has been reported that carA mutation reduces the expression of capsule operon promoter (P cps ) in medium lacking uracil in the exponential growth period 7 , but it remains unclear whether several other genes affect the expression of P cps in S. pneumoniae . PyrF is an OMP decarboxylase located on the pyrimidineregulon along with other genes encoding enzymes involved in the biosynthesis of pyrimidines 5 . Recent research has mainly suggested that de novo synthesis of pyrimidines is regulated by the pyrR operon regulatory protein. PyrR is a bifunctional protein that not only encodes uracil phosphoribosyl transferase (UPRTase) but also plays an important regulatory role in the transcriptional level of pyrimidine biosynthesis 6 . In the presence of the UMP coregulator, PyrR binds to the 5 ’ UTR of the pyrR mRNA transcript and disrupts the anti- terminator stem-loop, resulting in decreased downstream gene expression. In contrast, when the UMP concentration is low, PRPP antagonizes UMP termination by binding to the PyrR protein 10– 12 . The expression of de novo synthesis of pyrimidine genes is regulated by RNA-binding proteins, but it remains unclear whether it is regulated by DNA-binding proteins. Pyrimidine nucleotides play an important role in the expression and production of S. pneumoniae capsules 6 , 7 . The capsule is an exopolysaccharide layer, which is a carbohydrate layer that protects bacteria from various host destructive actions, such as complement deposition, opsonophagocytosis, mucous embedding, and neutrophil extracellular traps 13 – 15 . The capsule of the D39s strain, theserotype 2 strain used in the present study, is formed by repeating units of glucose (Glc), glucuronic acid (GlcUA), andrhamnose(Rha) in a ratio of 1:2:3 16 . The sugar component is activated by UTP to produce uridine diphosphate glucose (UDP-Glc), uridine diphosphate glucuronic acid (UDP-GlcUA), deoxythymidine triphosphate (dTTP), and deoxythymidine diphosphate rhamnose (dTDP-Rha) 16 . However, the mechanisms of pyrimidine and capsule synthesis remain unclear, and the role of central metabolism in pneumococcal virulence factors and pathogenesis is not fully understood. Mga Spn belongs to the Mga/AtxA family of global transcriptional regulators, and it plays an important role in host immune escape, bacterial biofilm formation, host environment adaptation, and invasive disease 17– 19 . We have previously confirmed that Mga Spn is a negative regulator of capsule biosynthesis. Deletion of Mga Spn increases the capsular content in whole bacterial lysate, and the increased content is comprised of small molecular weight proteins 20 . However, the cause of the increase in the small molecular weight capsular proteins remains unclear 20 . We conducted transcriptomic analysis to identify other factors that maybe regulated by Mga Spn . Among them, the significant upregulation of the pyrimidine de novo synthesis genes, pyrR , pyrF , carA , and carB , in Mga Spn -deficient strains attracted our attention. Thus, we explored the relationship between S. pneumoniae uracil metabolism and capsule synthesis and virulence. The present study demonstrated that Mga Spn regulates capsular production through the de novo synthesis pathway gene pyrR , thus affecting virulence. The study of the relationship between S. pneumoniae uracil metabolism and the mechanism of capsule synthesis is of great significance for understanding the biological characteristics of S. pneumoniae , developing new antibacterial drugs, and treating S. pneumoniae infections. 2 Materials and Methods 2.1. Bacterial strains and growth conditions The bacterial strains and plasmids used in the present study are listed in Table 1 . The S. pneumoniae D39s strain and its derivatives were cultured in semi-synthetic casein hydrolysate medium supplemented with 5% yeast extract (C + Y, pH 7.0) or C + Y medium without uracil 21 . E. coli strains were grown in lysogeny broth (LB) with shaking or on LB agar plates at 37°C 22 . When appropriate, antibiotics were added to the growth medium as shown in Table 1 . Antibiotic selection was used at the following concentrations: streptomycin, 150 ug/ml; kanamycin; 200 ug/ml; chloramphenicol, 200 ug/ml; spectinomycin, 50 ug/ml for Escherichia coli and 200 ug/ml for S.pneumoniae . Table 1 Strains and plasmids Strain Relevant genotype and/or phenotype Resistance D39s S.pneumoniae D39 strain,Capsulated Strainserotype2,rpsl K56T Sm R D39Δ MgaSpn D39, Δ MgaSpn Kan R D39Δ MgaSpn :: MgaSpn D39, Δ MgaSpn ::Δ MgaSpn Spec R D39Δ pyrR D39,Δ pyrR Kan R D39Δ MgaSpn Δ pyrR D39, Δ MgaSpn Δ pyrR Kan R D39 Δ MgaSpn Δ pyrR :: pyrR D39, Δ MgaSpn Δ pyrR :: pyrR Chl R D39-PTH D39,P cps -luc Chl R D39Δ MgaSpn -PTH D39Δ MgaSpn ,P cps -luc Chl R D39Δ pyrR -PTH D39,Δ pyrR , P cps -luc Chl R D39Δ MgaSpn Δ pyrR -PTH D39,Δ MgaSpn Δ pyrR ,P cps -luc Chl R Plasmid TH3937 P cps cloned into insertion vector Chl R PIB166 Contains pyrR Chl R 2.2. Strain construction All mutant strains originated from the D39s strain, a streptomycin-resistant derivative of D39 referred to as the wild type (WT) strain. Sequences of primers used in the present study are listed in Table 2 . The D39Δ pyrR strain was generated in a two-step transformation procedure 23 . The upstream and downstream homologous arms of the pyrR locus were amplified from D39s genomic DNA with the pyrR P1/ pyrR P2 and pyrR P3/ pyrR P4 primer pairs, respectively. The Janus cassette was amplified with the JC F and JC R primers from the genomic DNA of the ST588 strain. The Janus cassette, which has kanamycin resistance and a dominant rpsL allele, was utilized for the selection of kanamycin-resistant, streptomycin-sensitive colonies. The unmarked strains were kanamycin- sensitive and streptomycin-resistant. Fusion PCR was performed with the upstream arm, the Janus cassette, and the downstream arm with the pyrR P1 and pyrR P4 primer pairs, and the product was transformed into the D39s strain to generate D39Δ pyrR ::kan- rpsL (Δ pyrR ::JC). To generate unmarked deletions in the pyrR locus, the upstream and downstream sequences were amplified with the pyrR P1/JC F and pyrR P4/JC R primer pairs. The amplicons were ligated by fusion PCR with the pyrR P1 and pyrR P4 primer pairs, followed by transformation into the D39s strain to generate the pyrR unmarked deletion strain (D39Δ pyrR )(Figure S1 ). Table 2 Sequences of primers Primers Sequence(5’to 3’) PyrR P1 TCGCTTGGGATTGTATCGG PyrR P2 GGAGTTTTCAGCATTATCCTCTAGAGACAAACCTCCAAAAAGAAAAGTC PyrR P3 GCATAAGGAAAGGCTCGAGGTTAAAGGAGTAGCCATGTCAG PyrR P4 TCATGACATCAACCTGATCAATG JC F TCTAGAGGATAATGCTGAAAACTCCTTGAAG JC R CTCGAGCCTTTCCTTATGCTTTTGGAC gyrB F GTTCGTATGCGTCCAGGGAT gyrB F ATACCACGCCCATCATCCAC MgaSpn F AGTTGCTCCTAGTTACGAACC MgaSpn R ACCTTCTATTCCTTCTGCCTGC PyrR F ACTTCGCGGTCTGTCACATC PyrR R TGCCCACCGAATCCAAGAAC PyrF F GACACCAGGGATTCGTCCAG PyrF R TAAGCTGCAACAGGCTCCTC CarA F AGCAGGTTGGTATCTGTGGC CarA R TCAACCATGCGGTACGTGAA CarB F TGGCATCAACTTCGCACTCT CarB R CAGTTCTTGTCCGCCCATCT PyrR-BamHI CGCGGATCCGTGAAGTCTATACTGTGTGCAGT PyrR-XhoI CCGCTCGAGCCACATGGTTCAATGCTTGTTGA Pr1303 CGGGATCCAGGAGGAATAATGAGATCCG Pr1304 TTGCGGCCGCCTACGGGGATCTTACAATTT 2.3. Western blot analysis S. pneumoniae were grown statically to an OD620 0f 0.5 in 3 ml C + Ymedium at 37℃ under 5% CO 2 , and harvested by centrifugation at 4℃ for 15min at 10,000×g. Removed supernatant, added 200 µL of SEDS lysis buffer (150 mM NaCl, 0.1% deoxycholate, 15 mM EDTA, and 0.2% sodium dodecyl sulfate; pH = 8). The samples were separated by 10% SDS‒PAGE and subsequently transferred to PVDF membranes by wet transfer, and blocked for 30 min at room temperature in blocking buffer (PBS with 0.05% [vol/vol] Tween 20 and 5% [wt/vol] skimmed milk). The membranes were incubated with the following primary antibodies overnight at 4°C: type 2 CPS (1:5000 Pneumococcus Type 2 serum; States Serum Institut, København,Denmark). Washed the membranes four times with PBST for six minutes each(PBS with 0.05% [vol/vol] Tween 20). The membranes were then incubated with the following secondary antibodies at 37°C for 1 h: goat anti-mouse IgG (1:10000; KPL,USA) or goat anti-mouse IgA (1:8000; SantaCruz,USA). 2.4. Expression and purification of 6×His-Mga Spn Mga Spn protein expression and purification were performed as previously described 20 . The constructed pET28-Mga Spn plasmid was transformed into BL21 cells for cloning and expression. Protein purity in the eluent was analyzed by Coomassie brilliant blue staining, and the appropriate concentration was selected for ultrafiltration. 2.5. Electrophoretic mobility shift assay (EMSA) The probes used in the present study were the pyrR promoter (P pyrR ) fragments amplified from the D39s strain labeled with 5'-biotin. First, 10 µL of reaction buffer, consisting of 1 µL 10x binding buffer, 0–2.6 µgprotein, 0.5 µg poly (dI-dC), and 0.5 ng of the labeled probe, was incubated at 25°C for 20 min. After incubation, the unlabeled probe in 100-fold excess was added as a specific competitor in the cold probe reaction system. Following incubation, binding reaction mixtures were analyzed by electrophoresis in 6% native TBE polyacrylamide gels at constant 100 V for 60min. EMSA was then carried out using the LightShift R Chemiluminescent EMSA Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. 2.6. DNase I footprinting assay DNase I footprintingassays were performed as described previously 20 . Briefly, fluorescent FAM-labeled probes for the P cps and P pyrR were amplified using D39s as the template with the pyrR -FAM and pyrR R primers, respectively. Then, 390 ng of probes was incubated with differing concentrations of Mga Spn protein in a 40 µL reaction system at 25°C for 30 min. Then, 10 µL of a solution containing 0.015 units of DNase I (Promega, Madison, WI, USA) and freshly prepared 100 nM CaCl 2 was added to the system. After incubation at 37°C for 1 min, the reaction was stopped by adding 140 µL of terminator solution. The obtained samples were extracted with phenol and precipitated with ethanol. The pellets were dissolved in 30 µL of nuclease-free water. The GeneScan-LIZ600 size standard (Applied Biosystems, Foster City, CA, USA) was used for electrophoresis. 2.7. Enzyme-linked immunosorbent assay (ELISA) Samples were prepared as described above. Dilution samples of 1:800 were used to coat 96-well microtiter plates, which were subsequently reacted with the type-2 CPS polyclonal antibody (1:5000) (Statens Serum Institut, København, Denmark). The bound primary antibodies were detected following incubation with goat anti-rabbit IgG-HRP antibody (1:8000) (KPL, Gaithersburg, MD, USA), and the absorbance at 450 nm was recorded. The experimental results are expressed as the mean ± standard deviation (SD) of three replicates. 2.8. RNA extraction and RT‒PCR Total RNA was extracted from 2 mL of log-phase bacterial culture grown statically to an OD620 0f 0.5 in normal C + Y medium. Bacterial cells were treated with 200 µL of 15 mg/mL lysozyme and 10 µL proteinase K for 30 min at 25°C. The RNAprotect bacterial reagent and RNeasy Protect bacterial kit (Qiagen, Hilden,Germany) were employed for RNA extraction according to the manufacturer ’s instructions. The RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher, Pittsburg, PA, USA), and its integrity was confirmed by agarose gel electrophoresis. PrimeScript RT master mix (TaKaRa, Beijing) was used to prepare cDNAaccording to the manufacturer’s instructions. RT- PCR was carried out using a CFX ConnectTM (Bio-Rad). The primers used for PCR are listed in Table 2 . gyrB was used as an internal control. The results of representative experiments are expressed as the mean ± standard deviation of three replicates. 2.9. Transcriptome sequencing and metabolome sequencing Strains for transcriptome sequencing were collected in the OD620 of 0.5 period in normal C + Y medium containing 15mg L- 1 pyrimidines. Pneumococcal cultures were pre-treated with ammonium sulfate to terminate protein-dependent transcription and degradation,then were centrifuged at 10,000g for 10 min. Bacterial sediment was transported at -20℃ temperature to the Beijing Novogene Company laboratory for transcriptome sequencing and differential expression analyzation. The DESeq software was used for differential expression analysis between the two comparison combinations (two biological replicates per group). Prior to differential gene expression analysis, for each sequenced library, the read count was adjusted by a proportional normalization factor via the DEGSeq package. Differential expression analysis of the two conditions was performed using the DEGseq software package (1.20.0). P value was adjusted by Benjamini and Hochberg methods, and P value 1.5 were used as the threshold of significant differential expression. Strains for metabolome sequencing were prepared as above. Bacterial sediment was transported at -20℃ temperature to Suzhou PANOMIX Biomedical Tech Co. LTD for metabolite quantification and identification and differential expression analysis (four biological replicates per group).By screening metabolites, differential metabolites (biomarkers) were found.The screening criteria for relevant differential metabolites were p-value ≤ 0.05 and fold_change ≥ 1.5 or ≤ 0.667. Finally, statistical map and heat map of differential metabolites were obtained. 2.10. Adhesion and antiphagocytic assays Bacterial adhesion experiments was carried according to Zhang's method 24 : A549 human type II pneumocytes were cultured in a 24-well plate according to laboratory SOP with a density of about 2 ×10 5 cells/well. The bacteria were resuspended with Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher) to 1 ×10 8 colony forming units (CFU)/mL, and 500 µL volumes were usedata multiplicity of infection (MOI) of 100 and incubated with A549 cells at 37°C for 1h. Following incubation, the cells were gently rinsed five times with PBS. The input bacteria were enumerated by plating serial dilutions.For adhesion assays, cells were lysed in sterile ddH 2 O. The lysates were serially diluted and plated on blood agar plates to determine intracellular and extracellular CFUs. For invasion assays, extracellular bacteria were treated with gentamicin (100ug/ml) and penicillin G (10ug/ml). Phagocytosis of pneumococci was determined with mouse peritoneal primary macrophages. The remaining steps were the same as described above. 2.11. Animal experiments All of the animals used in the present study were purchased from the Laboratory Animal Center of Chongqing Medical University. All animal experiments were approved by the Animal Care and Use Committee of Chongqing Medical University and were performed in strict accordance with the regulations of the Guide for the Care and Use of Laboratory Animals. For nasopharyngeal colonization model: themale C57BL/6 mice (6–8 weeks old, weighing 20–21 g) were randomly divided into five groups (n = 6 per group).Each mouse was inoculated with 2 ×10 7 CFU of bacteria through the nasal cavity. Nasal lavage fluid, heart blood, and lung tissues were collected and ground using a mechanical mortar and pestle. The samples were plated on blood agar plates after the appropriate dilutions to determine the CFU. For a model of infection: the mice were divided into six groups (n = 12 per group). Each mouse in five experiment group was infected intranasally with 1 ×10 7 CFU of bacteria. One group was injected the same amount sterile saline as control. The survival rate of the mice was monitored daily for 14 days. Moribund mice were not euthanized prior to the completion of the 14-day survival study. 2.12. Statistical analysis All analyses were performed using GraphPad Prism 8 (GraphPad Software). Statistical differences between groups were compared using Student’st test, and statistical significance was defined as P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). 3 Results 3.1 Transcriptome sequencing analysis of Mga Spn regulatory proteins Mga Spn is a member of the Mga/AtxA family and has high homology with the Mga protein of group A streptococcus (GAS). Mga transcribes and activates many virulence genes in Streptococcus pyogenes 25 , 26 . We previously found that Mga Spn participates in the transcriptional regulation of the cps gene cluster in the capsule and the lic1 teichoic acid synthesis-related gene cluster 20 . To explore whether the Mga Spn global transcriptional regulator mediates other surface virulence factors in addition to the above gene clusters, we conducted transcriptomic sequencing of the WT (D39s) and mgaSpn -deficient (D39Δ mgaSpn ) strains. The transcriptome results showed that there were 149 differential genes, with 113 upregulated genes and 36 downregulated genes above a change cutoff of 1.5-fold.(Table 3 ). The up-regulated genes mainly involved genes related to PTS sugar transporter, bacteriocin and de novo synthesis of uracil, the downregulated genes mainly involved ribosomal proteins and ABC transporters. What attracted us was pyrR , pyrF , carA , and carB pyrimidinede novo synthesis genes were significantly upregulated in mgaSpn -deficient strains. qRT-PCR confirmed higher transcript levels of pyrF , pyrR , carA , and carB , about 2- to 3-fold compared to WT bacteria (Fig. 1 ). These results were consistent with the transcriptome sequencing results, suggesting that Mga Spn maybe involved in the regulation of the uracil synthesis pathway. Table 3 Differential gene expression detected by transcriptome sequencing a Gene id log2FoldChange Gene name Gene description Decreased SPD_RS00470 3.540844073 - sugar ABC transporter permease && PF00528:Binding-protein-dependent transport system inner membrane component SPD_RS00555 3.076620487 - LysM peptidoglycan-binding domain-containing protein && PF01476:LysM domain SPD_RS00580 2.577646712 - transporter substrate-binding domain-containing protein SPD_RS00780 1.758994401 - glycosyltransferase family 2 protein SPD_RS00815 1.794268401 - hypothetical protein SPD_RS00825 1.569853425 - CPBP family intramembrane metalloprotease SPD_RS00830 1.739055601 - MFS transporter SPD_RS01055 1.65768181 nrdG anaerobic ribonucleoside-triphosphate reductase activating protein SPD_RS01450 1.60393733 - Cof-type HAD-IIB family hydrolase SPD_RS01455 1.975312512 - NCS2 family permease SPD_RS01460 1.980699182 - CPBP family intramembrane metalloprotease SPD_RS02415 2.44806895 - hypothetical protein SPD_RS03785 2.792999234 - DUF3270 domain-containing protein SPD_RS04545 2.381183909 - hypothetical protein SPD_RS04555 1.539280353 - ABC transporter permease SPD_RS04770 1.699542615 - amino acid permease SPD_RS05420 1.609153602 - YueI family protein SPD_RS06320 3.167578317 - hypothetical protein SPD_RS06330 2.473874795 - replication initiator protein A SPD_RS07150 1.870233206 - GNAT family N-acetyltransferase SPD_RS07300 1.682021275 - DUF5590 domain-containing protein SPD_RS07520 3.016370291 - OFA family MFS transporter SPD_RS08460 7.007076022 - helix-turn-helix domain-containing protein SPD_RS08685 1.956176742 - xanthine phosphoribosyltransferase SPD_RS08690 1.766305707 - purine permease SPD_RS09375 1.56279407 - HlyC/CorC family transporter SPD_RS09575 1.66801798 - response regulator transcription factor SPD_RS09580 2.278029835 - sensor histidine kinase SPD_RS09585 1.847650149 - ABC transporter permease SPD_RS09590 2.663425036 - ABC transporter ATP-binding protein SPD_RS09600 3.955948867 - hypothetical protein SPD_RS09605 3.485912843 - tRNA-Pro SPD_RS09945 2.148818732 - LysM peptidoglycan-binding domain-containing protein SPD_RS10435 1.674119829 - glycoside hydrolase family 125 protein SPD_RS10725 1.571533956 - thiamine-binding protein SPD_RS10810 2.255949489 - CHAP domain-containing protein Increased SPD_RS00145 -1.879533021 - carbonic anhydrase SPD_RS00185 -2.721181153 - CoA-binding protein SPD_RS00320 -1.556051641 - beta-N-acetylhexosaminidase SPD_RS00330 -1.917993406 - beta-galactosidase SPD_RS00495 -1.989274903 - DUF4299 domain-containing protein SPD_RS00565 -1.859739364 - lactococcin 972 family bacteriocin SPD_RS00600 -4.011238736 - hypothetical protein SPD_RS00605 -3.919982428 - peptidase domain-containing ABC transporter SPD_RS00610 -4.240295599 - GyrI-like domain-containing protein SPD_RS00615 -4.294808957 - HlyD family efflux transporter periplasmic adaptor subunit SPD_RS00620 -4.697260495 - SP_0115 family bacteriocin-like peptide SPD_RS00635 -2.722559642 - SPH_0218 family bacteriocin-like peptide SPD_RS00925 -1.600528958 - 6%2C7-dimethyl-8-ribityllumazine synthase SPD_RS01525 -2.291662237 - PTS cellobiose transporter subunit IIB SPD_RS01530 -1.988115702 - BglG family transcription antiterminator SPD_RS01535 -2.749896408 - PTS cellobiose transporter subunit IIA SPD_RS01605 -2.34063299 - gluconate 5-dehydrogenase SPD_RS01645 -1.570803847 - LacI family DNA-binding transcriptional regulator SPD_RS01850 -1.508244909 rnpB RNase P RNA component class B SPD_RS02045 -2.339857524 - enoyl-CoA hydratase SPD_RS02060 -2.200698741 - acyl carrier protein SPD_RS02065 -2.26950146 fabK enoyl-[acyl-carrier-protein] reductase FabK SPD_RS02070 -2.833084477 fabD ACP S-malonyltransferase SPD_RS02075 -3.246698575 fabG 3-oxoacyl-[acyl-carrier-protein] reductase SPD_RS02080 -3.45560506 fabF beta-ketoacyl-ACP synthase II SPD_RS02085 -3.11639046 - acetyl-CoA carboxylase biotin carboxyl carrier protein SPD_RS02090 -3.773105326 fabZ 3-hydroxyacyl-ACP dehydratase FabZ SPD_RS02095 -3.111535703 accC acetyl-CoA carboxylase biotin carboxylase subunit SPD_RS02100 -2.954741497 - acetyl-CoA carboxylase carboxyltransferase subunit beta SPD_RS02105 -3.004358554 - acetyl-CoA carboxylase carboxyl transferase subunit alpha SPD_RS02115 -3.264746808 briC biofilm-regulating peptide BriC SPD_RS02125 -3.445879425 - CPBP family intramembrane metalloprotease SPD_RS02375 -1.797354206 - CTP synthase SPD_RS02470 -1.54647738 grpE nucleotide exchange factor GrpE SPD_RS02475 -1.953093782 dnaK molecular chaperone DnaK SPD_RS02480 -2.229957944 - hypothetical protein SPD_RS02510 -4.32306266 - hypothetical protein SPD_RS02530 -4.34995027 blpC quorum-sensing system pheromone BlpC SPD_RS02545 -3.257627149 - hypothetical protein SPD_RS02550 -3.335877819 - CPBP family intramembrane metalloprotease SPD_RS02555 -3.112489772 - hypothetical protein SPD_RS02565 -3.22119207 - CPBP family intramembrane metalloprotease SPD_RS03010 -1.953581792 - S8 family serine peptidase SPD_RS03025 -2.389396089 - PTS galactitol transporter subunit IIC SPD_RS03275 -1.702707644 pyrF orotidine-5'-phosphate decarboxylase SPD_RS03280 -1.569197499 - orotate phosphoribosyltransferase SPD_RS03475 -2.511480332 - DegV family protein SPD_RS03535 -2.413688835 - CBS domain-containing protein SPD_RS03675 -2.271067339 - hypothetical protein SPD_RS03695 -1.625360236 gor glutathione-disulfide reductase SPD_RS03840 -2.086302291 - DUF1827 family protein SPD_RS03940 -2.152351916 - DNA topology modulation protein SPD_RS04120 -2.042616047 ssrA transfer-messenger RNA SPD_RS04130 -1.710636931 - DeoR/GlpR transcriptional regulator SPD_RS04150 -1.831943834 - hypothetical protein SPD_RS04305 -2.105403339 - PspC domain-containing protein SPD_RS04310 -4.676754015 - hypothetical protein SPD_RS04580 -2.184672578 - dihydroorotate dehydrogenase electron transfer subunit SPD_RS04585 -2.022003954 - dihydroorotate dehydrogenase SPD_RS04590 -1.737808776 - endo-beta-N-acetylglucosaminidase SPD_RS04815 -1.786395769 hemH ferrochelatase SPD_RS04930 -1.505667841 - iron-siderophore ABC transporter substrate-binding protein SPD_RS05010 -1.918930761 - helix-turn-helix transcriptional regulator SPD_RS05165 -1.503059453 - metal-sulfur cluster assembly factor SPD_RS05630 -1.759193213 - transcription antiterminator SPD_RS05635 -2.003549678 lacD tagatose-bisphosphate aldolase SPD_RS05640 -2.458126365 - tagatose-6-phosphate kinase SPD_RS05645 -2.994294299 lacB galactose-6-phosphate isomerase subunit LacB SPD_RS05650 -2.8056828 lacA galactose-6-phosphate isomerase subunit LacA SPD_RS06035 -1.535668533 carB carbamoyl-phosphate synthase large subunit SPD_RS06040 -1.600881094 carA glutamine-hydrolyzing carbamoyl-phosphate synthase small subunit SPD_RS06050 -1.644316117 pyrR bifunctional pyr operon transcriptional regulator/uracil phosphoribosyltransferase PyrR SPD_RS06070 -1.732221049 - ABC-F family ATP-binding cassette domain-containing protein SPD_RS06090 -1.712128317 - uracil transporter SPD_RS06305 -1.590012213 - lanthionine synthetase SPD_RS06490 -1.939313287 - ABC transporter ATP-binding protein SPD_RS06650 -1.526791226 - 30S ribosomal protein S21 SPD_RS06750 -1.568313221 - ABC transporter ATP-binding protein SPD_RS06910 -1.806831456 - DUF1836 domain-containing protein SPD_RS06915 -2.426500081 - hemolysin III family protein SPD_RS07065 -1.548217135 - thioredoxin SPD_RS07525 -2.25529155 - FAD-containing oxidoreductase SPD_RS08060 -1.610646583 - hypothetical protein SPD_RS08110 -1.939427257 - ABC transporter ATP-binding protein SPD_RS08115 -1.655062375 - membrane protein SPD_RS08150 -1.793486751 - PTS beta-glucoside transporter subunit IIBC SPD_RS08330 -1.862798757 - DUF4649 family protein SPD_RS08335 -1.549051733 trxA thioredoxin SPD_RS08400 -2.034277727 - type II toxin-antitoxin system HicA family toxin SPD_RS08485 -1.805631069 - CsbD family protein SPD_RS08875 -2.139807301 treP PTS system trehalose-specific EIIBC component SPD_RS09185 -2.567762138 ply cholesterol-dependent cytolysin pneumolysin SPD_RS09190 -2.192158071 - hypothetical protein SPD_RS09195 -2.092791382 - hypothetical protein SPD_RS09200 -2.39262 - DUF4231 domain-containing protein SPD_RS09435 -2.318458004 - acylphosphatase SPD_RS09550 -2.260043723 - universal stress protein SPD_RS09790 -2.812594241 ulaG L-ascorbate 6-phosphate lactonase SPD_RS09825 -3.524245923 - PTS ascorbate transporter subunit IIC SPD_RS10125 -2.768053314 - phosphate-binding protein SPD_RS10225 -1.72876586 - membrane protein SPD_RS10245 -1.947416517 - extracellular solute-binding protein SPD_RS10405 -3.798523107 pcpA choline-binding protein PcpA SPD_RS10500 -1.757549455 - SPFH domain-containing protein SPD_RS10525 -2.529211255 - PTS mannose/fructose/sorbose transporter family subunit IID SPD_RS10530 -3.863757933 - PTS mannose/fructose/sorbose/N-acetylgalactosamine transporter subunit IIC SPD_RS10535 -4.532680442 - PTS sugar transporter subunit IIB SPD_RS10540 -4.727078471 - PTS sugar transporter subunit IIA SPD_RS10555 -3.778092141 - rhamnulokinase SPD_RS10630 -3.069439605 - hypothetical protein SPD_RS10755 -2.298327647 raiA ribosome-associated translation inhibitor SPD_RS10905 -1.809492243 - TetR/AcrR family transcriptional regulator SPD_RS10945 -1.897430199 - trypsin-like peptidase domain-containing protein a The reference genome comes from NCBI RefSeq assembly GCF_000014365.2. https://www.ncbi.nlm.nih.gov/nuccore/NC_008533 3.2. Mga Spn is involved in the regulation of the uracil synthesis pathway To investigate if Mga Spn participates in the uracil synthesis pathway, we examined the metabolism of WT and mgaSpn-deficient bacteria cultured in C + Y medium to an OD620 of 0.5 by metabolome sequencing(Fig. 2 ). There were 31 different metabolites in the two strains (Table 4 ). Compared to WT bacteria, the contents of 15 metabolites, including limonene- 1,2-diol and L-histidine, in mgaSpn - deficient bacteria were increased. The contents of 16 metabolites, including N-acetyl-neuraminic acid, gulonic acid, UMP, pyrimidodiazepine, and D-ribose, were decreased. Of note, UMP and pyrimidodiazepine metabolites in the uracil synthesis pathway were significantly reduced by 6- to 7- fold in mgaSpn -deficient bacteria. We previously confirmed that Mga Spn is a transcription suppressor in capsule biosynthesis 20 .Deletion of Mga Spn increases the capsular content in the whole bacterial lysate, and the small molecular weight proteins are concentrated in the increased capsular content 20 . However, the reason for the increase in the small molecular weight capsular proteins remains unclear. The uracil synthesis pathway has been shown to affect capsular polysaccharides (CPS) promoter expression and CPS production in the S. pneumoniae D39 strain 7 . After Mga Spn deletion, the transcriptomic results showed that the expression of uracil synthesis genes was increased, and the metabolomics results showed that the uracil synthesis pathway metabolites were decreased. These findings suggested that Mga Spn may affect capsule synthesis by regulating the uracil synthesis pathway. Table 4 Differential metabolites detected by metabolome sequencing VIP log2(FC_M/D) p.value N-Acetyl-a-neuraminic acid 1.87893 -19.098 0.021071 Gulonic acid 1.860573 -1.7987 0.030383 UMP 1.836096 -15.964 0.021071 5-Acetamidovalerate 1.778424 -3.8623 0.030383 Dimethyl sulfone 1.774266 -1.8729 0.030383 Limonene-1,2-diol 1.714941 2.7051 0.030383 D-Ribose 1.713519 -1.0715 0.030383 Guanosine 1.689839 1.8766 0.030383 Tetracosanoic acid 1.68122 0.92923 0.030383 Lanosterin 1.637613 -1.5964 0.030383 (-)-Epigallocatechin 1.610466 -16.227 0.021071 5-Guanidino-3-methyl-2-oxopentanoate 1.589845 -2.3838 0.030383 Pyrimidodiazepine 1.57903 -2.8171 0.030383 L-Histidine 1.577359 0.96313 0.030383 Phenyl acetate 1.575216 0.47183 0.030383 Acetylcholine chloride 1.554901 2.5472 0.030383 (S)-1-Phenylethanol 1.547117 1.1176 0.030383 Imidazol-5-yl-pyruvate 1.544352 0.31394 0.030383 D-Glucuronic Acid 1.52796 1.379 0.030383 12,13-DHOME 1.503511 -0.89095 0.030383 Spermidine 1.493071 -1.1778 0.030383 L-Erythrulose 1.453958 0.79214 0.030383 Stearic acid 1.365478 -1.7833 0.030383 Adenosine diphosphate ribose 1.361712 -1.523 0.030383 L-2-Hydroxyglutaric acid 1.33444 -1.7077 0.030383 4-Oxoproline 1.306298 2.1815 0.030383 Maleimide 1.293903 -1.4198 0.030383 4-Pyridoxic acid 1.271316 2.5939 0.030383 11-Dehydrocorticosterone 1.196153 1.5698 0.030383 Dehydroepiandrosterone 1.189842 1.7868 0.030383 Methyl (indol-3-yl)acetate 1.185804 2.3809 0.030383 3.3. Mga Spn binds to the pyrR promoter region Mga Spn , a member of the Mga/AtxA family of global transcriptional regulators, directly binds to the regulatory regions of target genes to regulate target gene expression. We found some differentially expressed genes the pyrR , pyrF , carA , and carB uracil de novo synthesis genes by transcriptomic analysis. To identify the uracil metabolism genes that Mga Spn directly regulates, we investigated the expression of the pyrR , pyrF , carA , and carB genes in WT bacteria (D39s), mgaSpn -deficient bacteria (D39∆ mgaSpn ), mgaSpn complement bacteria (D39∆ mgaSpn :: mgaSpn ), and mgaSpn -overexpressing bacteria (D39:: mgaSpn ). As shown in Fig. 3 , all genes were upregulated after mgaSpn deletion, but only pyrR was about 3-fold downregulated during mgaSpn recovery and overexpression strains, indicating that Mga Spn maybe directly involved in the regulation of pyrR . Mga Spn is a transcriptional regulator that contains two conserved helix-turn-helix (HTH) domains, which are DNA-binding motifs, indicating that Mga Spn has the ability to bind DNA27. To explore whether Mga Sp n protein directly participates in the transcriptional regulation of pyrR , EMSAs were performed using a DNA fragment probe approximately 300 bp upstream of the pyrR gene to verify the specific binding of the Mga Spn protein to the pyrR promoter. As the concentration of protein added to the reaction system gradually increased, the binding bands of the probes and proteins shifted backward, indicating that the binding of Mga Spn to the pyrR promoter was concentration-dependent (Fig. 4 a). In the unlabeled probe competition lane, the unlabeled probes competed to bind to the mgaSpn - and pyrR -labeled probes. These results indicated that Mga Spn specifically binds to the pyrR promoter region. Because Mga Spn directly binds to the pyrR promoter region, we performed DNase Ⅰ footprinting analysis to determine the specific recognition site of Mga Spn on the P pyrR probe (Fig. 4 b). After adding protein (1.5 µg) to the 390 ng probe system, the following two protection areas were identified: 31 bp (5'-TAGCAATTTGTAAGATGCTACATTGAAACTT-3') and 53 bp (5'-TTGTTTAAGGAGACTTTTCTTTTTGGAGGTTTGTCATGAAAACAAAAGAAGTT-3') (Fig. 4 c). To further confirm that Mga Spn specifically binds to P pyrR , we designed eight mutations in the binding site of the P pyrR promoter. The mutant pyrR probe EMSA results showed that Mga Spn lost the ability to bind the mutant probe(Figure S2 ). However, additional experiments are required to determine if these two binding sites are functional sites for Mga Spn transcriptional regulation. These data suggested that Mga Spn may play a key role in the co-transcription of pyrR genes. 3.4. Mga Spn negatively regulates CPS production by pyrR We next evaluated the morphology of the capsules of each strain grown to an OD620 of 0.5 in normal C + Y medium containing 15mg L- 1 pyrimidines using transmission electron microscopy (TEM) (Fig. 5 a). There was no obvious difference in capsule thickness when comparing the D39s strain to the D39∆ mgaSpn and D39∆ pyrR strains, but the D39∆ mgaSpn ∆ pyrR strain had a thinner capsule than the D39s strain.The transmission electron microscopy (TEM) was used to measure the capsule thickness of 10 strain with ImageJ software, and the average capsule thicknesses of the D39s, D39∆ mgaSpn , D39∆ pyrR , and D39∆ mgaSpn ∆ pyrR strains were 57.03 ± 14.73 nm, 56.51 ± 10.52 nm, 53.84 ± 17.45nm, and 30.32 ± 6.97nm, respectively (Fig. 5 a). We also detected the capsular content of the bacteria using ELISAs. When the bacteria grew to an OD620 of 0.5, the same amount of bacteria was collected by centrifugation for ELISA detection. Figure 5 b shows that there was no obvious difference in the capsular content between the D39s and D39∆ mgaSpn strains grown in normal C + Y medium contains 15mg L- 1 pyrimidines, and the capsular content of the D39∆ pyrR -( grown in uracil-free C + Y medium) and ∆D39∆ mgaSpn ∆ pyrR strains was 2 to 3 times lower than that of the D39s and D39∆ mgaSpn strains, which was consistent with the TEM results. In addition, capsular polysaccharides were quantified by the uronic acid method. Glucuronic acid is a specific component of S. pneumoniae capsules regulated by CpsT/F/G/L, and it exists in the form of glucuronic acid in type II capsules, with each repeating unit containing one glucuronic acid. The content of the capsule in bacteria can be measured by detecting the content of uronic acid. The capsular content of the whole bacteria (Bacteria-CPS) was detected, and the results showed that the capsular content of the D39∆ mgaSpn ∆ pyrR strain was significantly lower than that of the WT strain and mgaSpn -deficient strains (Fig. 5 c). There were fewer pods of the D39∆ pyrR -deficient strain grown in uracil-free C + Y than the WT and mgaSpn -deficient strains. We next collected the whole bacterial lysate of each strain and detected the expression of capsules by Western blot analysis (Fig. 5 d). The total capsular content of the D39∆ mgaSpn strain was significantly increased compared to that of the D39s strain, and the small molecular weight capsular proteins of the D39∆ pyrR strain were slightly decreased compared to that of the D39s strain, which maybe due to the presence of a remedial synthesis pathway in uracil C + Y. Therefore, we detected the expression of capsules in the D39∆ pyrR strain grown in uracil-free C + Y medium (D39∆ pyrR -). The expression of capsules in the D39∆ pyrR - and D39∆ mgaSpn ∆ pyrR strains was significantly decreased, but the expression of capsules was recovered after supplementing the D39∆ mgaSpn ∆ pyrR strain with pyrR . These results indicated that both mgaSpn and pyrR are involved in the regulation of capsule synthesis and mgaSpn has a positive synergistic effect on the regulation of the capsule by pyrR . 3.5. pyrR influences adhesion and pathogenicity of S. pneumoniae To understand the effect of pyrR deficiency on the virulence of S. pneumoniae , we used the A549 lung epithelial cell line to detect the adhesion and invasion ability of the D39s, D39∆ pyrR , D39∆ pyrR -, D39∆ mgaSpn , and D39∆ mgaSpn ∆ pyrR :: pyrR strains. As shown in Fig. 6 a, the D39s and D39∆ pyrR strains adhered to epithelial cells 2–3 fold greater than other strains, and the D39∆ mgaSpn strain had a stronger invasion ability than the other strains. In addition, the D39∆ pyrR - and D39∆ mgaSpn ∆ pyrR strains had significantly reduced invasion and adhesion abilities compared to the other strains (Fig. 6 b). Previous studies have suggested that during colonization, S. pneumoniae express low levels of CPS to enhance the exposure of cell surface proteins and promote binding to epithelial cells 28 . However, the invasion and adhesion of low-level capsularstrains, such as the D39∆ pyrR - and D39∆ mgaSpn ∆ pyrR strains, were reduced. These findings suggested that the simultaneous loss of mgaSpn and pyrR may also lead to changes in other adherence-related virulence factors. Because thicker capsules help S. pneumoniae escape phagocytosis, we evaluated the anti-phagocytic ability of the strains after incubation with mouse macrophages in the absence of serum. The damaged capsules of the D39∆ pyrR - and D39∆ mgaSpn ∆ pyrR strains led to significantly reduced anti-phagocytic effects on macrophages(Fig. 6 C). 3.6. pyrR is involved in systemic virulence and nasopharyngeal colonization To explore the role of pyrR in systemic infection, the experimental mice were divided into five groups, with 6 mice in each group, and were treated with the D39s, D39∆ pyrR , D39∆ mgaSpn , D39∆ mgaSpn ∆ pyrR , and D39∆ mgaSpn ∆ pyrR :: pyrR strains through nasal drops. At 48 h after bacterial infection, nasal lavage fluid, heart blood, and lung tissues were harvested and used for colony counting (Fig. 7 a-c). In addition, another five groups of mice, with 12 mice in each group, were infected with the D39s, D39∆ pyrR , D39∆ mgaSpn ,D39∆ mgaSpn ∆ pyrR and D39∆ mgaSpn ∆ pyrR :: pyrR strains through the nasal passage, and the survival time of each group of mice was recorded for 14 days (Fig. 7 d). In vivo virulence tests showed that the bacterial load of the double deficient strain was significantly reduced in thenasopharyngeal lavage solution, lung tissues, and heart blood. In the double deficient strain, the colonization ability of the nasal cavity and lung was significantly reduced, and the survival rate was significantly increased. All abilities of pyrR were recovered after pyrR replenishment. These results indicated that Mga Spn affects the pathogenicity of S. pneumoniae through pyrR . 4 Discussion In many cases, the production of pathogen capsules is related to central metabolism, such as in S. pyogenes , and zinc interferes with central metabolism and capsule biosynthesis4. Deficiency of spxB in S. pneumonia e causes peptidoglycan and fatty acid biosynthesis metabolic imbalance, resulting in reduced capsule synthesis 29 , and impaired polyamine synthesis affects the availability of the serum type 4 CPS precursor, UDP-galactose, and the nucleotide sugar precursor, UDP-N-acetylglucosamine (UDP-GLcNAc), for the biosynthesis of CPS and peptidoglycan repeat units 30 . The interaction between capsule synthesis and metabolism is complex, and there are many unknowns. In the present study, we investigated the association between S. pneumoniae uracil metabolism and capsule synthesis. We previously found that Mga Spn protein negatively regulates the synthesis of capsular polysaccharides, and transcriptome and metabolome sequencing indicated changes in the metabolism of uracil and related molecules. Subsequently, we screened the uracil de novo synthesis gene, pyrR , by qRT‒PCR. PyrR is a bifunctional protein that not only encodes UPRTase but also regulates the de novo synthesis pathway of organisms through a transcriptional attenuation regulation mechanism. Recent studies on PyrR have focused on Bacillus subtilis 31 – 33 , and no DNA-binding proteins regulating the expression of the bacterial de novo synthesis gene have been reported. In the present study, an EMSA demonstrated that Mga Spn specifically binds P pyrR , and a DNase I footprinting assay identified potential binding sites. Uracil metabolism plays an important role in the growth and energy production of S. pneumoniae . The growth rate and maximum biomass of pyrR -deficient bacteria significantly differ in the C + Y medium presence or absence of uracil. Pyrimidine biosynthesis can be divided into remedial synthesis and de novo synthesis 5 . In medium with uracil C + Y, remedial synthesis compensates for part of the energy loss caused by pyrimidine deficiency. In uracil‒free C + Y medium, the growth rate and maximum biomass of pyrR -deficient bacteria are significantly reduced, and both remedial synthesis and de novo synthesis are blocked. Deficiency in the pyrimidine metabolic pathway leads to growth retardation and metabolic disorders in S. pneumoniae . The growth rate and maximum biomass of mgaSpn - and pyrR -double deficient bacteria were not significantly different in medium with or without uracil (Figure S3 ). These findings suggested that there maybe a compensatory mechanism. When mgaSpn and pyrR are absent and bacteria are in a relatively nutrient-deficient environment,bacteria reduce unnecessary energy burden by decreasing capsules. Gram staining also identified a shorter chain length(Figure S4 ), which would allow more energy to be available for growth and reproduction. In the present study, the expression of small molecular weight capsular proteins slightly decreased when pyrR was deficient, which may have been due to the presence of remedial synthesis to compensate for the absence of pyrR . Therefore, we detected the expression of the capsule in the absence of uracil, and the results showed that the expression of the capsule was significantly reduced. However, defects of mgaSpn and pyrR at the sametime significantly reduced capsule expression, and capsule expression was recovered with pyrR replacement. The above results suggested that Mga Spn affects capsule synthesis by regulating pyrR . Because the biosynthesis of capsular polysaccharides is regulated by cps operons, many studies have used cps operons as the starting point to explore the biosynthesis mechanism of capsular polysaccharides 34 , 35 . The transcriptional activity of P cps plays an important role in the transcriptional regulation of genes downstream of the capsule. However, we observed that pyrR -deficient bacteria had no effect on P cps in medium with and without uracil (data not shown). In addition, the P cps activity of the D39∆ mgaSpn strain was significantly higher than that of the D39s strain, which was consistent with a previous study, demonstrating that Mga Spn is a transcriptional suppressor of capsule biosynthesis. Compared to the presence of uracil, the promoter of the four strains decreased in the absence of uracil (data not shown). The capsule is considered to be a large energy burden, and its production is thought to directly compete with central metabolism for energy 36 . The present findings suggested that in medium without uracil, the low yield of capsule is associated with less energy demand, and in the nutrient-deficient environment, more energy is used for growth and reproduction. The P cps activity of the D39∆ mgaSpn ∆ pyrR strain was higher than that of the D39s strain. However, Western blot analysis, ELISAs, uronic acid assays, and TEM observations showed that the capsular content of the D39∆ mgaSpn ∆ pyrR strain was lower than that of the D39s strain. The polysaccharide synthesis of S. pneumoniae capsule is divided into several stages as follows: synthesis of capsule precursors, polymerization of repeat units, polymer flipping, and final localization 37 . Ghim and Neuhard 38 studied pyrR in thermosoluble Bacillus SPP and found that it catalyzes the UPRTase reaction. The present study suggested that the function of PyrR UPRTase plays a major role. The decrease of capsules caused by PyrR deficiency does not directly affect P cps activity, but the decrease of UPRTase PyrR affects capsule precursor synthesis, similar to a "raw material" loss. Previous studies have suggested that S. pneumoniae expresses low levels of CPS during colonization to enhance cell surface protein exposure and promote binding to epithelial cells 39 , 40 . In contrast, S. pneumoniae expresses high levels of CPS during systemic infection to escape complement-mediated opsonophagy 41 . Based on the CPS reduction phenotypes observed in the D39∆ pyrR - and D39∆ mgaSpn ∆ pyrR strains, we speculated that the D39∆ pyrR - and D39∆ mgaSpn ∆ pyrR strains have increased adhesion to epithelial cells and decreased anti-phagocytic ability. As expected, the D39∆ pyrR - and D39∆ mgaSpn ∆ pyrR strains invaded the A549 lung epithelial cell line, and the anti- phagocytotic ability of mouse macrophages was decreased. In contrast to expectations, there was a decrease in adhesion. The adhesion ability of S. pneumoniae is related to the SpsA choline-binding protein, the PfbA plasminase-binding protein, and the PavA S. pneumoniae adhesion factor 42 – 44 . Thus, the D39∆ pyrR - and D39∆ mgaSpn ∆ pyrR strains may also cause changes in other virulence factors in addition to the reduction of capsular content, causing defects in bacterial cell adhesion. The significantly shorter chain length observed by gram staining may also be responsible for the reduced adhesion, warranting additional studies(Figure S4 ). According to the site of bacterial colonization and the stage of infection, the thickness and density of the capsule are dynamic 45 . In vivo experiments demonstrated that the D39∆ mgaSpn and D39∆ mgaSpn ∆ pyrR :: pyrR strains with thicker capsulates were more aggressive and more likely to circulate to the spleen to cause systemic infection in mice. Mga Spn regulates virulence and metabolism-related genes responding to various environmental changes 19 . Because the content of uracil varies with different niches 46 , Mga Spn may regulate the virulence and adaptability of S. pneumoniae by sensing environmental changes, such as uracil content, during infection. Overall, the in vivo and in vitro experiments showed that Mga Spn regulates capsule production through pyrR , thereby affecting its virulence. An increasing number of studies have shown that there is a close relationship between the bacterial metabolic network and toxicity. The perception of nutritional availability of pathogenic bacteria drives the regulation and production of various virulence factors. The energy obtained by bacteria can be used to synthesize carbohydrate capsules, or it can be transferred to glycolysis to support replication 36 . Therefore, it is not surprising that capsule biosynthesis is integrated into the metabolic regulatory network of S. pneumoniae . For the first time, the present study demonstrated that the pyrR de novo synthesis gene of S. pneumoniae is regulated by the Mga Spn transcriptional regulator. The present results suggested that Mga Spn inhibits the expression of pyrR , leading to a decrease in polysaccharide synthesis in capsules. The decrease of polysaccharide in the capsule is due to the decrease of the "raw material" for capsule synthesis due to the decrease of PyrR UPRTase rather than affecting P cps activity, thus indicating the link between uracil metabolism and capsule production. Exploring the mechanism of pyrimidine metabolism and capsule synthesis, especially how pyrimidine intermediates affect the regulation of capsule expression at the molecular level, will help to better understand the regulatory phenomena controlling S. pneumoniae capsule synthesis and identify new therapeutic strategies affecting its virulence. Abbreviations CPS: Capsular polysaccharide; E.coli: Escherichia coli; EMSA: Electrophoretic mobility shift assay; S.pn: Streptococcus pneumoniae; UMP: Uridylic acid; UTP: Uridine triphosphate Declarations Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Consent for publication Not applicable. Author details 1 Department of Medicine Laboratory, Children’s Hospital of Chongqing Medical University;National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders; and Chongqing Key Laboratory of Pediatrics, Chongqing, People’s Republic of China; 2 Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Chongqing Medical University, Chongqing, People’s Republic of China; 3 Department of Pediatrics, Shengli Oil Field Central Hospital, Dongying 257034, China; 4 Department of Laboratory Medicine, The First Hospital of Changsha, 311 Yingpan Road, Changsha 410005, Hunan, China Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Authors’ contributions Yuqiang Zheng, Xuemei Zhang, andYibing Yin conceived and designed the experiments. Xinlin Guo, Shuhui Wang and Ye Tao performed the experiments. Xinlin Guo and Weicai Suo analyzed the data. Li lei and Yapeng Zhang contributed the reagents, materials, and analysis tools. Xinlin Guo and Yapeng Zhang wrote the paper. All authors read and approved the final manuscript. Acknowledgments This work was supported by Projects of the National Natural Science Foundation of China (No. 81871698 and No. 81772153). Funding This work was supported by Projects of the National Natural Science Foundation of China (No. 81871698 and No. 81772153). References Prina, E., Ranzani, O. T. & Torres, A. Community-acquired pneumonia. Lancet 386 , 1097–1108 (2015). Suaya, J. A. et al Identification of Streptococcus pneumoniae in hospital-acquired pneumonia in adults. J Hosp Infect 108 , 146–157 (2021). Weiser, J. N., Ferreira, D. M. & Paton, J. C. 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Capsule type of Streptococcus pneumoniae determines growth phenotype. PLOS Pathog 8 , e1002574 (2012). Cartee, R. T., Forsee, W. T., Bender, M. H., Ambrose, K. D. & Yother, J. CpsE from Type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating Type 2 capsule repeat unit formation. J Bacteriol 187 , 7425–7433 (2005). Ghim, S. Y. & Neuhard, J. The pyrimidine biosynthesis operon of the thermophile Bacillus caldolyticus includes genes for uracil phosphoribosyltransferase and uracil permease. J Bacteriol 176 , 3698–3707 (1994). Wartha, F. et al Capsule and d-alanylated lipoteichoic acids protect Streptococcus pneumoniae against neutrophil extracellular traps. Cell Microbiol 9 , 1162–1171 (2007). de Vos, A. F. et al The polysaccharide capsule of Streptococcus pneumonia partially impedes MyD88-mediated immunity during pneumonia in mice. PLOS ONE 10 , e0118181 (2015). Engholm, D. H., Kilian, M., Goodsell, D. S., Andersen, E. S. & Kjærgaard, R. S. A visual review of the human pathogen Streptococcus pneumoniae. FEMS Microbiol Rev 41 , 854–879 (2017). Pracht, D. et al. PavA of Streptococcus pneumoniae modulates adherence, invasion, and meningeal inflammation. Infect Immun 73 , 2680–2689 (2005). Iovino, F., Molema, G. & Bijlsma, J. J. E. Platelet endothelial cell adhesion Molecule-1, a putative receptor for the adhesion of Streptococcus pneumoniae to the vascular endothelium of the blood-brain barrier. Infect Immun 82 , 3555–3566 (2014). Hammerschmidt, S. et al The host immune regulator factor H interacts via two contact sites with the PspC protein of Streptococcus pneumoniae and mediates adhesion to host epithelial cells. J Immunol 178 , 5848–5858 (2007). Shak, J. R., Vidal, J. E. & Klugman, K. P. Influence of bacterial interactions on pneumococcal colonization of the nasopharynx. Trends Microbiol 21 , 129–135 (2013). Aprianto, R., Slager, J., Holsappel, S. & Veening, J. W. High-resolution analysis of the pneumococcal transcriptome under a wide range of infection-relevant conditions. Nucleic Acids Res 46, 9990–10006 (2018) doi:10.1093/nar/gky750. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Additional file 1: Figure S1. Verification of D39∆pyrR mutant. The pyrR mRNA of D39s, D39Δ pyrR, D39 ∆MgaSpn∆pyrR and D39Δ MgaSpn Δ pyrR :: pyrR were determined by real-time quantitative PCR (qPCR). qPCR was conducted in triplicate. P < 0.05 ( ), P < 0.01 ( ), and P < 0.001 ( ). Additionalfile2.docx Additional file 2: Figure S2. EMSA of Mga Spn protein with P pyrR mutation probes. (a)P pyrR is the original pyrR promoter sequence, P pyrR mutation is the mutant promoter sequence. The yellow background region is the specific recognition site of Mga Spn in P pyrR , while the blue background region is the mutation sequence. (b) EMSA of 6 × His-MgaSpn protein with P pyrR mutation probes. Additionalfile3.docx Additional file 3: Figure S3. Growth profies of D39s(circles), D39∆Mga Spn (squares), D39∆ pyrR (triangles) and D39ΔMga Spn Δ pyrR :: pyrR (inverted triangles) strains in C+Y medium(closed symbols) and uracil-free C+Y medium (open symbols). The growth rates (h− 1) for each strain are indicated in the graph and the values shown are means at 5h from three biological replicates ± SD. “-”represents grown in uracil-free C+Y medium. Additionalfile4.docx Additional file 4: Figure S4. Bacterial morphology. Gram staining was used to observe the Chain length (×40 objective). Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 01 Jul, 2024 Submission checks completed at journal 01 Jul, 2024 First submitted to journal 21 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4618066","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":321173255,"identity":"fe87d060-4bff-400e-a46b-f1dee1a2c6e7","order_by":0,"name":"Xinlin Guo","email":"","orcid":"","institution":"Department of Medicine Laboratory, Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinlin","middleName":"","lastName":"Guo","suffix":""},{"id":321173256,"identity":"e10494da-7165-45d5-a36d-4b49e149ccc0","order_by":1,"name":"shuhui wang","email":"","orcid":"","institution":"Department of Medicine Laboratory, Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"shuhui","middleName":"","lastName":"wang","suffix":""},{"id":321173257,"identity":"4e1305f2-661b-4688-92f1-6a5d47734495","order_by":2,"name":"Ye Tao","email":"","orcid":"","institution":"Department of Medicine Laboratory, Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Tao","suffix":""},{"id":321173258,"identity":"b25bf6f8-8399-4189-aaec-b28927b3a36f","order_by":3,"name":"Xuemei Zhang","email":"","orcid":"","institution":"Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuemei","middleName":"","lastName":"Zhang","suffix":""},{"id":321173259,"identity":"e23be4be-1085-4c21-9199-7378e147d9d8","order_by":4,"name":"Weicai Suo","email":"","orcid":"","institution":"Department of Pediatrics, Shengli Oil Field Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weicai","middleName":"","lastName":"Suo","suffix":""},{"id":321173260,"identity":"f9155166-33e1-408a-98ab-3972291fcceb","order_by":5,"name":"Yapeng Zhang","email":"","orcid":"","institution":"Department of Laboratory Medicine, The First Hospital of Changsha","correspondingAuthor":false,"prefix":"","firstName":"Yapeng","middleName":"","lastName":"Zhang","suffix":""},{"id":321173261,"identity":"20ac3ca6-628c-4a2c-9471-0a653c4e797f","order_by":6,"name":"Li Lei","email":"","orcid":"","institution":"Department of Medicine Laboratory, Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Lei","suffix":""},{"id":321173262,"identity":"5792d26e-ad12-4e4a-8258-145bd7ff6130","order_by":7,"name":"Yibing Yin","email":"","orcid":"","institution":"Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yibing","middleName":"","lastName":"Yin","suffix":""},{"id":321173263,"identity":"f2d1910a-9bf4-44b4-93a6-d610f07f578a","order_by":8,"name":"Yuqiang Zheng","email":"data:image/png;base64,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","orcid":"","institution":"Department of Medicine Laboratory, Children’s Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yuqiang","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2024-06-21 16:06:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4618066/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4618066/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60932546,"identity":"df8a20d0-6cef-4346-b269-3e1eed2b787f","added_by":"auto","created_at":"2024-07-23 17:30:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20420,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eqRT‒PCR analysis of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecarA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecarB\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epyrR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epyrF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in the D39s and D39Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMgaSpn\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e derivative strains. \u003c/strong\u003eThe expression of the \u003cem\u003ecarA\u003c/em\u003e, \u003cem\u003ecarB\u003c/em\u003e, \u003cem\u003epyrR\u003c/em\u003e, and \u003cem\u003epyrF\u003c/em\u003e genes in \u003cem\u003eMgaSpn\u003c/em\u003e-deficient bacteria was increased by 2–3 times compared to that in the D39s strain. mRNA levels are expressed relative to that of gyrB. Data are presented as the mean ± SD of three independent experiments, with biological duplicates. ***P \u0026lt; 0.001, **P \u0026lt; 0.01, and not significant (NS) as analyzed by unpaired two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/1bb2e83619651e531acc2939.jpg"},{"id":60932877,"identity":"2670d36a-a739-4dff-a229-592283a4a588","added_by":"auto","created_at":"2024-07-23 17:38:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap of metabolites. \u003c/strong\u003eM represents \u003cem\u003eMgaSpn\u003c/em\u003e-deficient bacteria, and D represents D39s.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/3999ff4415a8670159047570.jpg"},{"id":60932878,"identity":"893758a4-0a79-4dfb-821b-3201684c9396","added_by":"auto","created_at":"2024-07-23 17:38:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of uracil-related genes. \u003c/strong\u003eDetermination of the expression the \u003cem\u003epyrR\u003c/em\u003e, \u003cem\u003epyrF\u003c/em\u003e, \u003cem\u003ecarA\u003c/em\u003e, and \u003cem\u003ecarB\u003c/em\u003e in the D39s, D39Δ\u003cem\u003eMgaSpn\u003c/em\u003e, D39Δ\u003cem\u003eMgaSpn\u003c/em\u003e::\u003cem\u003eMgaSpn\u003c/em\u003e, and D39::\u003cem\u003eMgaSpn\u003c/em\u003e strains by quantitative RT-PCR. mRNA levels are expressed relative to that of gyrB. Data are presented as the mean ± SD of three independent experiments, with biological duplicates. ***P \u0026lt; 0.001, **P \u0026lt; 0.01, and not significant (NS) as analyzed by unpaired two-tailed Student’s t test.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/bc1c5489dea82dc8a42a40d8.jpg"},{"id":60933138,"identity":"bdf162b7-fa66-4fda-b4cc-e4867d84421b","added_by":"auto","created_at":"2024-07-23 17:46:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":40642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMga\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSpn\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e binds to the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epyrR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e probe. \u003c/strong\u003e(a) EMSA of P\u003cem\u003epyrR\u003c/em\u003e using the Mga\u003cem\u003eSpn\u003c/em\u003e protein. (b) DNase I footprinting protection assay of Mga\u003cem\u003eSpn\u003c/em\u003e. (c) Structural organization of the \u003cem\u003ecps\u003c/em\u003epromoter-proximal region. The binding sites for Mga\u003cem\u003eSpn\u003c/em\u003e are shown in yellow.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/1069a4480750cae4e7e88f09.jpg"},{"id":60932556,"identity":"39d5ab99-d492-49c1-a308-f78a503de9e5","added_by":"auto","created_at":"2024-07-23 17:30:36","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55771,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of CPS production. \u003c/strong\u003e(a)TEM analysis of strains. Representative TEM images of the D39s, D39∆\u003cem\u003eMgaSpn\u003c/em\u003e, D39∆\u003cem\u003epyrR\u003c/em\u003e, and D39∆\u003cem\u003eMgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003estrains. The mean capsule widths of the D39s (57.03 nm), D39∆\u003cem\u003eMgaSpn\u003c/em\u003e (56.51 nm), D39∆\u003cem\u003epyrR\u003c/em\u003e (53.84 nm), and D39∆\u003cem\u003eMgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e (30.32 nm) strains are indicated. (b)and(c) Bacterial capsular polysaccharides were measured by an ELISA and uronic acid assay. (d) CPS-banding patterns of whole-cell lysates for the indicated strains as evaluated by western blot analysis. GAPDH was used as a sample loading control. ELISA and western blots were probed with a rabbit anti-serotype 2 CPS polyclonal antibody. The results of representative experiments are presented as the mean ± SD of three replicates.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/9da1094a97b4ad8f6dc77e1a.jpg"},{"id":60932550,"identity":"55ef4a5c-eaaf-4258-acf8-e857be5ee1f4","added_by":"auto","created_at":"2024-07-23 17:30:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfection of epithelial cells and mouse peritoneal primary macrophages. \u003c/strong\u003eBacterial adherence and invasion of A549 cells. (a, b) Infection of mouse peritoneal primary macrophages. (c) The cells were infected at an MOI of 100 with the indicated strains. ***P \u0026lt; 0.001, **P \u0026lt; 0.01, *P \u0026lt; 0.05, and not significant (NS) as analyzed by unpaired two-tailed Student’s t test.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/4f3b8aba9340d8ad3fcda7b1.jpg"},{"id":60932881,"identity":"22a2685f-e37f-475d-92c6-6f490a868e7b","added_by":"auto","created_at":"2024-07-23 17:38:36","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":40146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBacterial colonization and survival of infected mice. \u003c/strong\u003e(a) Nasopharyngeal colonization of mice (n = 6 per group) infected with the indicated strains at 2 × 10\u003csup\u003e7\u003c/sup\u003e CFU intranasally in nasopharyngeal lavage fluid collected 48 h postinfection. CFU of infected bacteria present in (b) lung homogenates and (c) blood. (d) Survival of mice (n = 12 for each group) after intranasal infection with 1 × 10\u003csup\u003e7\u003c/sup\u003e CFU. Survival was analyzed using log-rank comparisons. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/b81098aab27049b0352dce20.jpg"},{"id":60933452,"identity":"bf2d7b7a-bd3f-4063-912f-fd74221c8d81","added_by":"auto","created_at":"2024-07-23 17:54:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1954378,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/7476aaf7-38da-4957-b2fa-15f1b81bb0ac.pdf"},{"id":60932547,"identity":"d05c9ab8-e86b-498a-ba81-a4b924b39eac","added_by":"auto","created_at":"2024-07-23 17:30:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":94480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Figure S1.\u003c/strong\u003e Verification of D39∆pyrR mutant. The \u003cem\u003epyrR \u003c/em\u003emRNA of D39s, D39Δ\u003cem\u003epyrR, \u003c/em\u003eD39\u003cem\u003e∆MgaSpn∆pyrR and \u003c/em\u003eD39Δ\u003cem\u003eMgaSpn\u003c/em\u003eΔ\u003cem\u003epyrR\u003c/em\u003e::\u003cem\u003epyrR \u003c/em\u003ewere determined by real-time quantitative PCR (qPCR). qPCR was conducted in triplicate. P \u0026lt; 0.05 (*), P \u0026lt; 0.01 (**), and P \u0026lt; 0.001 (***).\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/0d98a9abe3d8c7b11d8a5730.docx"},{"id":60932553,"identity":"6440d4ea-373b-4a30-ba93-408c41662bec","added_by":"auto","created_at":"2024-07-23 17:30:36","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":462669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Figure S2. \u003c/strong\u003eEMSA of Mga\u003cem\u003eSpn \u003c/em\u003eprotein with P\u003cem\u003epyrR \u003c/em\u003emutation probes.\u003cstrong\u003e \u003c/strong\u003e(a)P\u003cem\u003epyrR \u003c/em\u003eis the original \u003cem\u003epyrR \u003c/em\u003epromoter sequence, P\u003cem\u003epyrR \u003c/em\u003emutation is the mutant promoter sequence. The yellow background region is the specific recognition site of Mga\u003cem\u003eSpn \u003c/em\u003ein P\u003cem\u003epyrR\u003c/em\u003e, while the blue background region is the mutation sequence. (b) EMSA of 6 × His-MgaSpn protein with P\u003cem\u003epyrR \u003c/em\u003emutation probes.\u003c/p\u003e","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/19ec88eb5c89a38500b831b4.docx"},{"id":60933139,"identity":"a241163a-7991-4e9c-883d-e2d6a54e075c","added_by":"auto","created_at":"2024-07-23 17:46:36","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":48193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3: Figure S3. \u003c/strong\u003eGrowth profies of D39s(circles), D39∆Mga\u003cem\u003eSpn\u003c/em\u003e(squares), D39∆\u003cem\u003epyrR \u003c/em\u003e(triangles) and D39ΔMga\u003cem\u003eSpn\u003c/em\u003eΔ\u003cem\u003epyrR\u003c/em\u003e::\u003cem\u003epyrR\u003c/em\u003e(inverted triangles) strains in C+Y medium(closed symbols) and uracil-free C+Y medium (open symbols). The growth rates (h− 1) for each strain are indicated in the graph and the values shown are means at 5h from three biological replicates ± SD. “-”represents grown in uracil-free C+Y medium.\u003c/p\u003e","description":"","filename":"Additionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/fb0d3f6c458ec8305679ff97.docx"},{"id":60932555,"identity":"80502f98-caae-466c-8d30-9037d8504be9","added_by":"auto","created_at":"2024-07-23 17:30:36","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":354428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4: Figure S4. \u003c/strong\u003eBacterial morphology. Gram staining was used to observe the Chain length (×40 objective).\u003c/p\u003e","description":"","filename":"Additionalfile4.docx","url":"https://assets-eu.researchsquare.com/files/rs-4618066/v1/cac19f190d5d43b544cf469c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The MgaSpn Global Transcriptional Regulator Mediates the Biosynthesis of Capsular Polysaccharides and Affects Virulence via the Uracil Synthesis Pathway in Streptococcus pneumoniae","fulltext":[{"header":"1 Background","content":"\u003cp\u003e \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e is a clinically common gram-positive pathogen that generally colonizes the human nasopharynx without symptoms, but it can also invade different ecological niches of the host and cause various diseases, such as acute otitis media, bronchitis, pneumonia, and meningitis\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Bacterial metabolic networks and virulence share an intimate relationship. Sensing nutrient availability drives the regulation and production of virulence factors in a multitude of bacterial pathogens\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eS. pneumoniae\u003c/em\u003e capsule metabolism is significant for its pathogenicity; however, the specific regulatory mechanism is not fully elucidated.\u003c/p\u003e \u003cp\u003eUracil plays an important role in metabolism and virulence of \u003cem\u003eS. pneumoniae\u003c/em\u003e. Uridine triphosphate is usually synthesized by the pyrimidine biosynthesis salvage pathway, and it is also endogenously synthesized through de novo synthesis pathway via pyrimidine biosynthesis genes\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e(such as \u003cem\u003epyrR\u003c/em\u003e, \u003cem\u003epyrF\u003c/em\u003e, \u003cem\u003ecarA\u003c/em\u003e, \u003cem\u003ecarB\u003c/em\u003e). De novo biosynthesis of pyrimidine begins with a pyrimidine ring assembled with aspartate, bicarbonate, and glutamine. The pyrimidine ring reacts with ribosyl pyrophosphate (PRPP) to form orotidine 5-monophosphate (OMP). Subsequently, OMP decarboxylates to form uridine monophosphoric acid (UMP), which is converted into other essential pyrimidine nucleotides. The pyrimidine de novo synthesis pathway consists of six enzymatic steps, and the genes encoding these enzymes (\u003cem\u003ecarA\u003c/em\u003e, \u003cem\u003ecarB\u003c/em\u003e, \u003cem\u003epyrB\u003c/em\u003e, \u003cem\u003epyrC\u003c/em\u003e, \u003cem\u003epyrD\u003c/em\u003e, \u003cem\u003epyrE\u003c/em\u003e, and \u003cem\u003epyrF\u003c/em\u003e) are widely conserved in bacteria \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. CarAB (\u003cem\u003espd_1132\u003c/em\u003e,\u003cem\u003espd_1131\u003c/em\u003e)encodes two subunits of carbamyl phosphate synthetase in \u003cem\u003eEscherichia coli\u003c/em\u003e, and this enzyme catalyzes the formation of carbamyl phosphate, an intermediate in the pyrimidine nucleotide and arginine biosynthetic pathways\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. It has been reported that \u003cem\u003ecarA\u003c/em\u003e mutation reduces the expression of capsule operon promoter (P\u003csub\u003e\u003cem\u003ecps\u003c/em\u003e\u003c/sub\u003e) in medium lacking uracil in the exponential growth period\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, but it remains unclear whether several other genes affect the expression of P\u003csub\u003e\u003cem\u003ecps\u003c/em\u003e\u003c/sub\u003e in \u003cem\u003eS. pneumoniae\u003c/em\u003e. PyrF is an OMP decarboxylase located on the pyrimidineregulon along with other genes encoding enzymes involved in the biosynthesis of pyrimidines\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Recent research has mainly suggested that de novo synthesis of pyrimidines is regulated by the \u003cem\u003epyrR\u003c/em\u003e operon regulatory protein. PyrR is a bifunctional protein that not only encodes uracil phosphoribosyl transferase (UPRTase) but also plays an important regulatory role in the transcriptional level of pyrimidine biosynthesis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In the presence of the UMP coregulator, PyrR binds to the 5 \u0026rsquo; UTR of the \u003cem\u003epyrR\u003c/em\u003e mRNA transcript and disrupts the anti- terminator stem-loop, resulting in decreased downstream gene expression. In contrast, when the UMP concentration is low, PRPP antagonizes UMP termination by binding to the PyrR protein\u003csup\u003e10\u0026ndash; \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The expression of \u003cem\u003ede novo\u003c/em\u003e synthesis of pyrimidine genes is regulated by RNA-binding proteins, but it remains unclear whether it is regulated by DNA-binding proteins.\u003c/p\u003e \u003cp\u003ePyrimidine nucleotides play an important role in the expression and production of \u003cem\u003eS. pneumoniae\u003c/em\u003e capsules \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The capsule is an exopolysaccharide layer, which is a carbohydrate layer that protects bacteria from various host destructive actions, such as complement deposition, opsonophagocytosis, mucous embedding, and neutrophil extracellular traps \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The capsule of the D39s strain, theserotype 2 strain used in the present study, is formed by repeating units of glucose (Glc), glucuronic acid (GlcUA), andrhamnose(Rha) in a ratio of 1:2:3\u003csup\u003e16\u003c/sup\u003e. The sugar component is activated by UTP to produce uridine diphosphate glucose (UDP-Glc), uridine diphosphate glucuronic acid (UDP-GlcUA), deoxythymidine triphosphate (dTTP), and deoxythymidine diphosphate rhamnose (dTDP-Rha)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, the mechanisms of pyrimidine and capsule synthesis remain unclear, and the role of central metabolism in pneumococcal virulence factors and pathogenesis is not fully understood.\u003c/p\u003e \u003cp\u003eMga\u003cem\u003eSpn\u003c/em\u003e belongs to the Mga/AtxA family of global transcriptional regulators, and it plays an important role in host immune escape, bacterial biofilm formation, host environment adaptation, and invasive disease\u003csup\u003e17\u0026ndash; \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We have previously confirmed that Mga\u003cem\u003eSpn\u003c/em\u003e is a negative regulator of capsule biosynthesis. Deletion of Mga\u003cem\u003eSpn\u003c/em\u003e increases the capsular content in whole bacterial lysate, and the increased content is comprised of small molecular weight proteins\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, the cause of the increase in the small molecular weight capsular proteins remains unclear\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. We conducted transcriptomic analysis to identify other factors that maybe regulated by Mga\u003cem\u003eSpn\u003c/em\u003e. Among them, the significant upregulation of the pyrimidine \u003cem\u003ede novo\u003c/em\u003e synthesis genes, \u003cem\u003epyrR\u003c/em\u003e, \u003cem\u003epyrF\u003c/em\u003e, \u003cem\u003ecarA\u003c/em\u003e, and \u003cem\u003ecarB\u003c/em\u003e, in Mga\u003cem\u003eSpn\u003c/em\u003e-deficient strains attracted our attention.\u003c/p\u003e \u003cp\u003eThus, we explored the relationship between \u003cem\u003eS. pneumoniae\u003c/em\u003e uracil metabolism and capsule synthesis and virulence. The present study demonstrated that Mga\u003cem\u003eSpn\u003c/em\u003e regulates capsular production through the \u003cem\u003ede novo\u003c/em\u003e synthesis pathway gene \u003cem\u003epyrR\u003c/em\u003e, thus affecting virulence. The study of the relationship between \u003cem\u003eS. pneumoniae\u003c/em\u003e uracil metabolism and the mechanism of capsule synthesis is of great significance for understanding the biological characteristics of \u003cem\u003eS. pneumoniae\u003c/em\u003e, developing new antibacterial drugs, and treating \u003cem\u003eS. pneumoniae\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cp\u003e2.1. Bacterial strains and growth conditions\u003c/p\u003e\n\u003cp\u003eThe bacterial strains and plasmids used in the present study are listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The \u003cem\u003eS. pneumoniae\u003c/em\u003e D39s strain and its derivatives were cultured in semi-synthetic casein hydrolysate medium supplemented with 5% yeast extract (C\u0026thinsp;+\u0026thinsp;Y, pH 7.0) or C\u0026thinsp;+\u0026thinsp;Y medium without uracil\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eE. coli\u003c/em\u003e strains were grown in lysogeny broth (LB) with shaking or on LB agar plates at 37\u0026deg;C\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. When appropriate, antibiotics were added to the growth medium as shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Antibiotic selection was used at the following concentrations: streptomycin, 150 ug/ml; kanamycin; 200 ug/ml; chloramphenicol, 200 ug/ml; spectinomycin, 50 ug/ml for \u003cem\u003eEscherichia coli\u003c/em\u003e and 200 ug/ml for\u0026nbsp;\u003cem\u003eS.pneumoniae\u003c/em\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStrains and plasmids\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRelevant genotype and/or phenotype\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResistance\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39s\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eS.pneumoniae\u003c/em\u003e D39 strain,Capsulated\u003c/p\u003e\n \u003cp\u003eStrainserotype2,rpsl K56T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39\u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39, \u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39\u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e::\u003cem\u003eMgaSpn\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39, \u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e::\u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpec\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39,\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39\u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39, \u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39 \u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e::\u003cem\u003epyrR\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39, \u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e::\u003cem\u003epyrR\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChl\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39-PTH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39,P\u003cem\u003ecps\u003c/em\u003e-luc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChl\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39\u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e-PTH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39\u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e,P\u003cem\u003ecps\u003c/em\u003e-luc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChl\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e-PTH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39,\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e, P\u003cem\u003ecps\u003c/em\u003e-luc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChl\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39\u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e-PTH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD39,\u0026Delta;\u003cem\u003eMgaSpn\u003c/em\u003e\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e,P\u003cem\u003ecps\u003c/em\u003e-luc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChl\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTH3937\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP\u003cem\u003ecps\u003c/em\u003e cloned into insertion vector\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChl\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePIB166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eContains \u003cem\u003epyrR\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChl\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Strain construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll mutant strains originated from the D39s strain, a streptomycin-resistant derivative of D39 referred to as the wild type (WT) strain. Sequences of primers used in the present study are listed in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The D39\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e strain was generated in a two-step transformation procedure\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The upstream and downstream homologous arms of the \u003cem\u003epyrR\u003c/em\u003e locus were amplified from D39s genomic DNA with the \u003cem\u003epyrR\u003c/em\u003e P1/\u003cem\u003epyrR\u003c/em\u003e P2 and \u003cem\u003epyrR\u003c/em\u003e P3/\u003cem\u003epyrR\u003c/em\u003e P4 primer pairs, respectively. The Janus cassette was amplified with the JC F and JC R primers from the genomic DNA of the ST588 strain. The Janus cassette, which has kanamycin resistance and a dominant \u003cem\u003erpsL\u003c/em\u003e allele, was utilized for the selection of kanamycin-resistant, streptomycin-sensitive colonies. The unmarked strains were kanamycin- sensitive and streptomycin-resistant. Fusion PCR was performed with the upstream arm, the Janus cassette, and the downstream arm with the \u003cem\u003epyrR\u003c/em\u003e P1 and \u003cem\u003epyrR\u003c/em\u003e P4 primer pairs, and the product was transformed into the D39s strain to generate D39\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e::kan-\u003cem\u003erpsL\u003c/em\u003e(\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e::JC). To generate unmarked deletions in the \u003cem\u003epyrR\u003c/em\u003e locus, the upstream and downstream sequences were amplified with the \u003cem\u003epyrR\u003c/em\u003e P1/JC F and \u003cem\u003epyrR\u003c/em\u003e P4/JC R primer pairs. The amplicons were ligated by fusion PCR with the \u003cem\u003epyrR\u003c/em\u003e P1 and \u003cem\u003epyrR\u003c/em\u003e P4 primer pairs, followed by transformation into the D39s strain to generate the \u003cem\u003epyrR\u003c/em\u003e unmarked deletion strain (D39\u0026Delta;\u003cem\u003epyrR\u003c/em\u003e)(Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eSequences of primers\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimers\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSequence(5\u0026rsquo;to 3\u0026rsquo;)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrR P1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTCGCTTGGGATTGTATCGG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrR P2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGGAGTTTTCAGCATTATCCTCTAGAGACAAACCTCCAAAAAGAAAAGTC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrR P3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGCATAAGGAAAGGCTCGAGGTTAAAGGAGTAGCCATGTCAG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrR P4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTCATGACATCAACCTGATCAATG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJC F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTCTAGAGGATAATGCTGAAAACTCCTTGAAG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJC R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCTCGAGCCTTTCCTTATGCTTTTGGAC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egyrB F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGTTCGTATGCGTCCAGGGAT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egyrB F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eATACCACGCCCATCATCCAC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMgaSpn F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAGTTGCTCCTAGTTACGAACC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMgaSpn R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACCTTCTATTCCTTCTGCCTGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrR F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACTTCGCGGTCTGTCACATC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrR R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTGCCCACCGAATCCAAGAAC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrF F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGACACCAGGGATTCGTCCAG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrF R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAAGCTGCAACAGGCTCCTC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCarA F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAGCAGGTTGGTATCTGTGGC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCarA R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTCAACCATGCGGTACGTGAA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCarB F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTGGCATCAACTTCGCACTCT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCarB R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCAGTTCTTGTCCGCCCATCT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrR-BamHI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCGCGGATCCGTGAAGTCTATACTGTGTGCAGT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrR-XhoI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCCGCTCGAGCCACATGGTTCAATGCTTGTTGA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePr1303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCGGGATCCAGGAGGAATAATGAGATCCG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePr1304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTTGCGGCCGCCTACGGGGATCTTACAATTT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Western blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. pneumoniae\u003c/em\u003e were grown statically to an OD620 0f 0.5 in 3 ml C\u0026thinsp;+\u0026thinsp;Ymedium at 37℃ under 5% CO\u003csub\u003e2\u003c/sub\u003e, and harvested by centrifugation at 4℃ for 15min at 10,000\u0026times;g. Removed supernatant, added 200 \u0026micro;L of SEDS lysis buffer (150 mM NaCl, 0.1% deoxycholate, 15 mM EDTA, and 0.2% sodium dodecyl sulfate; pH\u0026thinsp;=\u0026thinsp;8). The samples were separated by 10% SDS‒PAGE and subsequently transferred to PVDF membranes by wet transfer, and blocked for 30 min at room temperature in blocking buffer (PBS with 0.05% [vol/vol] Tween 20 and 5% [wt/vol] skimmed milk). The membranes were incubated with the following primary antibodies overnight at 4\u0026deg;C: type 2 CPS (1:5000 Pneumococcus Type 2 serum; States Serum Institut, K\u0026oslash;benhavn,Denmark). Washed the membranes four times with PBST for six minutes each(PBS with 0.05% [vol/vol] Tween 20). The membranes were then incubated with the following secondary antibodies at 37\u0026deg;C for 1 h: goat anti-mouse IgG (1:10000; KPL,USA) or goat anti-mouse IgA (1:8000; SantaCruz,USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Expression and purification of 6\u0026times;His-Mga\u003c/strong\u003e \u003cstrong\u003eSpn\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMga\u003cem\u003eSpn\u003c/em\u003e protein expression and purification were performed as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The constructed pET28-Mga\u003cem\u003eSpn\u003c/em\u003e plasmid was transformed into BL21 cells for cloning and expression. Protein purity in the eluent was analyzed by Coomassie brilliant blue staining, and the appropriate concentration was selected for ultrafiltration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Electrophoretic mobility shift assay (EMSA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe probes used in the present study were the \u003cem\u003epyrR\u003c/em\u003e promoter (P\u003csub\u003e\u003cem\u003epyrR\u003c/em\u003e\u003c/sub\u003e) fragments amplified from the D39s strain labeled with 5\u0026apos;-biotin. First, 10 \u0026micro;L of reaction buffer, consisting of 1 \u0026micro;L 10x binding buffer, 0\u0026ndash;2.6 \u0026micro;gprotein, 0.5 \u0026micro;g poly (dI-dC), and 0.5 ng of the labeled probe, was incubated at 25\u0026deg;C for 20 min. After incubation, the unlabeled probe in 100-fold excess was added as a specific competitor in the cold probe reaction system. Following incubation, binding reaction mixtures were analyzed by electrophoresis in 6% native TBE polyacrylamide gels at constant 100 V for 60min. EMSA was then carried out using the LightShift R Chemiluminescent EMSA Kit (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. DNase I footprinting assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNase I footprintingassays were performed as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Briefly, fluorescent FAM-labeled probes for the P\u003csub\u003e\u003cem\u003ecps\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003epyrR\u003c/em\u003e\u003c/sub\u003e were amplified using D39s as the template with the \u003cem\u003epyrR\u003c/em\u003e-FAM and \u003cem\u003epyrR\u003c/em\u003e R primers, respectively. Then, 390 ng of probes was incubated with differing concentrations of Mga\u003cem\u003eSpn\u003c/em\u003e protein in a 40 \u0026micro;L reaction system at 25\u0026deg;C for 30 min. Then, 10 \u0026micro;L of a solution containing 0.015 units of DNase I (Promega, Madison, WI, USA) and freshly prepared 100 nM CaCl\u003csub\u003e2\u003c/sub\u003e was added to the system. After incubation at 37\u0026deg;C for 1 min, the reaction was stopped by adding 140 \u0026micro;L of terminator solution. The obtained samples were extracted with phenol and precipitated with ethanol. The pellets were dissolved in 30 \u0026micro;L of nuclease-free water. The GeneScan-LIZ600 size standard (Applied Biosystems, Foster City, CA, USA) was used for electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Enzyme-linked immunosorbent assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were prepared as described above. Dilution samples of 1:800 were used to coat 96-well microtiter plates, which were subsequently reacted with the type-2 CPS polyclonal antibody (1:5000)\u003c/p\u003e\n\u003cp\u003e(Statens Serum Institut, K\u0026oslash;benhavn, Denmark). The bound primary antibodies were detected following incubation with goat anti-rabbit IgG-HRP antibody (1:8000) (KPL, Gaithersburg, MD, USA), and the absorbance at 450 nm was recorded. The experimental results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of three replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. RNA extraction and RT‒PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from 2 mL of log-phase bacterial culture grown statically to an OD620 0f 0.5 in normal C\u0026thinsp;+\u0026thinsp;Y medium. Bacterial cells were treated with 200 \u0026micro;L of 15 mg/mL lysozyme and 10 \u0026micro;L proteinase K for 30 min at 25\u0026deg;C. The RNAprotect bacterial reagent and RNeasy Protect bacterial kit (Qiagen, Hilden,Germany) were employed for RNA extraction according to the manufacturer \u0026rsquo;s instructions. The RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher, Pittsburg, PA, USA), and its integrity was confirmed by agarose gel electrophoresis. PrimeScript RT master mix (TaKaRa, Beijing) was used to prepare cDNAaccording to the manufacturer\u0026rsquo;s instructions. RT- PCR was carried out using a CFX ConnectTM (Bio-Rad). The primers used for PCR are listed in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. \u003cem\u003egyrB\u003c/em\u003e was used as an internal control. The results of representative experiments are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9. Transcriptome sequencing and metabolome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrains for transcriptome sequencing were collected in the OD620 of 0.5 period in normal C\u0026thinsp;+\u0026thinsp;Y medium containing 15mg L- 1 pyrimidines. Pneumococcal cultures were pre-treated with ammonium sulfate to terminate protein-dependent transcription and degradation,then were centrifuged at 10,000g for 10 min. Bacterial sediment was transported at -20℃ temperature to the Beijing Novogene Company laboratory for transcriptome sequencing and differential expression analyzation. The DESeq software was used for differential expression analysis between the two comparison combinations (two biological replicates per group). Prior to differential gene expression analysis, for each sequenced library, the read count was adjusted by a proportional normalization factor via the DEGSeq package. Differential expression analysis of the two conditions was performed using the DEGseq software package (1.20.0). P value was adjusted by Benjamini and Hochberg methods, and P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 after correction and |log2foldchange|\u0026gt;1.5 were used as the threshold of significant differential expression.\u003c/p\u003e\n\u003cp\u003eStrains for metabolome sequencing were prepared as above. Bacterial sediment was transported at -20℃ temperature to Suzhou PANOMIX Biomedical Tech Co. LTD for metabolite quantification and identification and differential expression analysis (four biological replicates per group).By screening metabolites, differential metabolites (biomarkers) were found.The screening criteria for relevant differential metabolites were p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 and fold_change\u0026thinsp;\u0026ge;\u0026thinsp;1.5 or \u0026le;\u0026thinsp;0.667. Finally, statistical map and heat map of differential metabolites were obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10. Adhesion and antiphagocytic assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial adhesion experiments was carried according to Zhang\u0026apos;s method\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e : A549 human type II pneumocytes were cultured in a 24-well plate according to laboratory SOP with a density of about 2 \u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well. The bacteria were resuspended with Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) (Thermo Fisher) to 1 \u0026times;10\u003csup\u003e8\u003c/sup\u003e colony forming units (CFU)/mL, and 500 \u0026micro;L volumes were usedata multiplicity of infection (MOI) of 100 and incubated with A549 cells at 37\u0026deg;C for 1h. Following incubation, the cells were gently rinsed five times with PBS. The input bacteria were enumerated by plating serial dilutions.For adhesion assays, cells were lysed in sterile ddH\u003csub\u003e2\u003c/sub\u003eO. The lysates were serially diluted and plated on blood agar plates to determine intracellular and extracellular CFUs. For invasion assays, extracellular bacteria were treated with gentamicin (100ug/ml) and penicillin G (10ug/ml). Phagocytosis of pneumococci was determined with mouse peritoneal primary macrophages. The remaining steps were the same as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11. Animal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the animals used in the present study were purchased from the Laboratory Animal Center of Chongqing Medical University. All animal experiments were approved by the Animal Care and Use Committee of Chongqing Medical University and were performed in strict accordance with the regulations of the Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003eFor nasopharyngeal colonization model: themale C57BL/6 mice (6\u0026ndash;8 weeks old, weighing 20\u0026ndash;21 g) were randomly divided into five groups (n\u0026thinsp;=\u0026thinsp;6 per group).Each mouse was inoculated with 2 \u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU of bacteria through the nasal cavity. Nasal lavage fluid, heart blood, and lung tissues were collected and ground using a mechanical mortar and pestle. The samples were plated on blood agar plates after the appropriate dilutions to determine the CFU.\u003c/p\u003e\n\u003cp\u003eFor a model of infection: the mice were divided into six groups (n\u0026thinsp;=\u0026thinsp;12 per group). Each mouse in five experiment group was infected intranasally with 1 \u0026times;10\u003csup\u003e7\u003c/sup\u003e CFU of bacteria. One group was injected the same amount sterile saline as control. The survival rate of the mice was monitored daily for 14 days. Moribund mice were not euthanized prior to the completion of the 14-day survival study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll analyses were performed using GraphPad Prism 8 (GraphPad Software). Statistical differences between groups were compared using Student\u0026rsquo;st test, and statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***).\u003c/p\u003e"},{"header":"3 Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 \u0026nbsp;Transcriptome sequencing analysis of Mga\u003c/strong\u003e \u003cstrong\u003eSpn\u003c/strong\u003e \u003cstrong\u003eregulatory proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMga\u003cem\u003eSpn\u003c/em\u003e is a member of the Mga/AtxA family and has high homology with the Mga protein of group A streptococcus (GAS). Mga transcribes and activates many virulence genes in \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We previously found that Mga\u003cem\u003eSpn\u003c/em\u003e participates in the transcriptional regulation of the \u003cem\u003ecps\u003c/em\u003e gene cluster in the capsule and the \u003cem\u003elic1\u003c/em\u003e teichoic acid synthesis-related gene cluster\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To explore whether the Mga\u003cem\u003eSpn\u003c/em\u003e global transcriptional regulator mediates other surface virulence factors in addition to the above gene clusters, we conducted transcriptomic sequencing of the WT (D39s) and \u003cem\u003emgaSpn\u003c/em\u003e-deficient (D39\u0026Delta;\u003cem\u003emgaSpn\u003c/em\u003e) strains.\u003c/p\u003e\n\u003cp\u003eThe transcriptome results showed that there were 149 differential genes, with 113 upregulated genes and 36 downregulated genes above a change cutoff of 1.5-fold.(Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The up-regulated genes mainly involved genes related to PTS sugar transporter, bacteriocin and de novo synthesis of uracil, the downregulated genes mainly involved ribosomal proteins and ABC transporters. What attracted us was \u003cem\u003epyrR\u003c/em\u003e, \u003cem\u003epyrF\u003c/em\u003e, \u003cem\u003ecarA\u003c/em\u003e, and \u003cem\u003ecarB\u003c/em\u003e pyrimidinede novo synthesis genes were significantly upregulated in \u003cem\u003emgaSpn\u003c/em\u003e-deficient strains. qRT-PCR confirmed higher transcript levels of \u003cem\u003epyrF\u003c/em\u003e, \u003cem\u003epyrR\u003c/em\u003e, \u003cem\u003ecarA\u003c/em\u003e, and \u003cem\u003ecarB\u003c/em\u003e, about 2- to 3-fold compared to WT bacteria (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). These results were consistent with the transcriptome sequencing results, suggesting that Mga\u003cem\u003eSpn\u003c/em\u003e maybe involved in the regulation of the uracil synthesis pathway.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eDifferential gene expression detected by transcriptome sequencing\u003c/strong\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene id\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elog2FoldChange\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene description\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDecreased\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.540844073\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esugar ABC transporter permease \u0026amp;\u0026amp; PF00528:Binding-protein-dependent transport system inner membrane component\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.076620487\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLysM peptidoglycan-binding domain-containing protein \u0026amp;\u0026amp; PF01476:LysM domain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.577646712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etransporter substrate-binding domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.758994401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglycosyltransferase family 2 protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00815\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.794268401\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00825\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.569853425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCPBP family intramembrane metalloprotease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00830\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.739055601\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMFS transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.65768181\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enrdG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eanaerobic ribonucleoside-triphosphate reductase activating protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.60393733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCof-type HAD-IIB family hydrolase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01455\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.975312512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNCS2 family permease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.980699182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCPBP family intramembrane metalloprotease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02415\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.44806895\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03785\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.792999234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF3270 domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04545\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.381183909\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.539280353\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABC transporter permease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04770\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.699542615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eamino acid permease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS05420\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.609153602\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYueI family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.167578317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.473874795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ereplication initiator protein A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS07150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.870233206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGNAT family N-acetyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS07300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.682021275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF5590 domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS07520\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.016370291\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOFA family MFS transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.007076022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehelix-turn-helix domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08685\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.956176742\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003exanthine phosphoribosyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.766305707\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epurine permease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.56279407\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHlyC/CorC family transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.66801798\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eresponse regulator transcription factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.278029835\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esensor histidine kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09585\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.847650149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABC transporter permease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09590\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.663425036\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABC transporter ATP-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.955948867\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.485912843\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etRNA-Pro\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09945\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.148818732\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLysM peptidoglycan-binding domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.674119829\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglycoside hydrolase family 125 protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10725\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.571533956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ethiamine-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10810\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.255949489\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCHAP domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIncreased\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.879533021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecarbonic anhydrase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.721181153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoA-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.556051641\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebeta-N-acetylhexosaminidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.917993406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebeta-galactosidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.989274903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF4299 domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00565\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.859739364\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003elactococcin 972 family bacteriocin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.011238736\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.919982428\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epeptidase domain-containing ABC transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00610\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.240295599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGyrI-like domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.294808957\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHlyD family efflux transporter periplasmic adaptor subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00620\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.697260495\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSP_0115 family bacteriocin-like peptide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00635\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.722559642\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPH_0218 family bacteriocin-like peptide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS00925\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.600528958\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6%2C7-dimethyl-8-ribityllumazine synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01525\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.291662237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS cellobiose transporter subunit IIB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01530\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.988115702\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBglG family transcription antiterminator\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01535\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.749896408\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS cellobiose transporter subunit IIA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.34063299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egluconate 5-dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.570803847\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLacI family DNA-binding transcriptional regulator\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS01850\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.508244909\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ernpB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRNase P RNA component class B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.339857524\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eenoyl-CoA hydratase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.200698741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacyl carrier protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.26950146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efabK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eenoyl-[acyl-carrier-protein] reductase FabK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02070\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.833084477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efabD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACP S-malonyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02075\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.246698575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efabG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-oxoacyl-[acyl-carrier-protein] reductase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.45560506\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efabF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebeta-ketoacyl-ACP synthase II\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.11639046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacetyl-CoA carboxylase biotin carboxyl carrier protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.773105326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efabZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-hydroxyacyl-ACP dehydratase FabZ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.111535703\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eaccC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacetyl-CoA carboxylase biotin carboxylase subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.954741497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacetyl-CoA carboxylase carboxyltransferase subunit beta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.004358554\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacetyl-CoA carboxylase carboxyl transferase subunit alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.264746808\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebriC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebiofilm-regulating peptide BriC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.445879425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCPBP family intramembrane metalloprotease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.797354206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCTP synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.54647738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egrpE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003enucleotide exchange factor GrpE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.953093782\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ednaK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emolecular chaperone DnaK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02480\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.229957944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02510\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.32306266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02530\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.34995027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eblpC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003equorum-sensing system pheromone BlpC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02545\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.257627149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.335877819\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCPBP family intramembrane metalloprotease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.112489772\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS02565\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.22119207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCPBP family intramembrane metalloprotease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.953581792\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS8 family serine peptidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.389396089\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS galactitol transporter subunit IIC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03275\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.702707644\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epyrF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eorotidine-5\u0026apos;-phosphate decarboxylase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.569197499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eorotate phosphoribosyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.511480332\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDegV family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03535\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.413688835\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCBS domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03675\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.271067339\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03695\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.625360236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglutathione-disulfide reductase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03840\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.086302291\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF1827 family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS03940\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.152351916\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDNA topology modulation protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.042616047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003essrA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etransfer-messenger RNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.710636931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDeoR/GlpR transcriptional regulator\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.831943834\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.105403339\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePspC domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.676754015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.184672578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edihydroorotate dehydrogenase electron transfer subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04585\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.022003954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edihydroorotate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04590\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.737808776\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eendo-beta-N-acetylglucosaminidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04815\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.786395769\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehemH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eferrochelatase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS04930\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.505667841\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eiron-siderophore ABC transporter substrate-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS05010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.918930761\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehelix-turn-helix transcriptional regulator\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS05165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.503059453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emetal-sulfur cluster assembly factor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS05630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.759193213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etranscription antiterminator\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS05635\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.003549678\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003elacD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etagatose-bisphosphate aldolase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS05640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.458126365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etagatose-6-phosphate kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS05645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.994294299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003elacB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egalactose-6-phosphate isomerase subunit LacB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS05650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.8056828\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003elacA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003egalactose-6-phosphate isomerase subunit LacA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.535668533\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecarB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecarbamoyl-phosphate synthase large subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.600881094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecarA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eglutamine-hydrolyzing carbamoyl-phosphate synthase small subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.644316117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epyrR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ebifunctional pyr operon transcriptional regulator/uracil phosphoribosyltransferase PyrR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06070\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.732221049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABC-F family ATP-binding cassette domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.712128317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003euracil transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.590012213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003elanthionine synthetase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06490\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.939313287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABC transporter ATP-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06650\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.526791226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30S ribosomal protein S21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.568313221\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABC transporter ATP-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06910\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.806831456\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF1836 domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS06915\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.426500081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehemolysin III family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS07065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.548217135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ethioredoxin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS07525\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.25529155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFAD-containing oxidoreductase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.610646583\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.939427257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eABC transporter ATP-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.655062375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emembrane protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.793486751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS beta-glucoside transporter subunit IIBC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.862798757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF4649 family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08335\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.549051733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etrxA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ethioredoxin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.034277727\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etype II toxin-antitoxin system HicA family toxin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08485\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.805631069\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCsbD family protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS08875\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.139807301\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etreP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS system trehalose-specific EIIBC component\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09185\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.567762138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eply\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echolesterol-dependent cytolysin pneumolysin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.192158071\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.092791382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.39262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF4231 domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.318458004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eacylphosphatase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.260043723\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003euniversal stress protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09790\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.812594241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eulaG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL-ascorbate 6-phosphate lactonase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS09825\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.524245923\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS ascorbate transporter subunit IIC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.768053314\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ephosphate-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.72876586\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003emembrane protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10245\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.947416517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eextracellular solute-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10405\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.798523107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epcpA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003echoline-binding protein PcpA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.757549455\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPFH domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10525\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.529211255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS mannose/fructose/sorbose transporter family subunit IID\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10530\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.863757933\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS mannose/fructose/sorbose/N-acetylgalactosamine transporter subunit IIC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10535\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.532680442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS sugar transporter subunit IIB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-4.727078471\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePTS sugar transporter subunit IIA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.778092141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003erhamnulokinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10630\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.069439605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ehypothetical protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10755\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.298327647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eraiA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eribosome-associated translation inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10905\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.809492243\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTetR/AcrR family transcriptional regulator\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSPD_RS10945\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.897430199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etrypsin-like peptidase domain-containing protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003e The reference genome comes from NCBI RefSeq assembly GCF_000014365.2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/nuccore/NC_008533\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Mga\u003c/strong\u003e \u003cstrong\u003eSpn\u003c/strong\u003e \u003cstrong\u003eis involved in the regulation of the uracil synthesis pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate if Mga\u003cem\u003eSpn\u003c/em\u003e participates in the uracil synthesis pathway, we examined the metabolism of WT and mgaSpn-deficient bacteria cultured in C\u0026thinsp;+\u0026thinsp;Y medium to an OD620 of 0.5 by metabolome sequencing(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). There were 31 different metabolites in the two strains (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Compared to WT bacteria, the contents of 15 metabolites, including limonene- 1,2-diol and L-histidine, in \u003cem\u003emgaSpn\u003c/em\u003e- deficient bacteria were increased. The contents of 16 metabolites, including N-acetyl-neuraminic acid, gulonic acid, UMP, pyrimidodiazepine, and D-ribose, were decreased. Of note, UMP and pyrimidodiazepine metabolites in the uracil synthesis pathway were significantly reduced by 6- to 7- fold in \u003cem\u003emgaSpn\u003c/em\u003e-deficient bacteria.\u003c/p\u003e\n\u003cp\u003eWe previously confirmed that Mga\u003cem\u003eSpn\u003c/em\u003e is a transcription suppressor in capsule biosynthesis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.Deletion of Mga\u003cem\u003eSpn\u003c/em\u003e increases the capsular content in the whole bacterial lysate, and the small molecular weight proteins are concentrated in the increased capsular content\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, the reason for the increase in the small molecular weight capsular proteins remains unclear. The uracil synthesis pathway has been shown to affect capsular polysaccharides (CPS) promoter expression and CPS production in the \u003cem\u003eS. pneumoniae\u003c/em\u003e D39 strain\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. After Mga\u003cem\u003eSpn\u003c/em\u003e deletion, the transcriptomic results showed that the expression of uracil synthesis genes was increased, and the metabolomics results showed that the uracil synthesis pathway metabolites were decreased. These findings suggested that Mga\u003cem\u003eSpn\u003c/em\u003e may affect capsule synthesis by regulating the uracil synthesis pathway.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDifferential metabolites detected by metabolome sequencing\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVIP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elog2(FC_M/D)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep.value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN-Acetyl-a-neuraminic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.87893\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-19.098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.021071\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGulonic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.860573\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.7987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUMP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.836096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-15.964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.021071\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5-Acetamidovalerate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.778424\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.8623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDimethyl sulfone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.774266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.8729\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLimonene-1,2-diol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.714941\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.7051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD-Ribose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.713519\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.0715\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGuanosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.689839\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.8766\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTetracosanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.68122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.92923\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLanosterin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.637613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.5964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(-)-Epigallocatechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.610466\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-16.227\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.021071\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5-Guanidino-3-methyl-2-oxopentanoate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.589845\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.3838\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyrimidodiazepine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.57903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.8171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL-Histidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.577359\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.96313\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhenyl acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.575216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.47183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetylcholine chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.554901\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.5472\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(S)-1-Phenylethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.547117\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.1176\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eImidazol-5-yl-pyruvate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.544352\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.31394\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD-Glucuronic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.52796\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.379\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12,13-DHOME\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.503511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-0.89095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpermidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.493071\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.1778\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL-Erythrulose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.453958\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.79214\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStearic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.365478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.7833\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAdenosine diphosphate ribose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.361712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.523\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL-2-Hydroxyglutaric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.33444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.7077\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-Oxoproline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.306298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.1815\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMaleimide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.293903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.4198\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-Pyridoxic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.271316\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.5939\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11-Dehydrocorticosterone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.196153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.5698\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDehydroepiandrosterone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.189842\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.7868\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethyl (indol-3-yl)acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.185804\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.3809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.030383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Mga\u003c/strong\u003e \u003cstrong\u003eSpn\u003c/strong\u003e \u003cstrong\u003ebinds to the\u003c/strong\u003e \u003cstrong\u003epyrR\u003c/strong\u003e \u003cstrong\u003epromoter region\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMga\u003cem\u003eSpn\u003c/em\u003e, a member of the Mga/AtxA family of global transcriptional regulators, directly binds to the regulatory regions of target genes to regulate target gene expression. We found some differentially expressed genes the \u003cem\u003epyrR\u003c/em\u003e, \u003cem\u003epyrF\u003c/em\u003e, \u003cem\u003ecarA\u003c/em\u003e, and \u003cem\u003ecarB\u003c/em\u003e uracil \u003cem\u003ede novo\u003c/em\u003e synthesis genes by transcriptomic analysis. To identify the uracil metabolism genes that Mga\u003cem\u003eSpn\u003c/em\u003e directly regulates, we investigated the expression of the \u003cem\u003epyrR\u003c/em\u003e, \u003cem\u003epyrF\u003c/em\u003e, \u003cem\u003ecarA\u003c/em\u003e, and \u003cem\u003ecarB\u003c/em\u003e genes in WT bacteria (D39s), \u003cem\u003emgaSpn\u003c/em\u003e-deficient bacteria (D39∆\u003cem\u003emgaSpn\u003c/em\u003e),\u003cem\u003emgaSpn\u003c/em\u003e complement bacteria (D39∆\u003cem\u003emgaSpn\u003c/em\u003e::\u003cem\u003emgaSpn\u003c/em\u003e), and \u003cem\u003emgaSpn\u003c/em\u003e-overexpressing bacteria (D39::\u003cem\u003emgaSpn\u003c/em\u003e). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, all genes were upregulated after \u003cem\u003emgaSpn\u003c/em\u003e deletion, but only \u003cem\u003epyrR\u003c/em\u003e was about 3-fold downregulated during \u003cem\u003emgaSpn\u003c/em\u003e recovery and overexpression strains, indicating that Mga\u003cem\u003eSpn\u003c/em\u003e maybe directly involved in the regulation of \u003cem\u003epyrR\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eMga\u003cem\u003eSpn\u003c/em\u003e is a transcriptional regulator that contains two conserved helix-turn-helix (HTH) domains, which are DNA-binding motifs, indicating that Mga\u003cem\u003eSpn\u003c/em\u003e has the ability to bind DNA27. To explore whether Mga\u003cem\u003eSp\u003c/em\u003en protein directly participates in the transcriptional regulation of \u003cem\u003epyrR\u003c/em\u003e, EMSAs were performed using a DNA fragment probe approximately 300 bp upstream of the \u003cem\u003epyrR\u003c/em\u003e gene to verify the specific binding of the Mga\u003cem\u003eSpn\u003c/em\u003e protein to the \u003cem\u003epyrR\u003c/em\u003e promoter. As the concentration of protein added to the reaction system gradually increased, the binding bands of the probes and proteins shifted backward, indicating that the binding of Mga\u003cem\u003eSpn\u003c/em\u003e to the \u003cem\u003epyrR\u003c/em\u003e promoter was concentration-dependent (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). In the unlabeled probe competition lane, the unlabeled probes competed to bind to the \u003cem\u003emgaSpn\u003c/em\u003e- and \u003cem\u003epyrR\u003c/em\u003e-labeled probes. These results indicated that Mga\u003cem\u003eSpn\u003c/em\u003e specifically binds to the \u003cem\u003epyrR\u003c/em\u003e promoter region.\u003c/p\u003e\n\u003cp\u003eBecause Mga\u003cem\u003eSpn\u003c/em\u003e directly binds to the \u003cem\u003epyrR\u003c/em\u003e promoter region, we performed DNase Ⅰ footprinting analysis to determine the specific recognition site of Mga\u003cem\u003eSpn\u003c/em\u003e on the P\u003csub\u003e\u003cem\u003epyrR\u003c/em\u003e\u003c/sub\u003e probe (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). After adding protein (1.5 \u0026micro;g) to the 390 ng probe system, the following two protection areas were identified: 31 bp (5\u0026apos;-TAGCAATTTGTAAGATGCTACATTGAAACTT-3\u0026apos;) and 53 bp (5\u0026apos;-TTGTTTAAGGAGACTTTTCTTTTTGGAGGTTTGTCATGAAAACAAAAGAAGTT-3\u0026apos;) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). To further confirm that Mga\u003cem\u003eSpn\u003c/em\u003e specifically binds to P\u003csub\u003e\u003cem\u003epyrR\u003c/em\u003e\u003c/sub\u003e, we designed eight mutations in the binding site of the P\u003csub\u003e\u003cem\u003epyrR\u003c/em\u003e\u003c/sub\u003e promoter. The mutant \u003cem\u003epyrR\u003c/em\u003e probe EMSA results showed that Mga\u003cem\u003eSpn\u003c/em\u003e lost the ability to bind the mutant probe(Figure \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). However, additional experiments are required to determine if these two binding sites are functional sites for Mga\u003cem\u003eSpn\u003c/em\u003e transcriptional regulation. These data suggested that Mga\u003cem\u003eSpn\u003c/em\u003e may play a key role in the co-transcription of \u003cem\u003epyrR\u003c/em\u003e genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Mga\u003c/strong\u003e \u003cstrong\u003eSpn\u003c/strong\u003e \u003cstrong\u003enegatively regulates CPS production by\u003c/strong\u003e \u003cstrong\u003epyrR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next evaluated the morphology of the capsules of each strain grown to an OD620 of 0.5 in normal C\u0026thinsp;+\u0026thinsp;Y medium containing 15mg L- 1 pyrimidines using transmission electron microscopy (TEM) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). There was no obvious difference in capsule thickness when comparing the D39s strain to the D39∆\u003cem\u003emgaSpn\u003c/em\u003e and D39∆\u003cem\u003epyrR\u003c/em\u003e strains, but the D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strain had a thinner capsule than the D39s strain.The transmission electron microscopy (TEM) was used to measure the capsule thickness of 10 strain with ImageJ software, and the average capsule thicknesses of the D39s, D39∆\u003cem\u003emgaSpn\u003c/em\u003e, D39∆\u003cem\u003epyrR\u003c/em\u003e, and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains were 57.03\u0026thinsp;\u0026plusmn;\u0026thinsp;14.73 nm, 56.51\u0026thinsp;\u0026plusmn;\u0026thinsp;10.52 nm, 53.84\u0026thinsp;\u0026plusmn;\u0026thinsp;17.45nm, and 30.32\u0026thinsp;\u0026plusmn;\u0026thinsp;6.97nm, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003eWe also detected the capsular content of the bacteria using ELISAs. When the bacteria grew to an OD620 of 0.5, the same amount of bacteria was collected by centrifugation for ELISA detection. Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb shows that there was no obvious difference in the capsular content between the D39s and D39∆\u003cem\u003emgaSpn\u003c/em\u003e strains grown in normal C\u0026thinsp;+\u0026thinsp;Y medium contains 15mg L- 1 pyrimidines, and the capsular content of the D39∆\u003cem\u003epyrR\u003c/em\u003e-( grown in uracil-free C\u0026thinsp;+\u0026thinsp;Y medium) and ∆D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains was 2 to 3 times lower than that of the D39s and D39∆\u003cem\u003emgaSpn\u003c/em\u003e strains, which was consistent with the TEM results.\u003c/p\u003e\n\u003cp\u003eIn addition, capsular polysaccharides were quantified by the uronic acid method. Glucuronic acid is a specific component of \u003cem\u003eS. pneumoniae\u003c/em\u003e capsules regulated by CpsT/F/G/L, and it exists in the form of glucuronic acid in type II capsules, with each repeating unit containing one glucuronic acid. The content of the capsule in bacteria can be measured by detecting the content of uronic acid. The capsular content of the whole bacteria (Bacteria-CPS) was detected, and the results showed that the capsular content of the D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strain was significantly lower than that of the WT strain and \u003cem\u003emgaSpn\u003c/em\u003e-deficient strains (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). There were fewer pods of the D39∆\u003cem\u003epyrR\u003c/em\u003e-deficient strain grown in uracil-free C\u0026thinsp;+\u0026thinsp;Y than the WT and \u003cem\u003emgaSpn\u003c/em\u003e-deficient strains.\u003c/p\u003e\n\u003cp\u003eWe next collected the whole bacterial lysate of each strain and detected the expression of capsules by Western blot analysis (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). The total capsular content of the D39∆\u003cem\u003emgaSpn\u003c/em\u003e strain was significantly increased compared to that of the D39s strain, and the small molecular weight capsular proteins of the D39∆\u003cem\u003epyrR\u003c/em\u003e strain were slightly decreased compared to that of the D39s strain, which maybe due to the presence of a remedial synthesis pathway in uracil C\u0026thinsp;+\u0026thinsp;Y. Therefore, we detected the expression of capsules in the D39∆\u003cem\u003epyrR\u003c/em\u003e strain grown in uracil-free C\u0026thinsp;+\u0026thinsp;Y medium (D39∆\u003cem\u003epyrR\u003c/em\u003e-). The expression of capsules in the D39∆\u003cem\u003epyrR\u003c/em\u003e- and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains was significantly decreased, but the expression of capsules was recovered after supplementing the D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strain with \u003cem\u003epyrR\u003c/em\u003e. These results indicated that both \u003cem\u003emgaSpn\u003c/em\u003e and \u003cem\u003epyrR\u003c/em\u003e are involved in the regulation of capsule synthesis and \u003cem\u003emgaSpn\u003c/em\u003e has a positive synergistic effect on the regulation of the capsule by \u003cem\u003epyrR\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. pyrR\u003c/strong\u003e \u003cstrong\u003einfluences adhesion and pathogenicity of\u003c/strong\u003e \u003cstrong\u003eS. pneumoniae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the effect of \u003cem\u003epyrR\u003c/em\u003e deficiency on the virulence of \u003cem\u003eS. pneumoniae\u003c/em\u003e, we used the A549 lung epithelial cell line to detect the adhesion and invasion ability of the D39s, D39∆\u003cem\u003epyrR\u003c/em\u003e, D39∆\u003cem\u003epyrR\u003c/em\u003e-, D39∆\u003cem\u003emgaSpn\u003c/em\u003e, and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e::\u003cem\u003epyrR\u003c/em\u003e strains. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, the D39s and D39∆\u003cem\u003epyrR\u003c/em\u003e strains adhered to epithelial cells 2\u0026ndash;3 fold greater than other strains, and the D39∆\u003cem\u003emgaSpn\u003c/em\u003e strain had a stronger invasion ability than the other strains. In addition, the D39∆\u003cem\u003epyrR\u003c/em\u003e- and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains had significantly reduced invasion and adhesion abilities compared to the other strains (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). Previous studies have suggested that during colonization, \u003cem\u003eS. pneumoniae\u003c/em\u003e express low levels of CPS to enhance the exposure of cell surface proteins and promote binding to epithelial cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, the invasion and adhesion of low-level capsularstrains, such as the D39∆\u003cem\u003epyrR\u003c/em\u003e- and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains, were reduced. These findings suggested that the simultaneous loss of \u003cem\u003emgaSpn\u003c/em\u003e and \u003cem\u003epyrR\u003c/em\u003e may also lead to changes in other adherence-related virulence factors.\u003c/p\u003e\n\u003cp\u003eBecause thicker capsules help \u003cem\u003eS. pneumoniae\u003c/em\u003e escape phagocytosis, we evaluated the anti-phagocytic ability of the strains after incubation with mouse macrophages in the absence of serum. The damaged capsules of the D39∆\u003cem\u003epyrR\u003c/em\u003e- and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains led to significantly reduced anti-phagocytic effects on macrophages(Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. pyrR\u003c/strong\u003e \u003cstrong\u003eis involved in systemic virulence and nasopharyngeal colonization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the role of \u003cem\u003epyrR\u003c/em\u003e in systemic infection, the experimental mice were divided into five groups, with 6 mice in each group, and were treated with the D39s, D39∆\u003cem\u003epyrR\u003c/em\u003e, D39∆\u003cem\u003emgaSpn\u003c/em\u003e, D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e, and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e::\u003cem\u003epyrR\u003c/em\u003e strains through nasal drops. At 48 h after bacterial infection, nasal lavage fluid, heart blood, and lung tissues were harvested and used for colony counting (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea-c). In addition, another five groups of mice, with 12 mice in each group, were infected with the D39s, D39∆\u003cem\u003epyrR\u003c/em\u003e, D39∆\u003cem\u003emgaSpn\u003c/em\u003e,D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e::\u003cem\u003epyrR\u003c/em\u003e strains through the nasal passage, and the survival time of each group of mice was recorded for 14 days (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003eIn vivo virulence tests showed that the bacterial load of the double deficient strain was significantly reduced in thenasopharyngeal lavage solution, lung tissues, and heart blood. In the double deficient strain, the colonization ability of the nasal cavity and lung was significantly reduced, and the survival rate was significantly increased. All abilities of \u003cem\u003epyrR\u003c/em\u003e were recovered after \u003cem\u003epyrR\u003c/em\u003e replenishment. These results indicated that Mga\u003cem\u003eSpn\u003c/em\u003e affects the pathogenicity of \u003cem\u003eS. pneumoniae\u003c/em\u003e through \u003cem\u003epyrR\u003c/em\u003e.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn many cases, the production of pathogen capsules is related to central metabolism, such as in \u003cem\u003eS. pyogenes\u003c/em\u003e, and zinc interferes with central metabolism and capsule biosynthesis4. Deficiency of \u003cem\u003espxB\u003c/em\u003e in \u003cem\u003eS. pneumonia\u003c/em\u003ee causes peptidoglycan and fatty acid biosynthesis metabolic imbalance, resulting in reduced capsule synthesis\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and impaired polyamine synthesis affects the availability of the serum type 4 CPS precursor, UDP-galactose, and the nucleotide sugar precursor, UDP-N-acetylglucosamine (UDP-GLcNAc), for the biosynthesis of CPS and peptidoglycan repeat units\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The interaction between capsule synthesis and metabolism is complex, and there are many unknowns. In the present study, we investigated the association between \u003cem\u003eS. pneumoniae\u003c/em\u003e uracil metabolism and capsule synthesis.\u003c/p\u003e \u003cp\u003eWe previously found that Mga\u003cem\u003eSpn\u003c/em\u003e protein negatively regulates the synthesis of capsular polysaccharides, and transcriptome and metabolome sequencing indicated changes in the metabolism of uracil and related molecules. Subsequently, we screened the uracil \u003cem\u003ede novo\u003c/em\u003e synthesis gene, \u003cem\u003epyrR\u003c/em\u003e, by qRT‒PCR. PyrR is a bifunctional protein that not only encodes UPRTase but also regulates the \u003cem\u003ede novo\u003c/em\u003e synthesis pathway of organisms through a transcriptional attenuation regulation mechanism. Recent studies on PyrR have focused on Bacillus subtilis\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and no DNA-binding proteins regulating the expression of the bacterial \u003cem\u003ede novo\u003c/em\u003e synthesis gene have been reported. In the present study, an EMSA demonstrated that Mga\u003cem\u003eSpn\u003c/em\u003e specifically binds P\u003csub\u003e\u003cem\u003epyrR\u003c/em\u003e\u003c/sub\u003e, and a DNase I footprinting assay identified potential binding sites.\u003c/p\u003e \u003cp\u003eUracil metabolism plays an important role in the growth and energy production of \u003cem\u003eS. pneumoniae\u003c/em\u003e. The growth rate and maximum biomass of \u003cem\u003epyrR\u003c/em\u003e-deficient bacteria significantly differ in the C\u0026thinsp;+\u0026thinsp;Y medium presence or absence of uracil. Pyrimidine biosynthesis can be divided into remedial synthesis and \u003cem\u003ede novo\u003c/em\u003e synthesis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In medium with uracil C\u0026thinsp;+\u0026thinsp;Y, remedial synthesis compensates for part of the energy loss caused by pyrimidine deficiency. In uracil‒free C\u0026thinsp;+\u0026thinsp;Y medium, the growth rate and maximum biomass of \u003cem\u003epyrR\u003c/em\u003e-deficient bacteria are significantly reduced, and both remedial synthesis and \u003cem\u003ede novo\u003c/em\u003e synthesis are blocked. Deficiency in the pyrimidine metabolic pathway leads to growth retardation and metabolic disorders in \u003cem\u003eS. pneumoniae\u003c/em\u003e. The growth rate and maximum biomass of \u003cem\u003emgaSpn\u003c/em\u003e- and \u003cem\u003epyrR\u003c/em\u003e-double deficient bacteria were not significantly different in medium with or without uracil (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). These findings suggested that there maybe a compensatory mechanism. When \u003cem\u003emgaSpn\u003c/em\u003e and \u003cem\u003epyrR\u003c/em\u003e are absent and bacteria are in a relatively nutrient-deficient environment,bacteria reduce unnecessary energy burden by decreasing capsules. Gram staining also identified a shorter chain length(Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), which would allow more energy to be available for growth and reproduction.\u003c/p\u003e \u003cp\u003eIn the present study, the expression of small molecular weight capsular proteins slightly decreased when \u003cem\u003epyrR\u003c/em\u003e was deficient, which may have been due to the presence of remedial synthesis to compensate for the absence of \u003cem\u003epyrR\u003c/em\u003e. Therefore, we detected the expression of the capsule in the absence of uracil, and the results showed that the expression of the capsule was significantly reduced. However, defects of \u003cem\u003emgaSpn\u003c/em\u003e and \u003cem\u003epyrR\u003c/em\u003e at the sametime significantly reduced capsule expression, and capsule expression was recovered with \u003cem\u003epyrR\u003c/em\u003e replacement. The above results suggested that Mga\u003cem\u003eSpn\u003c/em\u003e affects capsule synthesis by regulating \u003cem\u003epyrR\u003c/em\u003e. Because the biosynthesis of capsular polysaccharides is regulated by \u003cem\u003ecps\u003c/em\u003e operons, many studies have used cps operons as the starting point to explore the biosynthesis mechanism of capsular polysaccharides\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The transcriptional activity of P\u003csub\u003e\u003cem\u003ecps\u003c/em\u003e\u003c/sub\u003e plays an important role in the transcriptional regulation of genes downstream of the capsule. However, we observed that \u003cem\u003epyrR\u003c/em\u003e-deficient bacteria had no effect on P\u003csub\u003e\u003cem\u003ecps\u003c/em\u003e\u003c/sub\u003e in medium with and without uracil (data not shown). In addition, the P\u003csub\u003e\u003cem\u003ecps\u003c/em\u003e\u003c/sub\u003e activity of the D39∆\u003cem\u003emgaSpn\u003c/em\u003e strain was significantly higher than that of the D39s strain, which was consistent with a previous study, demonstrating that Mga\u003cem\u003eSpn\u003c/em\u003e is a transcriptional suppressor of capsule biosynthesis. Compared to the presence of uracil, the promoter of the four strains decreased in the absence of uracil (data not shown). The capsule is considered to be a large energy burden, and its production is thought to directly compete with central metabolism for energy \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The present findings suggested that in medium without uracil, the low yield of capsule is associated with less energy demand, and in the nutrient-deficient environment, more energy is used for growth and reproduction. The P\u003csub\u003e\u003cem\u003ecps\u003c/em\u003e\u003c/sub\u003e activity of the D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strain was higher than that of the D39s strain. However, Western blot analysis, ELISAs, uronic acid assays, and TEM observations showed that the capsular content of the D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strain was lower than that of the D39s strain. The polysaccharide synthesis of S. pneumoniae capsule is divided into several stages as follows: synthesis of capsule precursors, polymerization of repeat units, polymer flipping, and final localization\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Ghim and Neuhard\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e studied \u003cem\u003epyrR\u003c/em\u003e in thermosoluble \u003cem\u003eBacillus\u003c/em\u003e SPP and found that it catalyzes the UPRTase reaction. The present study suggested that the function of PyrR UPRTase plays a major role. The decrease of capsules caused by PyrR deficiency does not directly affect P\u003csub\u003e\u003cem\u003ecps\u003c/em\u003e\u003c/sub\u003e activity, but the decrease of UPRTase PyrR affects capsule precursor synthesis, similar to a \"raw material\" loss.\u003c/p\u003e \u003cp\u003ePrevious studies have suggested that \u003cem\u003eS. pneumoniae\u003c/em\u003e expresses low levels of CPS during colonization to enhance cell surface protein exposure and promote binding to epithelial cells\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In contrast, \u003cem\u003eS. pneumoniae\u003c/em\u003e expresses high levels of CPS during systemic infection to escape complement-mediated opsonophagy\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Based on the CPS reduction phenotypes observed in the D39∆\u003cem\u003epyrR\u003c/em\u003e- and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains, we speculated that the D39∆\u003cem\u003epyrR\u003c/em\u003e- and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains have increased adhesion to epithelial cells and decreased anti-phagocytic ability. As expected, the D39∆\u003cem\u003epyrR\u003c/em\u003e- and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains invaded the A549 lung epithelial cell line, and the anti- phagocytotic ability of mouse macrophages was decreased. In contrast to expectations, there was a decrease in adhesion. The adhesion ability of \u003cem\u003eS. pneumoniae\u003c/em\u003e is related to the SpsA choline-binding protein, the PfbA plasminase-binding protein, and the PavA \u003cem\u003eS. pneumoniae\u003c/em\u003e adhesion factor \u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Thus, the D39∆\u003cem\u003epyrR\u003c/em\u003e- and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e strains may also cause changes in other virulence factors in addition to the reduction of capsular content, causing defects in bacterial cell adhesion. The significantly shorter chain length observed by gram staining may also be responsible for the reduced adhesion, warranting additional studies(Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to the site of bacterial colonization and the stage of infection, the thickness and density of the capsule are dynamic\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In vivo experiments demonstrated that the D39∆\u003cem\u003emgaSpn\u003c/em\u003e and D39∆\u003cem\u003emgaSpn\u003c/em\u003e∆\u003cem\u003epyrR\u003c/em\u003e::\u003cem\u003epyrR\u003c/em\u003e strains with thicker capsulates were more aggressive and more likely to circulate to the spleen to cause systemic infection in mice. Mga\u003cem\u003eSpn\u003c/em\u003e regulates virulence and metabolism-related genes responding to various environmental changes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Because the content of uracil varies with different niches\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, Mga\u003cem\u003eSpn\u003c/em\u003e may regulate the virulence and adaptability of \u003cem\u003eS. pneumoniae\u003c/em\u003e by sensing environmental changes, such as uracil content, during infection. Overall, the in vivo and in vitro experiments showed that Mga\u003cem\u003eSpn\u003c/em\u003e regulates capsule production through \u003cem\u003epyrR\u003c/em\u003e, thereby affecting its virulence.\u003c/p\u003e \u003cp\u003eAn increasing number of studies have shown that there is a close relationship between the bacterial metabolic network and toxicity. The perception of nutritional availability of pathogenic bacteria drives the regulation and production of various virulence factors. The energy obtained by bacteria can be used to synthesize carbohydrate capsules, or it can be transferred to glycolysis to support replication\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Therefore, it is not surprising that capsule biosynthesis is integrated into the metabolic regulatory network of \u003cem\u003eS. pneumoniae\u003c/em\u003e. For the first time, the present study demonstrated that the \u003cem\u003epyrR de novo\u003c/em\u003e synthesis gene of \u003cem\u003eS. pneumoniae\u003c/em\u003e is regulated by the Mga\u003cem\u003eSpn\u003c/em\u003e transcriptional regulator. The present results suggested that Mga\u003cem\u003eSpn\u003c/em\u003e inhibits the expression of \u003cem\u003epyrR\u003c/em\u003e, leading to a decrease in polysaccharide synthesis in capsules. The decrease of polysaccharide in the capsule is due to the decrease of the \"raw material\" for capsule synthesis due to the decrease of PyrR UPRTase rather than affecting P\u003csub\u003e\u003cem\u003ecps\u003c/em\u003e\u003c/sub\u003e activity, thus indicating the link between uracil metabolism and capsule production. Exploring the mechanism of pyrimidine metabolism and capsule synthesis, especially how pyrimidine intermediates affect the regulation of capsule expression at the molecular level, will help to better understand the regulatory phenomena controlling \u003cem\u003eS. pneumoniae\u003c/em\u003e capsule synthesis and identify new therapeutic strategies affecting its virulence.\u003c/p\u003e"},{"header":"Abbreviations","content":"CPS: Capsular polysaccharide; E.coli: Escherichia coli; EMSA: Electrophoretic mobility shift assay; S.pn: Streptococcus pneumoniae; UMP: Uridylic acid; UTP: Uridine triphosphate "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Medicine Laboratory, Children\u0026rsquo;s Hospital of Chongqing Medical University;National Clinical Research Center for Child Health and Disorders, Ministry of Education Key\u0026nbsp;Laboratory of Child Development and Disorders; and Chongqing Key Laboratory of Pediatrics,\u0026nbsp;Chongqing, People\u0026rsquo;s Republic of China;\u003csup\u003e2\u003c/sup\u003eKey Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Chongqing\u0026nbsp;Medical University, Chongqing, People\u0026rsquo;s Republic of China;\u003csup\u003e3\u003c/sup\u003eDepartment of Pediatrics, Shengli Oil Field Central Hospital, Dongying 257034, China;\u003csup\u003e4\u003c/sup\u003eDepartment of Laboratory Medicine, The First Hospital of Changsha, 311 Yingpan Road, Changsha\u0026nbsp;410005, Hunan, China\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuqiang Zheng, Xuemei Zhang, andYibing Yin conceived and designed the experiments. \u0026nbsp;Xinlin Guo, Shuhui Wang and Ye Tao performed the experiments. Xinlin Guo and Weicai Suo analyzed the data. Li lei and Yapeng Zhang contributed the reagents, materials, and analysis tools. Xinlin Guo and Yapeng Zhang wrote the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Projects of the National Natural Science Foundation of China (No. 81871698 and No. 81772153).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Projects of the National Natural Science Foundation of China (No. 81871698 and No. 81772153).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePrina, E., Ranzani, O. T. \u0026amp; Torres, A. Community-acquired pneumonia. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e386\u003c/strong\u003e, 1097\u0026ndash;1108 (2015).\u003c/li\u003e\n\u003cli\u003eSuaya, J. 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Influence of bacterial interactions on pneumococcal colonization of the nasopharynx. \u003cem\u003eTrends \u003c/em\u003e\u003cem\u003eMicrobiol\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 129\u0026ndash;135 (2013).\u003c/li\u003e\n\u003cli\u003eAprianto, R., Slager, J., Holsappel, S. \u0026amp; Veening, J. W. High-resolution analysis of the pneumococcal transcriptome under a wide range of infection-relevant conditions. \u003cem\u003eNucleic Acids \u003c/em\u003e\u003cem\u003eRes\u003c/em\u003e 46, 9990\u0026ndash;10006 (2018) doi:10.1093/nar/gky750.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"streptococcus pneumoniae, capsular polysaccharides, MgaSpn, pyrR, uracil","lastPublishedDoi":"10.21203/rs.3.rs-4618066/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4618066/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUracil metabolism is an important step in the growth and metabolism of \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e, and pyrimidine nucleotides play an important role in the expression and production of \u003cem\u003eS. pneumoniae\u003c/em\u003e capsules. Mga\u003cem\u003eSpn\u003c/em\u003e(\u003cem\u003espd_1587\u003c/em\u003e),as a transcriptional ragulator of host environment adaptation, regulates the biosynthesis of the capsules and phosphorylcholine. However, the underlying regulation mechanism between uracil metabolism and biosynthesis of capsules remains incompletely understood. Here, we first described the relationship between uracil metabolism and capsule expression via the \u003cem\u003epyrR\u003c/em\u003e gene(\u003cem\u003espd_1134\u003c/em\u003e) in \u003cem\u003eS. pneumoniae\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eElectrophoretic mobility-shift assays (EMSAs) and DNase I footprinting assays showed a direct interaction between Mga\u003cem\u003eSpn\u003c/em\u003e and the \u003cem\u003epyrR\u003c/em\u003e promoter (P\u003csub\u003e\u003cem\u003epyrR\u003c/em\u003e\u003c/sub\u003e) at two specific binding sites. MgaSpn negatively regulated capsule production through \u003cem\u003epyrR\u003c/em\u003e as confirmed by complementing \u003cem\u003epyrR\u003c/em\u003e expression in D39Δ\u003cem\u003emgaSpn\u003c/em\u003eΔ\u003cem\u003epyrR\u003c/em\u003e. Virulence experiments showed that the Mga\u003cem\u003eSpn\u003c/em\u003e-\u003cem\u003epyrR\u003c/em\u003e interaction was necessary for both pneumococcal colonization and invasive infection.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eFor the first time, the present study demonstrated that the de novo synthesis gene \u003cem\u003epyrR\u003c/em\u003e of S. pneumoniae is regulated by the Mga\u003cem\u003eSpn\u003c/em\u003e transcriptional regulator.Taken together,these results provide an insight into the regulation of capsule production mediated by uracil metabolism and its important roles in pneumococcal pathogenesis.\u003c/p\u003e","manuscriptTitle":"The MgaSpn Global Transcriptional Regulator Mediates the Biosynthesis of Capsular Polysaccharides and Affects Virulence via the Uracil Synthesis Pathway in Streptococcus pneumoniae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-23 17:30:31","doi":"10.21203/rs.3.rs-4618066/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-07-01T11:21:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-01T11:20:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2024-06-21T14:46:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"85fb274e-112e-4cd5-8656-64cf6a0e75f4","owner":[],"postedDate":"July 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-23T17:30:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-23 17:30:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4618066","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4618066","identity":"rs-4618066","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}