Characteristic, genomics and transcriptomics comparation of phages Kp84B and Kp84S infecting Klebsiella pneumoniae

Characteristic, genomics and transcriptomics comparation of phages Kp84B and Kp84S infecting Klebsiella 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 Characteristic, genomics and transcriptomics comparation of phages Kp84B and Kp84S infecting Klebsiella pneumoniae Qingqing Sun, Wei Chen, Guangming Zhang, Yanmei Sun, Lixin Shen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7611342/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacteriophages have emerged as attractive alternatives to antibiotics for treating multidrug-resistant Klebsiella pneumoniae infections. Currently, numerous phage-host interaction factors remain to be discovered to better understand and optimize this therapeutic approach. In this study, we characterized two Klebsiella phages with highly similar genomes, Kp84B (40,452 bp) and Kp84S (40,466 bp), which share 99.46% sequence identity with 99% coverage. The general biological characteristics comparison showed that their host range, pH and temperature tolerance are same. However, their lysis ability was obvious different for the same host Kp84. Further comparative genome results showed that 21 different genes have 699 single nucleotide change. Five most promising protein were overexpressed in host Kp84, and lysis phenotypes of Kp84B and Kp84S were examined. As results, although lysis ability did not show obvious different between Kp84B and Kp84S, it was found that Gp4.5 and Gp5.5 genes can cause host cell abortive infection. RNA-seq analysis further revealed potential host interaction factors influencing their lysis ability, including some ABC transporters, propionate and vitamin B12 biosynthesis genes, insertion sequences, sRNA and their target genes. These provide further study target for phage-host interaction factors for lysis difference. Klebsiella pneumoniae bacteriophage(phage) biological characterization genome transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Klebsiella pneumoniae , a widespread Gram-negative opportunistic pathogen, was first isolated and named Friedlander bacterium in the late 19th century [ 1 , 2 ]. K. pneumoniae can cause a wide variety of hospital-acquired infections, including urinary and respiratory tract infections [ 3 ], liver abscesses [ 4 ], septicemia [ 5 ]. The emergence of hypervirulent carbapenem-resistant K. pneumoniae ( hv-CRKP ) has made the development of new and alternative therapies urgently needed [ 6 , 7 ]. Unlike other Enterobacteriaceae , K. pneumoniae possesses a thick capsular polysaccharide that plays a key role in host colonization and proliferation. Additionally, other virulence factors—such as lipopolysaccharide (LPS), siderophores, fimbriae, the type VI secretion system (T6SS) and outer membrane proteins-also contribute to its pathogenicity[ 8 – 10 ]. Notably, K pneumoniae is major contributor to antimicrobial resistance (AMR) epidemics [ 11 , 12 ]. Based on drug-resistant phenotypes, K. pneumoniae can be classified into classical ( cKp ) and hypervirulent ( hvKp ) strains [ 13 ]. In recent years, the infection rate of hvKp has risen annually, attracting widespread attention. The primary mechanisms of drug resistance in K. pneumoniae include horizontal gene transfer, biofilm formation, β-lactamase production, reduced membrane permeability, and enzyme modification [ 14 ]. Due to the excessive use of antibiotics, the drug resistance of K. pneumoniae has become a major concern [ 15 ]. Novel therapeutic approaches for drug-resistant K. pneumoniae remain to be developed. Bacteriophages (phages) are viruses that can specifically infect and may kill bacteria. They were independently discovered in 1915 [ 16 ]. Based on their life cycles, phages are classified into virulent/lytic phages (which directly lyse host bacteria) and temperate/lysogenic phages (which integrate into the bacterial genome). Phage therapy utilizes the bactericidal ability of phages to treat pathogenic infections. In the 1920s and 1930s, phage therapy gained attention and was widely used to combat bacterial infections [ 17 ]. However, some potential challenges for phage therapy also emerge. Such as, treatment failure because of limited host range [ 18 ], phage pollution, sustainable efficacy, and phage resistance [ 19 ]. Besides, some differences between the results of in vitro experiments and in vivo experiments are existed [ 20 ]. Following the discovery of chemical antibiotics, phage therapy was largely abandoned. However, with the rise of antibiotic resistance, it has reemerged as a promising complementary strategy [ 21 ]. Phage therapy now represents a potential alternative for treating drug-resistant K. pneumoniae [ 22 ]. For example, phage cocktail therapy (using mixed K. pneumoniae phages) significantly reduces bacterial load in colonized mice and alleviates liver injury [ 23 ]. Combining bacteriophages with antibiotics can achieve synergistic therapeutic effects [ 24 – 26 ]. During lytic phage infection of a host, phages can hijack host metabolic processes to enhance their own replication. Conversely, the bacterial strain can also trigger various anti-phage defense mechanisms, leading to complex phage-host interactions. For instance, Pseudomonas aeruginosa phages reprogram host amino acid metabolism to optimize viral production [ 27 ]. Additionally, at the phage adsorption stage, bacteria can inhibit phage attachment by modifying cell surface receptors, such as capsular polysaccharides (CPS), lipopolysaccharides (LPS), fimbriae, flagella, and outer membrane proteins [ 28 – 30 ]. For example, soluble alginases can degrade bacterial capsules. Restriction endonucleases can protect bacterial DNA by cleaving invading phage DNA [ 31 – 33 ]. Additionally, host cells can alter protein expression to resist phage infection, affecting genes involved in CPS and LPS synthesis, carbohydrate metabolism, elongation factors, and other critical pathways [ 34 ]. Once the phage injects its nucleic acids into the cell, the host employs targeted defense mechanisms to block phage replication, including CRISPR-Cas system [ 35 , 36 ], restriction modification system [ 37 ], toxin–antitoxin system (TA) and cyclic-oligonucleotide based antiphage signaling system (CBASS) [ 38 , 39 ]. With the advancement of sequencing technology, genomic and transcriptomic analysis has become increasingly widespread [ 40 ]. Additionally, genetic diversity in both phages and their hosts is promoted through coevolution [ 41 , 42 ]. Such diversity can lead to functional differences among phages. These phenomena have been explored in several studies using comparative (meta)genomics and transcriptomics [ 43 – 46 ]. However, the infection mechanisms of phages with a common origin remain unresolved. Furthermore, there are very few studies on host transcriptional responses to infections by two phages with highly similar genomes but different phenotypes. In this study, we compare two phages, Kp84B and Kp84S, which exhibit differences in lysis ability, to identify potential factors underlying these variations through genomic and transcriptomic analysis. These results will help elucidate key factors in phage-host interactions, offering promising insights for future research. Materials and methods 1.1 Bacterial strains and phages culture conditions All strains were cultured in Luria–Bertani (LB) broth at 37°C. K. pneumoniae 84 was used a host for phage. One hundred microliter phage solution was added to 10 mL K. pneumoniae 84 culture that grew to the exponential phase (OD 600 ≈ 0.6). The mixture was incubated at 37°C for 4–6 h with shaking at 200 rpm. Then, the culture was centrifuged at 8000 rpm for 10 min, filtered through a 0.22-µm filter. The phage titer was determined by the double-layer agar method. An aliquot of 100 µL phage filtrate and 100 µL of K. pneumoniae 84 were mixed with 0.7% LB ager medium, incubating 4–6 h or overnight at 37°C. Phage purification was performed by isolating single plaques and repeating the process three times. The collected phage preparation was subsequently stored at 4°C. 1.2 Biological characteristics 1.2.1 One-step growth curve The test method of one-step growth curve is described as follow. The cultures of Kp84 were sub-cultured in 50 mL of LB medium, culturing to the mid-logarithmic phase (OD 600 ≈ 0.5–0.6). Phage Kp84B and Kp84S were added at an MOI of 10 or 0.001, respectively. Next, the mixtures were incubated at 37°C, with shaking for 5–7 min. Subsequently, the bacterial pellets were collected through centrifuge for 5 min at 4°C. Finally, the collection was washed twice with sterile water, resuspending to 50 mL LB with shaking at 37°C. The suspension was cultured for 200 rpm at 37°C. Samples of mixture were taken at 0 to 60 min every 10 min. The collected sample centrifuged for 5 min at 4°C and determined phage titers. The one-step growth curve was drawn according phage titers as the function of time. 1.2.2 Lysis curve (infection curve) The phage Kp84B and Kp84S respectively were added to the mid-logarithmic phase Kp84 according to each MOI (0.0001, 0.001, 0.01, 0.1, 1,10, 100). Then, the mixtures were subpacked into 96-well plates, 100 µL in each well. Then, these were sealed using 50 µL Liquid Paraffin. Finally, the optical density was measured at 600 nm (OD 600 ) every 30 min using BioTek Epoch 2 (BioTek, Vermont, USA). The lysis curve was drawn according to OD 600 at different time. 1.2.3 Phage host range analysis The host range of phages Kp84B and Kp84S was determined against 220 different K. pneumoniae strains using spot testing. An aliquot of 5 µL purified phage suspension was spotted onto the surface of double-layer agar plates inoculated with the tested strains. The double-layer agar plates were incubated for 4–6 h at 37°C. Host bacteria were identified based on plaque formation. 1.2.4 Phage temperatures and pH stability tests The temperature and pH stability of two phages were experimented as follow described. About temperature stability, 1 mL of cell-free phages (10 8 PFU/mL) were incubated at various temperatures (4°C, 25°C, 37°C, 55°C, 65°C, 75°C, 85°C, 95°C, and 100°C) for 60 min. For pH stability, 100 µL phages were mixed with 900 µL of LB adjusted to different pH values (2, 4, 6, 7, 8, 10, 12, and 14). Immediately, the mixture was incubated at 37°C for 60 min. Finally, titers of these sample were determined using the double-layer agar method. 1.3 Phage genomic, sequencing, and bioinformatic analysis 1.3.1 Phage genomic DNA extraction Phage genomic DNA was extracted using the SDS method as described below. Firstly, 10 µL of phage lysates were centrifuged (12,000×g, 4°C, 15 min) and filtered (0.22-µm) to remove cellular debris. The purified lysates were mixed with 10 µL DNase I and 10 µL RNase I and incubated for 30 min at 37°C. Then, 20% buffer 1 (0.25% SDS, 20 mM Tris-Cl, 20mM EDTA) was added and the mixture was incubated at 80°C for 15 min. NaAc was added to a final concentration of 0.625 mM, the solution was mixed and incubated on ice for 1 hour. The suspension was centrifuged (12000×g, 4°C, 15 min) and collected pellets. Then, the pellets were resuspended and rinsed twice with pre-cooled absolute ethanol. After allowing the ethanol to evaporate, the genomic DNA was dissolved in an appropriate volume of nuclease-free water. Finally, DNA concentration was measured using a Nano Drop One (Thermo, Wyman Street, Waltham, MA, USA). 1.3.2 Phage whole-genome sequencing and bioinformatic analysis The fragmented genome was produced by Covaris M220 and detected concentration using Invitrogen Qubit 4.0. Then, the whole-genome was sequenced by the Illumina NovaSeq 6000 platform, after library was constructed using KAPA Hyper Prep Kit. Genome was assemble using MEGAHIT [ 47 ] or MetaSPADes [ 48 ]. Functional annotations of putative ORFs were performed using the NCBI online tool BLASTp in conjunction with conserved domains and Rapid Annotation using Subsystem Technology (RAST, https://rast.nmpdr.org/ ). tRNAs were predicted using the tRNAscan-SE program ( http://lowelab.ucsc.edu/tRNA scan-SE/). The antibiotic resistance and virulence factor genes were identified using the Antibiotic Resistance Genes Database ( http://card.mcmaster.ca/analyze/rgi ) and the Virulence Factor Database (VFDB, http://www.mgc.ac.cn/VFs/ ). The genome map was generated using the software SnapGene. Genome comparisons were performed using tBLASTx and visualized with Easyfig (version 2.2.5) [ 49 ] to describe the relationships among phage Kp84B, Kp84S, and their closest relatives. The related phylogenetic tree was constructed and displayed using the MEGA7 [ 50 ] program through the maximum likelihood method. 1.3.3 Plasmid construction and transformation pEXKp-B45, pEXKp-B50 and pEXKp-LysinB were constructed using the following procedures. The plasmid backbone containing pBR322_origin, lac promoter, and the apramycin resistance marker were amplified from the pUC19 plasmid. The B45 gene encoding peptidoglycan lytic exotransglycosylase was PCR amplified from the genomic DNA of the Kp84B. The B50 and LysinB gene encoding non-contractile tail tubular protein Gp12 and endolysin were amplified from the genomic DNA of the Kp84B. The fragment along with the linearized plasmid backbone were assembled together using In-Fusion cloning, resulting in the final plasmid pEXKp-B45, pEXKp-B50 and pEXKp-LysinB. pEX-Gp4.5B, pEX-Gp4.5S, pEX-Gp5.5B, pEX-Gp5.5S and pEX-LysinS were constructed using the following procedures. The plasmid backbone containing Rep101 origin, promoter of the L-arabinose operon of E. coli , and the apramycin resistance marker were amplified from the pCasKp-Apr plasmid. The Gp4.5B and Gp5.5B gene encoding inhibitor of host toxin/antitoxin system and suppressor of silencing were PCR amplified from the genomic DNA of the Kp84B. Gp4.5S, Gp5.5S and LysinS encoding inhibitor of host toxin/antitoxin system, suppressor of silencing and endolysin were PCR amplified from the genomic DNA of the Kp84B. The fragment along with the linearized plasmid backbone were assembled together using In-Fusion cloning, resulting in the final plasmid pEX-Gp4.5B, pEX-Gp4.5S, pEX-Gp5.5B, pEX-Gp5.5S and pEX-LysinS. 1.4 Transcriptome sequencing analysis RNA sequencing (RNA-Seq) was performed on an Illumina NovaSeqXPlus platform by Majorbio (Shanghai, China). According to one-step growth curves, samples were collected post- adsorption at 0, 20 and 30 min, representing the early, middle, and late phases of the Kp84B infection cycle, respectively. Samples for Kp84S were collected at 0, 20 and 40 min. 1.4.1 RNA Extraction and Quantification Total RNA was extracted using a Trizol kit (Life Technologies). RNA concentration and purity were evaluated using the Nanodrop 2000. RNA integrity and quality were measured using 1% Agarose gel electrophoresis. Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was employed for detecting RIN (RNA Integrity Number). 1.4.2 cDNA library construction cDNA library is prepared using TruSeq™ Stranded Total RNA Library Prep Kit (Illumina). The process outline is as follow. The enriched mRNA without rRNA was randomly interrupted by adding Fragmentation Buffer. The first cDNA strand was synthesized with random primers using mRNA as a template. Then, the second cDNA strand was synthesized by adding dUTP to replace dTTP, of which its bases contain A/U/C/G. The purified double-stranded cDNA was then end-repaired and A-tailed. Finally, the cDNA library was enriched by PCR. Sequencing was performed with the NovaSeqXPlus platform. 1.4.3 Quantification of Gene Expression Level Gene expression transcript expression levels are analyzed using RSEM. TPM was calculated based on the gene length. 1.4.4 Differential Expression Gene Analysis The differential gene expression analysis using DEGSeq. For comparison, a P-value of 0.001 and log 2 (fold change) of 1 were set as the thresholds for significantly differential expression [ 51 ]. 1.4.5 KEGG Enrichment Analysis Pathway enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genome) were performed using R script, referring to KEGG Pathway database ( http://www.kegg.jp/kegg/pathway.html ). Fisher's Exact Test is used as the calculation method. The KEGG pathway function is considered to be significantly enriched, when the P value < 0.05. Results 2.1 Biological characteristics examination of phages 2.1.1 Host range of Kp84B and Kp84S The number lysised hosts of both phages Kp84B and Kp84S is 47 out of 204 K. pneumoniae (Fig. 1 .a and Supplementary Information 1). The bacteriophage plaque with halo is successfully formed on LB double-layer plate when KP-YQ88, KP-YQ74, KP-YQ10, KP8-2 and KP7-25 was respectively mixed with Kp84B and Kp84S. However, the KP84, Kp7-20, Kp8-7, KP-YQ43 and KP-YQ79 had same phenomenon with Kp84B. All the other sensitive K. pneumoniae only formed bacteriophage plaque without halo when they were lysised by Kp84B and Kp84S. Specially, morphology of Kp84B and Kp84S phage plaque with Kp84 is showed that single phage Kp84B produce a large plaque with an opaque halo on a bacterial lawn (Fig. 1 .b). In contrast, single phage Kp84S only produce the small without halo plaque (Fig. 1 .c). 2.1.2 One-step growth curve The one-step growth curves showed as Fig. 1 .d. A latent period of Kp84S located between 0 and 10 min, a burst period was 10–30 min. After 30 min, Kp84S stayed in plateau period. About Kp84B, its latent period and burst period respectively was 0–10 min and 10–40 min. After 40 min, lytic cycle of Kp84B entered in plateau period. 2.1.3 Lysis curve (infection curve) After Kp84 were respectively mixed with Kp84B and Kp84S according to different MOI, the absorbance at 600 nm change with time of culture was measured to contrast those lysised regularly indirectly. As shown in the Fig. 1 .e-f, the optical density of Kp84B and Kp84 coculture reaches the peak at 1 hour and falls to minimum value at 2 h. The regularity is much the same on coculture Kp84S and Kp84. Nevertheless, the largest difference between of both phages is that optical density of Kp84B and Kp84 coculture is not obviously increase during 24 h after lysise but that of Kp84S is progressively increase from 6 h to 24 h. This suggests that it is more likely to produce anti-phage mutants when Kp84S infected host Kp84. 2.1.4 Phage temperature, pH and UV stability Both Kp84B and Kp84S can be maintain great activity below 45°C and reduce activity at 55°C, while be inactivated after incubation at temperatures above 65°C for 1 h (Fig. 1 .g). The two phages were inactivated above pH12 and below pH2. The activity of Kp84B and Kp84S was maintained out of 50% incubating 1 h at pH6 and pH10. The activity of Kp84B and Kp84S decreased two orders of magnitude at pH4 (Fig. 1 .h). The two phages are also equally sensitive to ultraviolet (UV) light. After being exposed to a 20 W ultraviolet lamp for 60 min, their titers decreased by nearly five orders of magnitude (Fig. 1 .i). 2.2 Genome analysis of phage Kp84B and Kp84S 2.2.1 Genome characteristic and phylogenetic analysis The genome length of phage Kp84B is 40,452bp, and GC content is 52%. Correspondingly, phage Kp84S is 40,466bp, and 52% GC. The function of 51 ORFs and 52 ORFs from phage Kp84B and Kp84S genome is predicted, respectively (Table 1 ). The annotated ORFs are classified into five functional modules: DNA replication, regulation and nucleotide metabolism, packaging, unknown functions, and lysis (Fig. 2 .a). No tRNA genes are identified in the genomes. No antibiotic resistance genes and virulence factors are predicted in the Kp84B and Kp84S genomes (Supplementary Information 2–3). Phylogenetic analysis of complete genome sequence suggests that the both of phages Kp84B and Kp84S are most closely related to the Autographiviridae family, Studiervirinae subfamily, Przondovirus genus, specifically Klebsiella phage SH-Kp152234 (Fig. 2 .b). Table 1 Genome summarize of Kp84B and Kp84S Phage Genome Size (bp) Type GC content % A% T% C% G% ORF Kp84B 40452 circle 52.36 25.63 22.01 24.68 27.69 51 Kp84S 40466 circle 52.34 25.61 22.04 24.65 27.70 52 2.2.2 Comparative genomes and Functional differential genes Nucleotide sequence identity of Kpg8B and Kp84S genome is 99.4% at 99% coverage (Fig. 2 .c). The comparison of two phage genome was visualled using Easyfig (Fig. 2 .b). Six hundred and ninety-nine bases of twenty-one proteins are different between Kp84B and Kp84S genome coding sequence (Supplementary Information 4). Particularly, inhibitor of host toxin/antitoxin system, non-contractile tail tubular protein Gp12, peptidoglycan lytic exotransglycosylase, phage protein Gp5.5 suppressor of silencing and endolysin are the biggest differences. Moreover, we note that a G→C transversion in the inhibitor of host toxin/antitoxin system gene (Phage protein Gp4.5), the base is located at 17,469 of the Kp84B genome. This change led to premature termination of Kp84B phage protein Gp4.5 translation than Kp84S. Phage non-contractile tail tubular protein Gp12 and peptidoglycan lytic exotransglycosylase gene of Kp84B is also premature termination comparing Kp84S. Therewith, those different gene were constructed into a plasmid containing L-Arabinose induced promoter or IPTG induced promoter. Those plasmids were transformed into Kp84 and build gene overexpression strains, named Kp84/pEX-Gp4.5B, Kp84/pEX-Gp4.5S, Kp84/pEX-Gp5.5B, Kp84/pEX-Gp5.5S, Kp84/pEX-B50, Kp84/pEX-B45, Kp84/pEX-LysinB, Kp84/pEX-LysinS. Notably, the strains lysis of Kp84/pEX-Gp4.5B, Kp84/pEX-Gp4.5S, Kp84/pEX-Gp5.5B, Kp84/pEX-Gp5.5S were weaken when they co-culture with Kp84B and Kp84S and were induced expression (Fig. 3 .g-j). The strains Kp84/pEX-B50, Kp84/pEX-B45, Kp84/pEX-LysinB, Kp84/pEX-LysinS were not changed comparing control Kp84/pUC19 when were lysised by Kp84B and Kp84S (Fig. 3 .a-h). Therefore, those genes were uninfluential to activity difference between Kp84B and Kp84S. However, Gp4.5B, Gp4.5S, Gp5.5B and Gp5.5S can cause abortive infection (Fig. 3 .g-j). 2.3 Transcriptome analysis of Kp84B and Kp84S infected Kp84 2.3.1 Overview of host gene expression changes and KEGG enrichment analysis According to one step growth curve, early (at 0 min), middle (at 20 min) and later (at 30 min for Kp84B, at 40 min for Kp84S) stage bacterial samples were collected after Kp84B and Kp84S infection. Those samples respectively correspond control group Kp84B-0, Kp84B-20 and experimental group Kp84S-0, Kp84S-20. As a whole, 5,780 genes of the Kp84 genome were differentially expressed. At early stage, 529 genes were upregulated expression and 402 genes were downregulated expression. At middle stage, 236 genes were upregulated expression and 247 genes were downregulated expression. At last stage, 236 genes were upregulated expression and 247 genes were downregulated expression (Fig. 4 .a). Early stage, the largest number of significantly differential expression genes by KEGG enrichment analysis were reflected in ABC transporters, next propanoate metabolism and butanoate metabolism etc. (Fig. 4 .b). Middle stage, those significantly differential expression genes are reflected in pyruvate metabolism, porphyrin and chlorophyll metabolism, butanoate metabolism, and TCA cycle etc. (Fig. 4 .c). Last stage, those significantly differently expressed genes are reflected in ABC transporters etc. (Fig. 4 .d). 2.3.2 ABC transporter response early infection Sixty-four significantly differently expressed genes were enriched onto pathway at early infection. However, they were almost no change at middle infection. These genes involved six classes of ABC transporters, and included 29 compound and molecule transporters (Fig. 5 .a-d). In detail, 10 monosaccharide transporters were upregulated significantly including phaseomannite ( IbpA , IatP , IatA ), methyl-galactoside ( mglA , mglB , mglC ), and erythritol ( eryE , eryG ) transporters (Fig. 5 A). NikABCDE operon belongs to nickel transporters, periplasmic binding protein NikA was upregulated, while nikC , nikD and nikE were downregulated (Fig. 5 .b). The ABC transporter DppABCDF matters dipeptide acquisition, and dipeptide transporter protein dppA was also upregulated (Fig. 5 .b). Cobalt transporter ATP binding domain cbiO was upregulated (Fig. 5 .b). Three ferric uptake genes afuA , afuB and afuC were high expression in Kp84S infection. Glutamic acid and aspartic acid transporter genes gltL , gltK , gltJ and gltI were upregulated (Fig. 5 .c). Finally, maltos transporters malK and malF , galactose and maltooligosaccharides transporters ganO and msmX , sorbitol transporter smoK were upregulated (Fig. 5 .c). Four genes rbsABCD of rbs operon were downregulated, involving ribose, Autoinducer2 and D- xylose uptake (Fig. 5 .a). Ferric hydroxamate uptake ( fhu ) genes fhuC and fhuD were downregulated. In addition, iron and manganese acquisition genes sitBCD , and ferrisiderophores uptake genes fepBCG were downregulated in Kp84S early infection (Fig. 5 .b). Phosphate transport system pstA , pstB and pstC , was also downregulated (Fig. 5 .c). Arginine, cystine, and branched-chain amino acid revealing transporters were downregulated, including artQ , artM , fliy , livK , livH , livG , livM and livF (Fig. 5 .c). Spermidine / putrescin transporter genes potB , potA and potC were low expression in Kp84S infection than Kp84B (Fig. 5 .d). Therefore, we infer that Kp84B life process may consume more spermidine/putrescin, ribose/D-xylose, phosphate, iron, arginine, cystine, and branched-chain amino acid. While Kp84S may consume more maltooligosaccharide, inositol, glutamic acid and aspartic acid. 2.3.3 Propionate synthesis, Porphyrin and chlorophyll metabolism response phage infection Propionate synthesis, porphyrin and chlorophyll metabolism pathway were significantly enriched different genes through KEGG analysis. Furthermore, propionate synthesis mainly reflected in early infection, porphyrin and chlorophyll metabolism mainly reflected in middle infection. As we known, butanoate metabolism can promote vitamin B12 biosynthesis. Even more, butanoate has ability to induce E. coil prophage to lysis, and produce more progeny phages [ 52 ]. Heme, chlorophyll, and vitamin B12 are essential for various metabolic pathways [ 53 ]. In early infection, 19 genes were significantly differential expression in propionate synthesis pathway. Among, pct , fadJ , fadB , puuE , mmsA , succC , sucD and atoB were upregulated expression in Kp84S infection. These genes involved acrylate and methylmanoyl-CoA metabolism to propionyl-CoA. Conversely, downregulated expression genes mostly belonged to pathway of propanediol to propionyl-CoA, including gldA , pduC , pduD , pduE , dhaT and pduQ . In addition, pct was also in charge of converting from propionyl-CoA to propionate (Fig. 6 .a). Sixteen different expressed genes involved vitamin B12 (cobalamin) biosynthesis, belonging to porphyrin and chlorophyll metabolism pathway. These genes were upregulated expression in middle infection, and fold change (log2FC) maintain it between 1.1 and 3.1 (Fig. 6 .b). In summary, host cell Kp84 may prefer to use propanediol to produce propionate when it was infected by Kp84B. While host cells may prefer to use acrylate and methylmanoyl-CoA to produce propionate in Kp84S infection. Moreover, Kp84S could promotes vitamin B12 biosynthesis in middle infection. 2.3.4 sRNA response phage infection The majority of sRNA bind target genes to regulate transcript stability, active or inhibit protein expression, and can regulate virulence factor, take part in defense responses etc. This report that prophage encode a sRNA can inhibit maltodextrin transport gene lamb to reduce lysis ability [ 54 ]. In our study, 211 sRNAs are significantly differential expression in early infection, while seven sRNA were differently expressed in middle stage (Fig. 7 .a-b). Among these sRNA, sRNA0122 can target eighteen genes, of which eight targeted genes were significantly differential expression (Fig. 7 .c). sRNA0576 can target 4 differently expression genes (Fig. 7 .d). According to targeted genes fold change, we concluded that sRNA0576 could influent Kp84B and Kp84S life process though negatively regulating gene4612 encoding hypothetical protein. sRNA0122 could positively regulate gene4286, gene4611 and gene3358, and negatively regulate gene2184, gene2972 and gene163 in early infection. Gene4286, gene4611 and gene3358 coded hypothetical protein, hypothetical protein and N-acetylmuramoyl-L-alanine amidase respectively. N-acetylmuramoyl-L-alanine amidase can participate in peptidoglycan catabolic process. Gene2184, gene2972 and gene163 coded hypothetical protein, phosphate acyltransferase and serine endoprotease, respectively. Specially, genes with a fold change exceeding 3 were summarized (Supplementary Information 5). Because the larger the difference, it is the more likely key factor. In early infection, 46x genes were upregulated expression, including Insertion Sequence genes, genes related to central carbon metabolism, and genes related to ABC transporters. Examples include IS5-like element ISKpn26 family transposase (gene1182), IS3 family transposase (gene1655), succinate dehydrogenase membrane anchor subunit (gene3739), succinyl glutamate-semialdehyde dehydrogenase (gene3111), 2-oxoglutarate dehydrogenase E1 component (gene3736), acetyl-CoA C-acyltransferase FadA (gene4941), LysR family transcriptional regulator (gene2316), ABC transporter ATP-binding protein (gene2184), iron ABC transporter permease (gene2183), and sugar ABC transporter substrate-binding protein (gene2021). Twenty-six genes was downregulated expression in the early stage, including DhaG protein (gene 633), the propanediol/glycerol family dehydratase medium subunit (gene635), the glycerol dehydratase reactivate beta/small subunit family protein (gene631), the glycerol dehydratase small subunit DhaB3 (gene636), 1,3-propanediol dehydrogenase (gene632), the diol dehydratase reactivate subunit alpha (gene637), the glycerol dehydratase large subunit (gene634), the phosphate ABC transporter substrate-binding protein PstS (gene5140), the ribose ABC transport system (gene5120), etc. In middle infection, eleven upregulated genes were annotated, including the IS5-like element ISKpn26 family transposase (gene12), the qaJ viral recombinase family protein (gene3036), the energy-coupling factor ABC transporter substrate-binding protein (gene999), the Rha family transcriptional regulator (gene1185), and NADH oxidoreductase (gene3525). Thirteen genes are downregulated, including oxidoreductase (gene3845), succinylglutamate desuccinylase (gene3109), malate: quinone oxidoreductase (gene2751), cytochrome c-type biogenesis protein CcmH (gene2196), and oxaloacetate decarboxylase subunit beta (gene4403), etc. In last infection, 6 genes exit upregulated expression, including the IS5-like element ISKpn26 family transposase (gene828), the IS5-like element ISKpn26 family transposase (gene120), the IS6-like element IS26 family transposase (gene3005), and the ssDNA-binding domain-containing protein (gene1796). The number of downregulated gene were more than 1000, for example, the O-antigen and lipid-linked capsular repeat unit polymerase (gene1721), the IS3-like element ISKpn1 family transposase (gene1762), the putative oligogalacturonide transporter (gene1868), dihydropteroate synthase (gene534), ABC transporter permease (gene1661), the methionine ABC transporter permease Met I (gene4214), etc. Discussion In this study, the biological characteristics differences were mainly manifested in the following, when phage Kp84B and Kp84S infected host Kp84. Phage Kp84B has stronger lysis ability than Kp84S to host Kp84. However, both phages genome is 99.46% identity at 99% coverage. Through comparing both genome different genes, and RNA-seq of infected host cell, potential major factors were predicted. In detail, 21 genes had 699 single base difference. Specially, we noted 5 most probable different genes, including inhibitor of host toxin/antitoxin system, non-contractile tail tubular protein Gp12, peptidoglycan lytic exotransglycosylase, phage protein Gp5.5 suppressor of silencing and endolysin. Further, these proteins were overexpressed in host cell Kp84 and compare lysis phenotype for Kp84B and Kp84S. Firstly, the nucleotide sequences of the capsid assembly scaffolding protein in Kp84B and Kp84S showed significant differences between the 160th and 360th base. Their portal proteins had 76 base mismatches. As reported in the study, the number of positively and negatively charged amino acid of major and minor coat protein also decides virion size, besides phage genome length [ 55 – 57 ]. In addition, capsid assembly scaffolding protein and portal protein can influence the shape and size of the phage pro head through regulating major and minor coat protein [ 58 – 60 ]. Secondly, nucleotide sequence of tail tubular protein gp12 of Kp84B and Kp84S had 93.53% identity (98% coverage), tail proteins are same. At mentioned article that tail proteins, tail tubular protein and a structural tail protein can encode depolymerases. These protein can recognize the host and bind to capsular polysaccharides (CPS), exopolysaccharides (EPS), or lipopolysaccharides (LPS) of the host bacteria [ 61 , 62 ]. It was important that these depolymerases cleave polysaccharide-repeating units, helping the phage reach the cell wall and inject its DNA to infect the cell. As well as depolymerases can produce opaque halo zones on a bacterial lawn. Thirdly, the peptidoglycan transglycosylase gp16 of Kp84B and Kp84S had 44 nucleotide difference, differed by 10 amino acids. As we all known, the peptidoglycan transglycosylase gp16 motif is essential for bacteriophage T7 growth at temperatures below 20°C. Further, glutamate residue mutation of peptidoglycan transglycosylase gp16 can increase infection efficiency of phage T7 for E. coli of high cell densities [ 63 ]. Although phage protein gp4.5 inhibitor of host toxin/antitoxin system and phage protein Gp5.5 suppressor of silencing can cause host cell abortive infection when they were overexpressed into Kp84. It was a pity that these genes whether were from Kp84B and Kp84S genome were no different for lysis phenotype. Therefore, major gene was unfound using this way. It was permuted that these different from genome are not solely enough to lysis activity. Through transcriptome analysis, some genes involved metabolic pathways, insertion sequences and ABC transporters were likely major factors. It can be known from KEGG enrichment that the most impact genes focus on ABC transporters and metabolic pathways. It was understandable that phage indeed can reprogram host cell carbon metabolism and nucleotide synthesis pathway to their reproduce offspring [ 64 ]. An example, cyanophage possessed the capacity that transfer carbon flux from carbon fixation to pentose phosphate pathway. This change increased the NADPH output that it was necessary energy to phage DNA replication [ 65 ]. In addition, phage also can reprogram host cell amino acid metabolism to promote themselves replication. Key amino acids and the arginine-derived polyamine putrescine levels were increased when phage protein Eht1 and Eht2, were expressed early in the infection cycle. More, putrescine played key actor for phage replication [ 66 ]. Amino acid transporters transcriptional level existed differences between Kp84B and Kp84S infection. Genes concerning spermidine/putrescin, arginine, cystine, and branched-chain amino acid transporter were higher expression in Kp84B early infection than Kp84S. However, glutamic acid and aspartic acid transporters were higher expression in Kp84S early infection. Besides, it was reported that DppBCDF ABC transporter substrate binding protein DppA can inhibit phage Pf5 lysis activity. The number of Pf5 progeny phages was increased when dppA is deleted [ 67 ]. As it happened, gene dppA of Kp84 was upregulated in Kp84S early infection. Kp84S had weak lysis ability than Kp84B. On the side, we noted that high fold changed genes were labeled in central carbon metabolism (CCM), insertion sequence transposase genes, beside ABC transporters in early and middle infection. Some phages can reprogram the metabolic pathways of their bacterial host, with variations among different phages. The siphovirus PSA-HS2 can suppress energy-consuming metabolisms, such as motility and translation through repressing expression of cycD , cycN , cysI and cycJ genes. Among these genes, cycD and cycN encode sulfate adenylyltransferase, cysI and cycJ encode assimilatory sulfite reductase (NADPH)[ 66 ]. During Kp84B and Kp84S infection, genes related to the Kp84 metabolic pathway change significantly, for example, NADH oxidoreductase (gene 3525) and ATP synthase and so on involved in energy metabolism. Insertion sequences (ISs) are small transposable elements that encode proteins required for their transposition and influence the evolution of the bacterial genome [ 68 ]. The IS family is defined as a group of insertion sequences (ISs) with related transposases, strong conservation of the catalytic site, conserved organization, and similar inverted terminal repeats (IRs) [ 69 ]. IS-mediated gene expression results in clinically significant increases in phage resistance. For example, IS903B and IS903-formed composite transposon can interrupt wcaJ or wbaZ genes associated with the capsular polysaccharide biosynthesis, thereby causing Klebsiella pneumoniae (CRKP) phage resistance [ 70 ]. In our study, the IS5-like element ISKpn26 family transposase, IS3 family transposase, and IS6-like element IS26 family transposase were the major upregulated genes. As well as, phage receptor-related genes were one class of the downregulated genes in the last stage. This may facilitate the emergence of Kp84 anti-phage Kp84S strains. In conclusion, although phage Kp84B and Kp84S had similar stability of temperature, pH and UV, lysis spectrum, they had different lysis ability. Homologous genes from two phages had same influence when they were expression into host cell. Although these homologous genes were the most likely influencing factors for lysis ability between Kp84B and Kp84S. However, comparative transcriptome results implied that some genes were significant differentially expressed. These genes related to ABC transporter pathways, molecular metabolic pathways, insertion sequences, sRNA and target genes, etc. We considered that they were potential interaction factor to make clear different lysis ability between Kp84B and Kp84S. In the future, the host differential expression genes can be verified. Conclusion In this study, phage Kp84B and Kp84S have similar host range, pH and temperature tolerance are same. However, Kp84B has stronger lysis ability than Kp84S to the same host Kp84. Five homologous genes are not factors leading to lysis difference. ABC transporters, propionate and vitamin B12 biosynthesis genes, insertion sequences, sRNA and their target genes are potential host interaction factors influencing their lysis ability. Declarations Conflict of Interest: The authors declare no conflict of interest. Author Contribution Qingqing Sun and Wei Chen, experiments design, data curation, writing original draft, review, and editing; Guangming Zhang and Yanmei Sun, review and editing; Linxin Shen and Shiwei Wang, experiments design, supervised the work progress and edited the manuscript. All authors agreed to be accountable for the content of the work. Acknowledgments: This work was supported in part by the grants from the Science & Technology Fundamental Resources Investigation Program (Grant No. 2022FY101100). This work was also supported by the National Natural Science Foundation of China (Grant No. 32170114 and 31770152). References Merino, S., Camprubí, S., Albertí, S., Benedí, V.J., and Tomás, J.M. (1992) Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing. Infection & Immunity 60 , 2529-2535. https://doi.org/10.1007/BF01960820 Bodey, G.P. (1988) Fungal infections in the compromised host. 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(2006) Insertion sequences in prokaryotic genomes. Current Opinion in Microbiology 9 , 526-531. https://doi.org/ 10.1016/j.mib.2006.08.005 Yin, X., Fang, Q., and Zong, Z. (2022) Interruption of capsular polysaccharide biosynthesis gene wbaZ by insertion sequence IS 903B mediates resistance to a lytic phage against ST11 K64 Carbapenem-resistant Klebsiella pneumoniae . msphere 7 , e00518-00522. https://doi.org/ 10.1128/msphere.00518-22 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation1.xlsx SupplementaryInformation2.xlsx SupplementaryInformation3.xlsx SupplementaryInformation4.xlsx SupplementaryInformation5.xlsx Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7611342","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":521844455,"identity":"7b170e25-a8d9-418b-aaaa-6c26ebed2b9c","order_by":0,"name":"Qingqing Sun","email":"","orcid":"","institution":"Ministry of Education, Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Qingqing","middleName":"","lastName":"Sun","suffix":""},{"id":521844456,"identity":"ebfe9fc3-ae89-49e9-a022-df9edb462ad9","order_by":1,"name":"Wei Chen","email":"","orcid":"","institution":"Nanjing University of Chinese 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UV stability.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7611342/v1/2fd22fca1e6b806978cc0a94.png"},{"id":92500188,"identity":"b2a425d3-c613-48a2-ba2a-d2bb4ceda4a7","added_by":"auto","created_at":"2025-09-30 11:12:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":327214,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of the genome of phage Kp84B and Kp84S (a), and Multiple genome alignments among Kp84B, Kp84S, and Klebsiella pneumoniae phage SH-Kp 152410 (b). B, different colored arrows represent predicted different functions module: purple, lysis; yellow, nucleic acid synthesis and metabolism function; pink, phage structure; green, host regulation related; blue, hypothetical protein. c, shows dot matrix view of similarity based upon the BLAST results.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7611342/v1/d6a5e07b595b9338617dc602.png"},{"id":92499462,"identity":"32acc57d-0b43-428d-9049-c4fa5ca04bb6","added_by":"auto","created_at":"2025-09-30 11:04:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":415301,"visible":true,"origin":"","legend":"\u003cp\u003eThe lysis curve of Kp84B and Kp84S to different gene overexpression strains. a-c, and g-h represent lysis curve of Kp84B. g-f, and i-j represent lysis curve of Kp84B. Kp84/pEX-B and Kp84/pEX-S, represent overexpression strain of which gene form Kp84B and Kp84S, respectively. a and d, represent peptidoglycan lytic exotransglycosylase gene. b and e, represent non-contractile tail tubular protein Gp12. c and f, represent endolysin gene overexpression. g and i, inhibitor of host toxin/antitoxin system gene. h and j, represent suppressor of silencing gene. Growth control, represent Kp84 bacteria no phage.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7611342/v1/cc8efd662025bc533e1a849a.png"},{"id":92498999,"identity":"034fd6e4-8f2c-4d4d-8d9f-9c95eea9a3c3","added_by":"auto","created_at":"2025-09-30 10:56:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":446422,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of different genes during Kp84B and Kp84S infection (a), and KEGG annotation of the different genes. b, c, d respectively represents early, middle and later stage.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7611342/v1/d487b8ff7204b247317111df.png"},{"id":92499463,"identity":"eabe2c37-71b1-437e-b7b1-983e5b4471e2","added_by":"auto","created_at":"2025-09-30 11:04:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":251934,"visible":true,"origin":"","legend":"\u003cp\u003eSignificantly differential expression genes of ABC transporters in Kp84B and Kp84S infection. a, represent monosaccharide transport proteins. b, represent peptides, nickel, metal ions and siderophore transporters. c, represent phosphoric acid and amino acid transporters. d, represent mineral and organic ion transporters, and oligosaccharides, polyols and lipid transporters.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7611342/v1/1f3a791b14a3216b149b891d.png"},{"id":92498076,"identity":"fee14bae-52fc-4042-a220-ec5c5c55f6d6","added_by":"auto","created_at":"2025-09-30 10:48:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":146952,"visible":true,"origin":"","legend":"\u003cp\u003eSignificantly differential expression genes of propionate and vitamin B12 synthesis pathway transporters in Kp84B and Kp84S infection. a, represent propionate synthesis pathway. b, represent vitamin B12 synthesis pathway.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7611342/v1/41712bf9decac42b138a865a.png"},{"id":92498097,"identity":"56250b87-3c3c-45be-8940-244ee58f767c","added_by":"auto","created_at":"2025-09-30 10:48:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":258595,"visible":true,"origin":"","legend":"\u003cp\u003eSignificantly differential expression sRNA and target genes in Kp84B and Kp84S infection. a, represent predicted sRNA number. b, represent differential expression sRNA. c and d, respectively represent differential expression target genes of sRNA0122 and 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10:48:12","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":39028,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7611342/v1/e427f460684fbd846eac8dae.xlsx"},{"id":92498079,"identity":"b7c41bbf-d4c3-418a-b274-e36bc5768e85","added_by":"auto","created_at":"2025-09-30 10:48:12","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":29639,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7611342/v1/64dc7a73606c3f3783286831.xlsx"},{"id":92498083,"identity":"0ac7ae5e-831e-4ed6-b9a1-7051e207950a","added_by":"auto","created_at":"2025-09-30 10:48:12","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":68801,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7611342/v1/2606a91ceb8fca28e5d3a30b.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characteristic, genomics and transcriptomics comparation of phages Kp84B and Kp84S infecting Klebsiella pneumoniae","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, a widespread Gram-negative opportunistic pathogen, was first isolated and named \u003cem\u003eFriedlander bacterium\u003c/em\u003e in the late 19th century [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eK. pneumoniae\u003c/em\u003e can cause a wide variety of hospital-acquired infections, including urinary and respiratory tract infections [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], liver abscesses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], septicemia [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The emergence of hypervirulent carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e (\u003cem\u003ehv-CRKP\u003c/em\u003e) has made the development of new and alternative therapies urgently needed [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Unlike other \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, \u003cem\u003eK. pneumoniae\u003c/em\u003e possesses a thick capsular polysaccharide that plays a key role in host colonization and proliferation. Additionally, other virulence factors\u0026mdash;such as lipopolysaccharide (LPS), siderophores, fimbriae, the type VI secretion system (T6SS) and outer membrane proteins-also contribute to its pathogenicity[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, \u003cem\u003eK pneumoniae\u003c/em\u003e is major contributor to antimicrobial resistance (AMR) epidemics [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Based on drug-resistant phenotypes, \u003cem\u003eK. pneumoniae\u003c/em\u003e can be classified into classical (\u003cem\u003ecKp\u003c/em\u003e) and hypervirulent (\u003cem\u003ehvKp\u003c/em\u003e) strains [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In recent years, the infection rate of \u003cem\u003ehvKp\u003c/em\u003e has risen annually, attracting widespread attention. The primary mechanisms of drug resistance in \u003cem\u003eK. pneumoniae\u003c/em\u003e include horizontal gene transfer, biofilm formation, β-lactamase production, reduced membrane permeability, and enzyme modification [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Due to the excessive use of antibiotics, the drug resistance of \u003cem\u003eK. pneumoniae\u003c/em\u003e has become a major concern [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Novel therapeutic approaches for drug-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e remain to be developed.\u003c/p\u003e\u003cp\u003eBacteriophages (phages) are viruses that can specifically infect and may kill bacteria. They were independently discovered in 1915 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Based on their life cycles, phages are classified into virulent/lytic phages (which directly lyse host bacteria) and temperate/lysogenic phages (which integrate into the bacterial genome). Phage therapy utilizes the bactericidal ability of phages to treat pathogenic infections. In the 1920s and 1930s, phage therapy gained attention and was widely used to combat bacterial infections [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, some potential challenges for phage therapy also emerge. Such as, treatment failure because of limited host range [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], phage pollution, sustainable efficacy, and phage resistance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Besides, some differences between the results of \u003cem\u003ein vitro\u003c/em\u003e experiments and \u003cem\u003ein vivo\u003c/em\u003e experiments are existed [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Following the discovery of chemical antibiotics, phage therapy was largely abandoned. However, with the rise of antibiotic resistance, it has reemerged as a promising complementary strategy [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Phage therapy now represents a potential alternative for treating drug-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For example, phage cocktail therapy (using mixed \u003cem\u003eK. pneumoniae\u003c/em\u003e phages) significantly reduces bacterial load in colonized mice and alleviates liver injury [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Combining bacteriophages with antibiotics can achieve synergistic therapeutic effects [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDuring lytic phage infection of a host, phages can hijack host metabolic processes to enhance their own replication. Conversely, the bacterial strain can also trigger various anti-phage defense mechanisms, leading to complex phage-host interactions. For instance, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e phages reprogram host amino acid metabolism to optimize viral production [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, at the phage adsorption stage, bacteria can inhibit phage attachment by modifying cell surface receptors, such as capsular polysaccharides (CPS), lipopolysaccharides (LPS), fimbriae, flagella, and outer membrane proteins [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For example, soluble alginases can degrade bacterial capsules. Restriction endonucleases can protect bacterial DNA by cleaving invading phage DNA [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, host cells can alter protein expression to resist phage infection, affecting genes involved in CPS and LPS synthesis, carbohydrate metabolism, elongation factors, and other critical pathways [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Once the phage injects its nucleic acids into the cell, the host employs targeted defense mechanisms to block phage replication, including CRISPR-Cas system [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], restriction modification system [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], toxin\u0026ndash;antitoxin system (TA) and cyclic-oligonucleotide based antiphage signaling system (CBASS) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWith the advancement of sequencing technology, genomic and transcriptomic analysis has become increasingly widespread [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, genetic diversity in both phages and their hosts is promoted through coevolution [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Such diversity can lead to functional differences among phages. These phenomena have been explored in several studies using comparative (meta)genomics and transcriptomics [\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, the infection mechanisms of phages with a common origin remain unresolved. Furthermore, there are very few studies on host transcriptional responses to infections by two phages with highly similar genomes but different phenotypes. In this study, we compare two phages, Kp84B and Kp84S, which exhibit differences in lysis ability, to identify potential factors underlying these variations through genomic and transcriptomic analysis. These results will help elucidate key factors in phage-host interactions, offering promising insights for future research.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.1 Bacterial strains and phages culture conditions\u003c/h2\u003e\u003cp\u003eAll strains were cultured in Luria\u0026ndash;Bertani (LB) broth at 37\u0026deg;C. \u003cem\u003eK. pneumoniae\u003c/em\u003e 84 was used a host for phage. One hundred microliter phage solution was added to 10 mL \u003cem\u003eK. pneumoniae\u003c/em\u003e 84 culture that grew to the exponential phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;0.6). The mixture was incubated at 37\u0026deg;C for 4\u0026ndash;6 h with shaking at 200 rpm. Then, the culture was centrifuged at 8000 rpm for 10 min, filtered through a 0.22-\u0026micro;m filter. The phage titer was determined by the double-layer agar method. An aliquot of 100 \u0026micro;L phage filtrate and 100 \u0026micro;L of \u003cem\u003eK. pneumoniae\u003c/em\u003e 84 were mixed with 0.7% LB ager medium, incubating 4\u0026ndash;6 h or overnight at 37\u0026deg;C. Phage purification was performed by isolating single plaques and repeating the process three times. The collected phage preparation was subsequently stored at 4\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.2 Biological characteristics\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e1.2.1 One-step growth curve\u003c/h2\u003e\u003cp\u003eThe test method of one-step growth curve is described as follow. The cultures of Kp84 were sub-cultured in 50 mL of LB medium, culturing to the mid-logarithmic phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;0.5\u0026ndash;0.6). Phage Kp84B and Kp84S were added at an MOI of 10 or 0.001, respectively. Next, the mixtures were incubated at 37\u0026deg;C, with shaking for 5\u0026ndash;7 min. Subsequently, the bacterial pellets were collected through centrifuge for 5 min at 4\u0026deg;C. Finally, the collection was washed twice with sterile water, resuspending to 50 mL LB with shaking at 37\u0026deg;C. The suspension was cultured for 200 rpm at 37\u0026deg;C. Samples of mixture were taken at 0 to 60 min every 10 min. The collected sample centrifuged for 5 min at 4\u0026deg;C and determined phage titers. The one-step growth curve was drawn according phage titers as the function of time.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e1.2.2 Lysis curve (infection curve)\u003c/h2\u003e\u003cp\u003eThe phage Kp84B and Kp84S respectively were added to the mid-logarithmic phase Kp84 according to each MOI (0.0001, 0.001, 0.01, 0.1, 1,10, 100). Then, the mixtures were subpacked into 96-well plates, 100 \u0026micro;L in each well. Then, these were sealed using 50 \u0026micro;L Liquid Paraffin. Finally, the optical density was measured at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) every 30 min using BioTek Epoch 2 (BioTek, Vermont, USA). The lysis curve was drawn according to OD\u003csub\u003e600\u003c/sub\u003e at different time.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e1.2.3 Phage host range analysis\u003c/h2\u003e\u003cp\u003eThe host range of phages Kp84B and Kp84S was determined against 220 different \u003cem\u003eK. pneumoniae\u003c/em\u003e strains using spot testing. An aliquot of 5 \u0026micro;L purified phage suspension was spotted onto the surface of double-layer agar plates inoculated with the tested strains. The double-layer agar plates were incubated for 4\u0026ndash;6 h at 37\u0026deg;C. Host bacteria were identified based on plaque formation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e1.2.4 Phage temperatures and pH stability tests\u003c/h2\u003e\u003cp\u003eThe temperature and pH stability of two phages were experimented as follow described. About temperature stability, 1 mL of cell-free phages (10\u003csup\u003e8\u003c/sup\u003e PFU/mL) were incubated at various temperatures (4\u0026deg;C, 25\u0026deg;C, 37\u0026deg;C, 55\u0026deg;C, 65\u0026deg;C, 75\u0026deg;C, 85\u0026deg;C, 95\u0026deg;C, and 100\u0026deg;C) for 60 min. For pH stability, 100 \u0026micro;L phages were mixed with 900 \u0026micro;L of LB adjusted to different pH values (2, 4, 6, 7, 8, 10, 12, and 14). Immediately, the mixture was incubated at 37\u0026deg;C for 60 min. Finally, titers of these sample were determined using the double-layer agar method.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e1.3 Phage genomic, sequencing, and bioinformatic analysis\u003c/h2\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e1.3.1 Phage genomic DNA extraction\u003c/h2\u003e\u003cp\u003ePhage genomic DNA was extracted using the SDS method as described below. Firstly, 10 \u0026micro;L of phage lysates were centrifuged (12,000\u0026times;g, 4\u0026deg;C, 15 min) and filtered (0.22-\u0026micro;m) to remove cellular debris. The purified lysates were mixed with 10 \u0026micro;L DNase I and 10 \u0026micro;L RNase I and incubated for 30 min at 37\u0026deg;C. Then, 20% buffer 1 (0.25% SDS, 20 mM Tris-Cl, 20mM EDTA) was added and the mixture was incubated at 80\u0026deg;C for 15 min. NaAc was added to a final concentration of 0.625 mM, the solution was mixed and incubated on ice for 1 hour. The suspension was centrifuged (12000\u0026times;g, 4\u0026deg;C, 15 min) and collected pellets. Then, the pellets were resuspended and rinsed twice with pre-cooled absolute ethanol. After allowing the ethanol to evaporate, the genomic DNA was dissolved in an appropriate volume of nuclease-free water. Finally, DNA concentration was measured using a Nano Drop One (Thermo, Wyman Street, Waltham, MA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e1.3.2 Phage whole-genome sequencing and bioinformatic analysis\u003c/h2\u003e\u003cp\u003eThe fragmented genome was produced by Covaris M220 and detected concentration using Invitrogen Qubit 4.0. Then, the whole-genome was sequenced by the Illumina NovaSeq 6000 platform, after library was constructed using KAPA Hyper Prep Kit. Genome was assemble using MEGAHIT [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] or MetaSPADes [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Functional annotations of putative ORFs were performed using the NCBI online tool BLASTp in conjunction with conserved domains and Rapid Annotation using Subsystem Technology (RAST, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rast.nmpdr.org/\u003c/span\u003e\u003cspan address=\"https://rast.nmpdr.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). tRNAs were predicted using the tRNAscan-SE program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://lowelab.ucsc.edu/tRNA\u003c/span\u003e\u003cspan address=\"http://lowelab.ucsc.edu/tRNA\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e scan-SE/). The antibiotic resistance and virulence factor genes were identified using the Antibiotic Resistance Genes Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://card.mcmaster.ca/analyze/rgi\u003c/span\u003e\u003cspan address=\"http://card.mcmaster.ca/analyze/rgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the Virulence Factor Database (VFDB, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.mgc.ac.cn/VFs/\u003c/span\u003e\u003cspan address=\"http://www.mgc.ac.cn/VFs/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The genome map was generated using the software SnapGene. Genome comparisons were performed using tBLASTx and visualized with Easyfig (version 2.2.5) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] to describe the relationships among phage Kp84B, Kp84S, and their closest relatives. The related phylogenetic tree was constructed and displayed using the MEGA7 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] program through the maximum likelihood method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e1.3.3 Plasmid construction and transformation\u003c/h2\u003e\u003cp\u003epEXKp-B45, pEXKp-B50 and pEXKp-LysinB were constructed using the following procedures. The plasmid backbone containing pBR322_origin, lac promoter, and the apramycin resistance marker were amplified from the pUC19 plasmid. The B45 gene encoding peptidoglycan lytic exotransglycosylase was PCR amplified from the genomic DNA of the Kp84B. The B50 and LysinB gene encoding non-contractile tail tubular protein Gp12 and endolysin were amplified from the genomic DNA of the Kp84B. The fragment along with the linearized plasmid backbone were assembled together using In-Fusion cloning, resulting in the final plasmid pEXKp-B45, pEXKp-B50 and pEXKp-LysinB.\u003c/p\u003e\u003cp\u003epEX-Gp4.5B, pEX-Gp4.5S, pEX-Gp5.5B, pEX-Gp5.5S and pEX-LysinS were constructed using the following procedures. The plasmid backbone containing Rep101 origin, promoter of the L-arabinose operon of \u003cem\u003eE. coli\u003c/em\u003e, and the apramycin resistance marker were amplified from the pCasKp-Apr plasmid. The Gp4.5B and Gp5.5B gene encoding inhibitor of host toxin/antitoxin system and suppressor of silencing were PCR amplified from the genomic DNA of the Kp84B. Gp4.5S, Gp5.5S and LysinS encoding inhibitor of host toxin/antitoxin system, suppressor of silencing and endolysin were PCR amplified from the genomic DNA of the Kp84B. The fragment along with the linearized plasmid backbone were assembled together using In-Fusion cloning, resulting in the final plasmid pEX-Gp4.5B, pEX-Gp4.5S, pEX-Gp5.5B, pEX-Gp5.5S and pEX-LysinS.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e1.4 Transcriptome sequencing analysis\u003c/h2\u003e\u003cp\u003eRNA sequencing (RNA-Seq) was performed on an Illumina NovaSeqXPlus platform by Majorbio (Shanghai, China). According to one-step growth curves, samples were collected post- adsorption at 0, 20 and 30 min, representing the early, middle, and late phases of the Kp84B infection cycle, respectively. Samples for Kp84S were collected at 0, 20 and 40 min.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e1.4.1 RNA Extraction and Quantification\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using a Trizol kit (Life Technologies). RNA concentration and purity were evaluated using the Nanodrop 2000. RNA integrity and quality were measured using 1% Agarose gel electrophoresis. Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was employed for detecting RIN (RNA Integrity Number).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e1.4.2 cDNA library construction\u003c/h2\u003e\u003cp\u003ecDNA library is prepared using TruSeq\u0026trade; Stranded Total RNA Library Prep Kit (Illumina). The process outline is as follow. The enriched mRNA without rRNA was randomly interrupted by adding Fragmentation Buffer. The first cDNA strand was synthesized with random primers using mRNA as a template. Then, the second cDNA strand was synthesized by adding dUTP to replace dTTP, of which its bases contain A/U/C/G. The purified double-stranded cDNA was then end-repaired and A-tailed. Finally, the cDNA library was enriched by PCR. Sequencing was performed with the NovaSeqXPlus platform.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e1.4.3 Quantification of Gene Expression Level\u003c/h2\u003e\u003cp\u003eGene expression transcript expression levels are analyzed using RSEM. TPM was calculated based on the gene length.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e1.4.4 Differential Expression Gene Analysis\u003c/h2\u003e\u003cp\u003eThe differential gene expression analysis using DEGSeq.\u0026nbsp;For comparison, a P-value of 0.001 and log 2 (fold change) of 1 were set as the thresholds for significantly differential expression [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e1.4.5 KEGG Enrichment Analysis\u003c/h2\u003e\u003cp\u003ePathway enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genome) were performed using R script, referring to KEGG Pathway database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.kegg.jp/kegg/pathway.html\u003c/span\u003e\u003cspan address=\"http://www.kegg.jp/kegg/pathway.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Fisher's Exact Test is used as the calculation method. The KEGG pathway function is considered to be significantly enriched, when the P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Biological characteristics examination of phages\u003c/h2\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1 Host range of Kp84B and Kp84S\u003c/h2\u003e\u003cp\u003eThe number lysised hosts of both phages Kp84B and Kp84S is 47 out of 204 \u003cem\u003eK. pneumoniae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.a and Supplementary Information 1). The bacteriophage plaque with halo is successfully formed on LB double-layer plate when KP-YQ88, KP-YQ74, KP-YQ10, KP8-2 and KP7-25 was respectively mixed with Kp84B and Kp84S. However, the KP84, Kp7-20, Kp8-7, KP-YQ43 and KP-YQ79 had same phenomenon with Kp84B. All the other sensitive \u003cem\u003eK. pneumoniae\u003c/em\u003e only formed bacteriophage plaque without halo when they were lysised by Kp84B and Kp84S. Specially, morphology of Kp84B and Kp84S phage plaque with Kp84 is showed that single phage Kp84B produce a large plaque with an opaque halo on a bacterial lawn (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.b). In contrast, single phage Kp84S only produce the small without halo plaque (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.c).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e2.1.2 One-step growth curve\u003c/h2\u003e\u003cp\u003eThe one-step growth curves showed as Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.d. A latent period of Kp84S located between 0 and 10 min, a burst period was 10\u0026ndash;30 min. After 30 min, Kp84S stayed in plateau period. About Kp84B, its latent period and burst period respectively was 0\u0026ndash;10 min and 10\u0026ndash;40 min. After 40 min, lytic cycle of Kp84B entered in plateau period.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e2.1.3 Lysis curve (infection curve)\u003c/h2\u003e\u003cp\u003eAfter Kp84 were respectively mixed with Kp84B and Kp84S according to different MOI, the absorbance at 600 nm change with time of culture was measured to contrast those lysised regularly indirectly. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.e-f, the optical density of Kp84B and Kp84 coculture reaches the peak at 1 hour and falls to minimum value at 2 h. The regularity is much the same on coculture Kp84S and Kp84. Nevertheless, the largest difference between of both phages is that optical density of Kp84B and Kp84 coculture is not obviously increase during 24 h after lysise but that of Kp84S is progressively increase from 6 h to 24 h. This suggests that it is more likely to produce anti-phage mutants when Kp84S infected host Kp84.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e2.1.4 Phage temperature, pH and UV stability\u003c/h2\u003e\u003cp\u003eBoth Kp84B and Kp84S can be maintain great activity below 45\u0026deg;C and reduce activity at 55\u0026deg;C, while be inactivated after incubation at temperatures above 65\u0026deg;C for 1 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.g).\u003c/p\u003e\u003cp\u003eThe two phages were inactivated above pH12 and below pH2. The activity of Kp84B and Kp84S was maintained out of 50% incubating 1 h at pH6 and pH10. The activity of Kp84B and Kp84S decreased two orders of magnitude at pH4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.h).\u003c/p\u003e\u003cp\u003eThe two phages are also equally sensitive to ultraviolet (UV) light. After being exposed to a 20 W ultraviolet lamp for 60 min, their titers decreased by nearly five orders of magnitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.i).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Genome analysis of phage Kp84B and Kp84S\u003c/h2\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Genome characteristic and phylogenetic analysis\u003c/h2\u003e\u003cp\u003eThe genome length of phage Kp84B is 40,452bp, and GC content is 52%. Correspondingly, phage Kp84S is 40,466bp, and 52% GC. The function of 51 ORFs and 52 ORFs from phage Kp84B and Kp84S genome is predicted, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The annotated ORFs are classified into five functional modules: DNA replication, regulation and nucleotide metabolism, packaging, unknown functions, and lysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.a). No tRNA genes are identified in the genomes. No antibiotic resistance genes and virulence factors are predicted in the Kp84B and Kp84S genomes (Supplementary Information 2\u0026ndash;3). Phylogenetic analysis of complete genome sequence suggests that the both of phages Kp84B and Kp84S are most closely related to the \u003cem\u003eAutographiviridae\u003c/em\u003e family, \u003cem\u003eStudiervirinae\u003c/em\u003e subfamily, \u003cem\u003ePrzondovirus\u003c/em\u003e genus, specifically \u003cem\u003eKlebsiella\u003c/em\u003e phage SH-Kp152234 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.b).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenome summarize of Kp84B and Kp84S\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenome Size (bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eType\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGC content %\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eA%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eT%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eC%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eG%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eORF\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKp84B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40452\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecircle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e52.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e25.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e22.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e24.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e27.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKp84S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e40466\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecircle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e52.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e25.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e22.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e24.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e27.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Comparative genomes and Functional differential genes\u003c/h2\u003e\u003cp\u003eNucleotide sequence identity of Kpg8B and Kp84S genome is 99.4% at 99% coverage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.c). The comparison of two phage genome was visualled using Easyfig (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.b). Six hundred and ninety-nine bases of twenty-one proteins are different between Kp84B and Kp84S genome coding sequence (Supplementary Information 4). Particularly, inhibitor of host toxin/antitoxin system, non-contractile tail tubular protein Gp12, peptidoglycan lytic exotransglycosylase, phage protein Gp5.5 suppressor of silencing and endolysin are the biggest differences. Moreover, we note that a G\u0026rarr;C transversion in the inhibitor of host toxin/antitoxin system gene (Phage protein Gp4.5), the base is located at 17,469 of the Kp84B genome. This change led to premature termination of Kp84B phage protein Gp4.5 translation than Kp84S. Phage non-contractile tail tubular protein Gp12 and peptidoglycan lytic exotransglycosylase gene of Kp84B is also premature termination comparing Kp84S.\u003c/p\u003e\u003cp\u003eTherewith, those different gene were constructed into a plasmid containing L-Arabinose induced promoter or IPTG induced promoter. Those plasmids were transformed into Kp84 and build gene overexpression strains, named Kp84/pEX-Gp4.5B, Kp84/pEX-Gp4.5S, Kp84/pEX-Gp5.5B, Kp84/pEX-Gp5.5S, Kp84/pEX-B50, Kp84/pEX-B45, Kp84/pEX-LysinB, Kp84/pEX-LysinS.\u003c/p\u003e\u003cp\u003eNotably, the strains lysis of Kp84/pEX-Gp4.5B, Kp84/pEX-Gp4.5S, Kp84/pEX-Gp5.5B, Kp84/pEX-Gp5.5S were weaken when they co-culture with Kp84B and Kp84S and were induced expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.g-j). The strains Kp84/pEX-B50, Kp84/pEX-B45, Kp84/pEX-LysinB, Kp84/pEX-LysinS were not changed comparing control Kp84/pUC19 when were lysised by Kp84B and Kp84S (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.a-h). Therefore, those genes were uninfluential to activity difference between Kp84B and Kp84S. However, Gp4.5B, Gp4.5S, Gp5.5B and Gp5.5S can cause abortive infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.g-j).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Transcriptome analysis of Kp84B and Kp84S infected Kp84\u003c/h2\u003e\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Overview of host gene expression changes and KEGG enrichment analysis\u003c/h2\u003e\u003cp\u003eAccording to one step growth curve, early (at 0 min), middle (at 20 min) and later (at 30 min for Kp84B, at 40 min for Kp84S) stage bacterial samples were collected after Kp84B and Kp84S infection. Those samples respectively correspond control group Kp84B-0, Kp84B-20 and experimental group Kp84S-0, Kp84S-20. As a whole, 5,780 genes of the Kp84 genome were differentially expressed. At early stage, 529 genes were upregulated expression and 402 genes were downregulated expression. At middle stage, 236 genes were upregulated expression and 247 genes were downregulated expression. At last stage, 236 genes were upregulated expression and 247 genes were downregulated expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.a).\u003c/p\u003e\u003cp\u003eEarly stage, the largest number of significantly differential expression genes by KEGG enrichment analysis were reflected in ABC transporters, next propanoate metabolism and butanoate metabolism etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.b). Middle stage, those significantly differential expression genes are reflected in pyruvate metabolism, porphyrin and chlorophyll metabolism, butanoate metabolism, and TCA cycle etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.c). Last stage, those significantly differently expressed genes are reflected in ABC transporters etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.d).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 ABC transporter response early infection\u003c/h2\u003e\u003cp\u003eSixty-four significantly differently expressed genes were enriched onto pathway at early infection. However, they were almost no change at middle infection. These genes involved six classes of ABC transporters, and included 29 compound and molecule transporters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.a-d).\u003c/p\u003e\u003cp\u003eIn detail, 10 monosaccharide transporters were upregulated significantly including phaseomannite (\u003cem\u003eIbpA\u003c/em\u003e, \u003cem\u003eIatP\u003c/em\u003e, \u003cem\u003eIatA\u003c/em\u003e), methyl-galactoside (\u003cem\u003emglA\u003c/em\u003e, \u003cem\u003emglB\u003c/em\u003e, \u003cem\u003emglC\u003c/em\u003e), and erythritol (\u003cem\u003eeryE\u003c/em\u003e, \u003cem\u003eeryG\u003c/em\u003e) transporters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). \u003cem\u003eNikABCDE\u003c/em\u003e operon belongs to nickel transporters, periplasmic binding protein \u003cem\u003eNikA\u003c/em\u003e was upregulated, while \u003cem\u003enikC\u003c/em\u003e, \u003cem\u003enikD\u003c/em\u003e and \u003cem\u003enikE\u003c/em\u003e were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.b). The ABC transporter \u003cem\u003eDppABCDF\u003c/em\u003e matters dipeptide acquisition, and dipeptide transporter protein \u003cem\u003edppA\u003c/em\u003e was also upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.b). Cobalt transporter ATP binding domain \u003cem\u003ecbiO\u003c/em\u003e was upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.b). Three ferric uptake genes \u003cem\u003eafuA\u003c/em\u003e, \u003cem\u003eafuB\u003c/em\u003e and \u003cem\u003eafuC\u003c/em\u003e were high expression in Kp84S infection. Glutamic acid and aspartic acid transporter genes \u003cem\u003egltL\u003c/em\u003e, \u003cem\u003egltK\u003c/em\u003e, \u003cem\u003egltJ\u003c/em\u003e and \u003cem\u003egltI\u003c/em\u003e were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.c). Finally, maltos transporters \u003cem\u003emalK\u003c/em\u003e and \u003cem\u003emalF\u003c/em\u003e, galactose and maltooligosaccharides transporters \u003cem\u003eganO\u003c/em\u003e and \u003cem\u003emsmX\u003c/em\u003e, sorbitol transporter \u003cem\u003esmoK\u003c/em\u003e were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.c).\u003c/p\u003e\u003cp\u003eFour genes \u003cem\u003erbsABCD\u003c/em\u003e of \u003cem\u003erbs\u003c/em\u003e operon were downregulated, involving ribose, Autoinducer2 and D- xylose uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.a). Ferric hydroxamate uptake (\u003cem\u003efhu\u003c/em\u003e) genes \u003cem\u003efhuC\u003c/em\u003e and \u003cem\u003efhuD\u003c/em\u003e were downregulated. In addition, iron and manganese acquisition genes \u003cem\u003esitBCD\u003c/em\u003e, and ferrisiderophores uptake genes \u003cem\u003efepBCG\u003c/em\u003e were downregulated in Kp84S early infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.b). Phosphate transport system \u003cem\u003epstA\u003c/em\u003e, \u003cem\u003epstB\u003c/em\u003e and \u003cem\u003epstC\u003c/em\u003e, was also downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.c). Arginine, cystine, and branched-chain amino acid revealing transporters were downregulated, including \u003cem\u003eartQ\u003c/em\u003e, \u003cem\u003eartM\u003c/em\u003e, \u003cem\u003efliy\u003c/em\u003e, \u003cem\u003elivK\u003c/em\u003e, \u003cem\u003elivH\u003c/em\u003e, \u003cem\u003elivG\u003c/em\u003e, \u003cem\u003elivM\u003c/em\u003e and \u003cem\u003elivF\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.c). Spermidine / putrescin transporter genes \u003cem\u003epotB\u003c/em\u003e, \u003cem\u003epotA\u003c/em\u003e and \u003cem\u003epotC\u003c/em\u003e were low expression in Kp84S infection than Kp84B (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.d).\u003c/p\u003e\u003cp\u003eTherefore, we infer that Kp84B life process may consume more spermidine/putrescin, ribose/D-xylose, phosphate, iron, arginine, cystine, and branched-chain amino acid. While Kp84S may consume more maltooligosaccharide, inositol, glutamic acid and aspartic acid.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Propionate synthesis, Porphyrin and chlorophyll metabolism response phage infection\u003c/h2\u003e\u003cp\u003ePropionate synthesis, porphyrin and chlorophyll metabolism pathway were significantly enriched different genes through KEGG analysis. Furthermore, propionate synthesis mainly reflected in early infection, porphyrin and chlorophyll metabolism mainly reflected in middle infection. As we known, butanoate metabolism can promote vitamin B12 biosynthesis. Even more, butanoate has ability to induce \u003cem\u003eE. coil\u003c/em\u003e prophage to lysis, and produce more progeny phages [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Heme, chlorophyll, and vitamin B12 are essential for various metabolic pathways [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn early infection, 19 genes were significantly differential expression in propionate synthesis pathway. Among, \u003cem\u003epct\u003c/em\u003e, \u003cem\u003efadJ\u003c/em\u003e, \u003cem\u003efadB\u003c/em\u003e, \u003cem\u003epuuE\u003c/em\u003e, \u003cem\u003emmsA\u003c/em\u003e, \u003cem\u003esuccC\u003c/em\u003e, \u003cem\u003esucD\u003c/em\u003e and \u003cem\u003eatoB\u003c/em\u003e were upregulated expression in Kp84S infection. These genes involved acrylate and methylmanoyl-CoA metabolism to propionyl-CoA. Conversely, downregulated expression genes mostly belonged to pathway of propanediol to propionyl-CoA, including \u003cem\u003egldA\u003c/em\u003e, \u003cem\u003epduC\u003c/em\u003e, \u003cem\u003epduD\u003c/em\u003e, \u003cem\u003epduE\u003c/em\u003e, \u003cem\u003edhaT\u003c/em\u003e and \u003cem\u003epduQ\u003c/em\u003e. In addition, \u003cem\u003epct\u003c/em\u003e was also in charge of converting from propionyl-CoA to propionate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.a).\u003c/p\u003e\u003cp\u003eSixteen different expressed genes involved vitamin B12 (cobalamin) biosynthesis, belonging to porphyrin and chlorophyll metabolism pathway. These genes were upregulated expression in middle infection, and fold change (log2FC) maintain it between 1.1 and 3.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.b).\u003c/p\u003e\u003cp\u003eIn summary, host cell Kp84 may prefer to use propanediol to produce propionate when it was infected by Kp84B. While host cells may prefer to use acrylate and methylmanoyl-CoA to produce propionate in Kp84S infection. Moreover, Kp84S could promotes vitamin B12 biosynthesis in middle infection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4 sRNA response phage infection\u003c/h2\u003e\u003cp\u003eThe majority of sRNA bind target genes to regulate transcript stability, active or inhibit protein expression, and can regulate virulence factor, take part in defense responses etc. This report that prophage encode a sRNA can inhibit maltodextrin transport gene \u003cem\u003elamb\u003c/em\u003e to reduce lysis ability [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn our study, 211 sRNAs are significantly differential expression in early infection, while seven sRNA were differently expressed in middle stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.a-b). Among these sRNA, sRNA0122 can target eighteen genes, of which eight targeted genes were significantly differential expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.c). sRNA0576 can target 4 differently expression genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.d).\u003c/p\u003e\u003cp\u003eAccording to targeted genes fold change, we concluded that sRNA0576 could influent Kp84B and Kp84S life process though negatively regulating gene4612 encoding hypothetical protein. sRNA0122 could positively regulate gene4286, gene4611 and gene3358, and negatively regulate gene2184, gene2972 and gene163 in early infection. Gene4286, gene4611 and gene3358 coded hypothetical protein, hypothetical protein and N-acetylmuramoyl-L-alanine amidase respectively. N-acetylmuramoyl-L-alanine amidase can participate in peptidoglycan catabolic process. Gene2184, gene2972 and gene163 coded hypothetical protein, phosphate acyltransferase and serine endoprotease, respectively.\u003c/p\u003e\u003cp\u003eSpecially, genes with a fold change exceeding 3 were summarized (Supplementary Information 5). Because the larger the difference, it is the more likely key factor. In early infection, 46x genes were upregulated expression, including Insertion Sequence genes, genes related to central carbon metabolism, and genes related to ABC transporters. Examples include IS5-like element ISKpn26 family transposase (gene1182), IS3 family transposase (gene1655), succinate dehydrogenase membrane anchor subunit (gene3739), succinyl glutamate-semialdehyde dehydrogenase (gene3111), 2-oxoglutarate dehydrogenase E1 component (gene3736), acetyl-CoA C-acyltransferase FadA (gene4941), LysR family transcriptional regulator (gene2316), ABC transporter ATP-binding protein (gene2184), iron ABC transporter permease (gene2183), and sugar ABC transporter substrate-binding protein (gene2021). Twenty-six genes was downregulated expression in the early stage, including DhaG protein (gene 633), the propanediol/glycerol family dehydratase medium subunit (gene635), the glycerol dehydratase reactivate beta/small subunit family protein (gene631), the glycerol dehydratase small subunit DhaB3 (gene636), 1,3-propanediol dehydrogenase (gene632), the diol dehydratase reactivate subunit alpha (gene637), the glycerol dehydratase large subunit (gene634), the phosphate ABC transporter substrate-binding protein PstS (gene5140), the ribose ABC transport system (gene5120), etc.\u003c/p\u003e\u003cp\u003eIn middle infection, eleven upregulated genes were annotated, including the IS5-like element ISKpn26 family transposase (gene12), the \u003cem\u003eqaJ\u003c/em\u003e viral recombinase family protein (gene3036), the energy-coupling factor ABC transporter substrate-binding protein (gene999), the Rha family transcriptional regulator (gene1185), and NADH oxidoreductase (gene3525). Thirteen genes are downregulated, including oxidoreductase (gene3845), succinylglutamate desuccinylase (gene3109), malate: quinone oxidoreductase (gene2751), cytochrome c-type biogenesis protein CcmH (gene2196), and oxaloacetate decarboxylase subunit beta (gene4403), etc.\u003c/p\u003e\u003cp\u003eIn last infection, 6 genes exit upregulated expression, including the IS5-like element ISKpn26 family transposase (gene828), the IS5-like element ISKpn26 family transposase (gene120), the IS6-like element IS26 family transposase (gene3005), and the ssDNA-binding domain-containing protein (gene1796). The number of downregulated gene were more than 1000, for example, the O-antigen and lipid-linked capsular repeat unit polymerase (gene1721), the IS3-like element ISKpn1 family transposase (gene1762), the putative oligogalacturonide transporter (gene1868), dihydropteroate synthase (gene534), ABC transporter permease (gene1661), the methionine ABC transporter permease Met I (gene4214), etc.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, the biological characteristics differences were mainly manifested in the following, when phage Kp84B and Kp84S infected host Kp84. Phage Kp84B has stronger lysis ability than Kp84S to host Kp84. However, both phages genome is 99.46% identity at 99% coverage. Through comparing both genome different genes, and RNA-seq of infected host cell, potential major factors were predicted.\u003c/p\u003e\u003cp\u003eIn detail, 21 genes had 699 single base difference. Specially, we noted 5 most probable different genes, including inhibitor of host toxin/antitoxin system, non-contractile tail tubular protein Gp12, peptidoglycan lytic exotransglycosylase, phage protein Gp5.5 suppressor of silencing and endolysin. Further, these proteins were overexpressed in host cell Kp84 and compare lysis phenotype for Kp84B and Kp84S.\u003c/p\u003e\u003cp\u003eFirstly, the nucleotide sequences of the capsid assembly scaffolding protein in Kp84B and Kp84S showed significant differences between the 160th and 360th base. Their portal proteins had 76 base mismatches. As reported in the study, the number of positively and negatively charged amino acid of major and minor coat protein also decides virion size, besides phage genome length [\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In addition, capsid assembly scaffolding protein and portal protein can influence the shape and size of the phage pro head through regulating major and minor coat protein [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Secondly, nucleotide sequence of tail tubular protein gp12 of Kp84B and Kp84S had 93.53% identity (98% coverage), tail proteins are same. At mentioned article that tail proteins, tail tubular protein and a structural tail protein can encode depolymerases. These protein can recognize the host and bind to capsular polysaccharides (CPS), exopolysaccharides (EPS), or lipopolysaccharides (LPS) of the host bacteria [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. It was important that these depolymerases cleave polysaccharide-repeating units, helping the phage reach the cell wall and inject its DNA to infect the cell. As well as depolymerases can produce opaque halo zones on a bacterial lawn. Thirdly, the peptidoglycan transglycosylase gp16 of Kp84B and Kp84S had 44 nucleotide difference, differed by 10 amino acids. As we all known, the peptidoglycan transglycosylase gp16 motif is essential for bacteriophage T7 growth at temperatures below 20\u0026deg;C. Further, glutamate residue mutation of peptidoglycan transglycosylase gp16 can increase infection efficiency of phage T7 for \u003cem\u003eE. coli\u003c/em\u003e of high cell densities [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough phage protein gp4.5 inhibitor of host toxin/antitoxin system and phage protein Gp5.5 suppressor of silencing can cause host cell abortive infection when they were overexpressed into Kp84. It was a pity that these genes whether were from Kp84B and Kp84S genome were no different for lysis phenotype. Therefore, major gene was unfound using this way. It was permuted that these different from genome are not solely enough to lysis activity.\u003c/p\u003e\u003cp\u003eThrough transcriptome analysis, some genes involved metabolic pathways, insertion sequences and ABC transporters were likely major factors. It can be known from KEGG enrichment that the most impact genes focus on ABC transporters and metabolic pathways. It was understandable that phage indeed can reprogram host cell carbon metabolism and nucleotide synthesis pathway to their reproduce offspring [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. An example, cyanophage possessed the capacity that transfer carbon flux from carbon fixation to pentose phosphate pathway. This change increased the NADPH output that it was necessary energy to phage DNA replication [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In addition, phage also can reprogram host cell amino acid metabolism to promote themselves replication. Key amino acids and the arginine-derived polyamine putrescine levels were increased when phage protein Eht1 and Eht2, were expressed early in the infection cycle. More, putrescine played key actor for phage replication [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Amino acid transporters transcriptional level existed differences between Kp84B and Kp84S infection. Genes concerning spermidine/putrescin, arginine, cystine, and branched-chain amino acid transporter were higher expression in Kp84B early infection than Kp84S. However, glutamic acid and aspartic acid transporters were higher expression in Kp84S early infection. Besides, it was reported that DppBCDF ABC transporter substrate binding protein DppA can inhibit phage Pf5 lysis activity. The number of Pf5 progeny phages was increased when \u003cem\u003edppA\u003c/em\u003e is deleted [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. As it happened, gene \u003cem\u003edppA\u003c/em\u003e of Kp84 was upregulated in Kp84S early infection. Kp84S had weak lysis ability than Kp84B.\u003c/p\u003e\u003cp\u003eOn the side, we noted that high fold changed genes were labeled in central carbon metabolism (CCM), insertion sequence transposase genes, beside ABC transporters in early and middle infection. Some phages can reprogram the metabolic pathways of their bacterial host, with variations among different phages. The \u003cem\u003esiphovirus\u003c/em\u003e PSA-HS2 can suppress energy-consuming metabolisms, such as motility and translation through repressing expression of \u003cem\u003ecycD\u003c/em\u003e, \u003cem\u003ecycN\u003c/em\u003e, \u003cem\u003ecysI\u003c/em\u003e and \u003cem\u003ecycJ\u003c/em\u003e genes. Among these genes, \u003cem\u003ecycD\u003c/em\u003e and \u003cem\u003ecycN\u003c/em\u003e encode sulfate adenylyltransferase, \u003cem\u003ecysI\u003c/em\u003e and \u003cem\u003ecycJ\u003c/em\u003e encode assimilatory sulfite reductase (NADPH)[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. During Kp84B and Kp84S infection, genes related to the Kp84 metabolic pathway change significantly, for example, NADH oxidoreductase (gene 3525) and ATP synthase and so on involved in energy metabolism. Insertion sequences (ISs) are small transposable elements that encode proteins required for their transposition and influence the evolution of the bacterial genome [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The IS family is defined as a group of insertion sequences (ISs) with related transposases, strong conservation of the catalytic site, conserved organization, and similar inverted terminal repeats (IRs) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. IS-mediated gene expression results in clinically significant increases in phage resistance. For example, IS903B and IS903-formed composite transposon can interrupt \u003cem\u003ewcaJ\u003c/em\u003e or \u003cem\u003ewbaZ\u003c/em\u003e genes associated with the capsular polysaccharide biosynthesis, thereby causing \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (CRKP) phage resistance [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. In our study, the IS5-like element ISKpn26 family transposase, IS3 family transposase, and IS6-like element IS26 family transposase were the major upregulated genes. As well as, phage receptor-related genes were one class of the downregulated genes in the last stage. This may facilitate the emergence of Kp84 anti-phage Kp84S strains.\u003c/p\u003e\u003cp\u003eIn conclusion, although phage Kp84B and Kp84S had similar stability of temperature, pH and UV, lysis spectrum, they had different lysis ability. Homologous genes from two phages had same influence when they were expression into host cell. Although these homologous genes were the most likely influencing factors for lysis ability between Kp84B and Kp84S. However, comparative transcriptome results implied that some genes were significant differentially expressed. These genes related to ABC transporter pathways, molecular metabolic pathways, insertion sequences, sRNA and target genes, etc. We considered that they were potential interaction factor to make clear different lysis ability between Kp84B and Kp84S. In the future, the host differential expression genes can be verified.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, phage Kp84B and Kp84S have similar host range, pH and temperature tolerance are same. However, Kp84B has stronger lysis ability than Kp84S to the same host Kp84. Five homologous genes are not factors leading to lysis difference. ABC transporters, propionate and vitamin B12 biosynthesis genes, insertion sequences, sRNA and their target genes are potential host interaction factors influencing their lysis ability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQingqing Sun and Wei Chen, experiments design, data curation, writing original draft, review, and editing; Guangming Zhang and Yanmei Sun, review and editing; Linxin Shen and Shiwei Wang, experiments design, supervised the work progress and edited the manuscript. All authors agreed to be accountable for the content of the work.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\u003cp\u003eThis work was supported in part by the grants from the Science \u0026amp; Technology Fundamental Resources Investigation Program (Grant No. 2022FY101100). This work was also supported by the National Natural Science Foundation of China (Grant No. 32170114 and 31770152).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMerino, S., Camprub\u0026iacute;, S., Albert\u0026iacute;, S., Bened\u0026iacute;, V.J., and Tom\u0026aacute;s, J.M. (1992) Mechanisms of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e resistance to complement-mediated killing. Infection \u0026amp; Immunity \u003cem\u003e60\u003c/em\u003e, 2529-2535. https://doi.org/10.1007/BF01960820\u003c/li\u003e\n\u003cli\u003eBodey, G.P. (1988) Fungal infections in the compromised host. 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(2022) Interruption of capsular polysaccharide biosynthesis gene wbaZ by insertion sequence IS 903B mediates resistance to a lytic phage against ST11 K64 Carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. msphere \u003cem\u003e7\u003c/em\u003e, e00518-00522. https://doi.org/ 10.1128/msphere.00518-22\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Klebsiella pneumoniae, bacteriophage(phage), biological characterization, genome, transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-7611342/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7611342/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacteriophages have emerged as attractive alternatives to antibiotics for treating multidrug-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e infections. Currently, numerous phage-host interaction factors remain to be discovered to better understand and optimize this therapeutic approach. In this study, we characterized two \u003cem\u003eKlebsiella\u003c/em\u003e phages with highly similar genomes, Kp84B (40,452 bp) and Kp84S (40,466 bp), which share 99.46% sequence identity with 99% coverage. The general biological characteristics comparison showed that their host range, pH and temperature tolerance are same. However, their lysis ability was obvious different for the same host Kp84. Further comparative genome results showed that 21 different genes have 699 single nucleotide change. Five most promising protein were overexpressed in host Kp84, and lysis phenotypes of Kp84B and Kp84S were examined. As results, although lysis ability did not show obvious different between Kp84B and Kp84S, it was found that Gp4.5 and Gp5.5 genes can cause host cell abortive infection. RNA-seq analysis further revealed potential host interaction factors influencing their lysis ability, including some ABC transporters, propionate and vitamin B12 biosynthesis genes, insertion sequences, sRNA and their target genes. These provide further study target for phage-host interaction factors for lysis difference.\u003c/p\u003e","manuscriptTitle":"Characteristic, genomics and transcriptomics comparation of phages Kp84B and Kp84S infecting Klebsiella pneumoniae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-30 10:48:07","doi":"10.21203/rs.3.rs-7611342/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"45431a25-115b-4df3-a5a8-49cbe9582519","owner":[],"postedDate":"September 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-12T13:39:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-30 10:48:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7611342","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7611342","identity":"rs-7611342","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}