Disruption of Protein BA_3317 Affects Asymmetric Division as an Exporter of Sporulation Signal Molecules in Bacillus anthracis

Disruption of Protein BA_3317 Affects Asymmetric Division as an Exporter of Sporulation Signal Molecules in Bacillus anthracis | 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 Disruption of Protein BA_3317 Affects Asymmetric Division as an Exporter of Sporulation Signal Molecules in Bacillus anthracis Yufei Lyu, Dongshu Wang, Jiefan Jiao, Meijie Feng, Meng Chen, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8551494/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 Background Bacillus anthracis forms dormant spores that constitute the primary infectious agent of anthrax. BA_3317, a membrane protein harboring a quorum-sensing (QS)-related AgrB domain, is essential for sporulation in B. anthracis . Methods We constructed an in-frame deletion mutant of BA_3317 in B. anthracis vaccine strain A16R. Sporulation efficiency was quantified, and mutant morphology was observed via confocal microscopy. To investigate the role of BA_3317 in spore germination, we performed secretion exchange experiments between A16R and ΔBA_3317 at T 0.5 , analyzed the transcriptional activity of spoIIE , and determined lecithinase activity after activating the plcR-papR QS system. Additionally, we identified the upstream regulators of BA_3317 using in vitro promoter pull-down assays. Results Deletion of BA_3317 severely reduced sporulation efficiency and ΔBA_3317 mutant was partially arrested at the asymmetric cell division stage. Cells secretion exchange experiments and spatial reporter assays revealed that BA_3317 exports a signal molecule required for sporulation, and its loss downregulated key sporulation gene spoIIE . The mutant also lacked lecithinase activity via the ectopically activated the plcR-papR QS system, which was restored by adding the PapR heptapeptide, indicating BA_3317 mediates peptide signal transport. BA_3317 expression is repressed by SpoVG prior to asymmetric division and positively regulated by GerE during late sporulation. Conclusion Our findings identify BA_3317 as a critical regulator of B. anthracis sporulation that functions as an exporter of sporulation signaling molecules. This study advances understanding of species-specific sporulation mechanisms in B. anthracis and provides a potential target for anthrax prevention and control. Bacillus anthracis transporter sporulation asymmetric cell division spoIIE Quorum-sensing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Like other members of the Bacillus genus, Bacillus anthracis initiates sporulation under nutrient-limiting conditions. While the spore serves as the primary infectious agent for anthrax, the specific molecular pathways that regulate its formation in this pathogen are not fully elucidated. The mechanistic framework for B. anthracis sporulation heavily relies on that established for the model bacterium Bacillus subtilis . Endospore formation is a complex developmental process involving asymmetric cell division, forespore engulfment, and spore morphogenesis in B. subtilis [ 1 ]. Asymmetric cell division is a critical early event that generates two cellular compartments with distinct sizes and developmental fates. This process, essential for establishing sporulation polarity, requires SpoIIE [ 2 , 3 ] and is partly repressed by SpoVG [ 4 ]. The resulting cellular asymmetry directly enables the activation of the forespore-specific sigma factor, σ F [ 5 ], which in turn regulates the expression of genes involved in forespore engulfment [ 1 , 6 ]. During this engulfment process, the membrane-associated proteins SpoIID, SpoIIM, and SpoIIP are essential for membrane migration, and their expression is indirectly regulated by σ F via σ E [ 1 ]. As an auxiliary transcriptional regulator, SpoIIID exerts both positive and negative control over a broad range of genes within the σ E regulon during early mother cell development [ 7 ]. The mother cell-specific sigma factor σ K regulates the transcription factor GerE, which controls the expression of numerous spore coat proteins and cell wall hydrolases responsible for mother cell lysis and spore release [ 1 , 7 ]. A key interface between environmental cues and cellular response is quorum sensing (QS). Sporulation is initiated when Spo0A, the master regulator of sporulation, is activated via a “phosphorelay” system governed by autophosphorylating histidine kinases that respond to various environmental stresses. In B. subtilis , the Rap protein dephosphorylates Spo0F ~ P, thereby blocking the transfer of ATP-derived phosphate to Spo0A via Spo0B and Spo0F. Notably, Rap activity is inhibited by a QS peptide exported by secretory proteins and reimported via an oligopeptide permease (Opp) [ 8 ]. The critical role of QS systems in regulating sporulation in Bacillus spp. is well established: the absence of functional App and Opp transporters leads to a marked reduction in sporulation in B. subtilis [ 9 ], and modulates the activity of the Spo0A response regulator [ 10 ]. Despite this, compared with other QS components, little is known about the secretion and maturation of small QS signaling molecules. In the Gram-positive bacterium Staphylococcus aureus, the accessory gene regulator ( agr ) operon ( agrA , agrB , agrC , agrD ) encodes a canonical QS system [ 11 ], where AgrB—a transmembrane protein—is essential for the maturation of QS pheromones [ 12 ]. However, it remains unclear whether AgrB-like proteins are involved in the import/export of quorum-signaling peptides that regulate endospore formation in B. anthracis A16R. In this study, we investigated the role of the putative BA_3317 protein in B. anthracis A16R (pXO1⁺, pXO2⁻). The B. anthracis vaccine strain A16R is derived from strain A16 via UV irradiation[ 13 ]. BA_3317 is annotated to contain two functional domains (Semialdehyde_dh and AgrB) and is predicted to act as an amino acid permease. We constructed an in-frame deletion mutant of BA_3317 and confirmed that disruption of this locus significantly reduced sporulation efficiency. In addition, the BA_3317 protein likely functions as a sporulation signal molecule secretory transporter protein, suggesting that the impaired sporulation in the ΔBA_3317 mutant is associated with disrupted sporulation signal transduction. Materials and methods Determination of sporulation efficiency The material and methods of bacterial strains and plasmids used in this study are listed in Table S5 (Supplementary Materials). The oligonucleotides used in this study are listed in Table S6 (Supplementary Materials). Strains A16R, ΔBA_3317, and HΔBA_3317 were grown in liquid DSM [ 14 ] at 37°C for 120 h with vigorous shaking. The number of viable cells in each culture was defined as the total number of colony-forming units, and was determined by serial dilution plate counts on LB agar medium. The number of spores per culture was defined as the number of heat-resistant cells in each culture, and was determined by plating serial dilutions of heat-treated (70℃ for 30 min) cells onto LB agar plates. Sporulation efficiency was defined as the ratio of the number of spores to the number of viable cells. At least three biological replicates were included for each sporulation efficiency assay. The data were analyzed by analysis of Chi-square in SPSS (version 19.0). Virulence assays. The DBA_2 mouse was purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. for virulence assay of these strains and housed in the Laboratory Animal Center which was constant ambient temperature (23 ± 3°C) and humidity (55 ± 5%). Food, bedding, and water were replaced every four days. Mice received an intraperitoneal injection of bacterial spores (~ 2 × 10⁶ CFU). The animals were monitored at least hourly, with predefined humane endpoints (e.g., 15–20% body weight loss, severe lethargy) established to trigger prompt euthanasia if necessary, thereby minimizing undue suffering. Euthanasia was performed using the CO₂ inhalation method. All animal experiments were conducted in accordance with the recommendations and approvals of the Animal Care and Use Committee of the Academy of Military Sciences (IACUC-DWZX-2021-037). Laser scanning confocal microscopy. FM4-64 (Molecular Probes Inc., Eugene, OR, USA), a lipophilic membrane stain, was dissolved in dimethyl sulfoxide at a final concentration of 100 µM. Aliquots (1 mL) of bacterial cultures were collected at designated time points ( T 2 , T 4 , T 8 , where T 0 is the end of the exponential phase and T n is n hours after T 0 ) and centrifuged. Cell pellets were resuspended in 100 µL of PBS and then incubated with an equal volume of FM4-64 (100 µM) for 1 min on ice. The stained cells were observed using a laser scanning confocal microscope (Carl Zeiss, Germany). Localization of the BA_3317 protein To examine the localization of BA_3317-GFP in sporulating cells and dormant spores, we used fluorescence microscopy to observe the morphology of the various B. anthracis strains, as described in the laser scanning confocal microscopy analysis. Additionally, spores and de-coated spores were observed using a normal fluorescence inverted microscope (Nikon, Tokyo, Japan) equipped with an Endow GFP filter. Spores were de-coated by treatment for 30 min at 70°C with 0.1 M NaCl, 0.1 M NaOH, 1% sodium dodecyl sulfate (SDS), and 0.1 M dithiothreitol before being washed, as described previously [ 15 ]. Construction of promoter fusions Construction of lacZ promoter fusions The gerE, spoIIID , and spoVG deletion strains were constructed as described previously [ 16 ]. To analyze the transcriptional activity of the BA_3317 promoter in B. anthracis cells, the putative PBA_3317 (500 bp) promoter fragment was PCR amplified from A16R genomic DNA using BA_3317P-F-specific primers (containing a HindIII restriction site and 15 bp of sequence homologous to the vector) and BA_3317P-R (containing a BamHI restriction site and 15 bp of sequence homologous to the vector). The P BA_3317 fragment was then integrated into vector pHT304-18Z, harboring a promoterless lacZ gene, using the CloneEZ PCR Cloning Kit (Genscript Biotech Co., Nanjing, China). The demethylated pHT-PBA_3317-lacZ plasmid was electroporated into various B. anthracis strains to generate A16R(PBA_3317- lacZ ), A16R ΔgerE (PBA_3317- lacZ ), and A16R ΔspoVG (PBA_3317- lacZ ) strains. Transformants were selected on plates containing erythromycin and confirmed by PCR and sequencing. To analyze the transcriptional activity of the spoIIE promoter in B. anthracis cells, the putative P spoIIE (500 bp) promoter fragment was PCR amplified from A16R genomic DNA using P spoIIE -F/R primers in Table S6 . The demethylated pHT-P spoIIE -lacZ plasmid was electroporated into various B. anthracis strains to generate A16R(P spoIIE -lacZ) and A16RΔBA_3317(P spoIIE -lacZ) strains. β-Galactosidase assays The B. anthracis strains containing lacZ transcriptional fusions were grown in liquid DSM at 37°C. Culture samples (1.5 ml) were collected every hour from the post-exponential ( T - 2 ) phase until post-stationary phase ( T 7 ). The β-galactosidase activities of the samples were measured with O-Nitrophenyl β-D-galactopyranoside (ONPG) as the substrate previously described[ 17 ] and expressed as Miller units per mg of protein. At least three independent cultures were assayed for enzyme activity. Transcriptic and qRT-PCR analysis Total RNA was extracted from B. anthracis A16R, ΔBA_3317 and HΔBA_3317 cells cultured in DSM at time points T 0.5 . Sequencing data were aligned to Bacillus anthracis Ames Ancestor reference genome (assembly: AE017334, AE017336). The qRT-PCR was performed to identify differences in the expression of genes between the wild-type and mutant strains. Genomic DNA contamination was removed using the HiFiScript gDNA Removal Kit. Prior to reverse transcription, total RNA was subjected to PCR using primers specific for the 16S rRNA and 23S rRNA genes to exclude the possibility of genomic DNA contamination. Double-stranded cDNA was generated using a cDNA synthesis kit, and qRT-PCR analysis was performed with UltraSYBR Mix (CoWin Biosciences, Beijing, China) and the BioRad (Hercules, CA, USA) CFX96 Connect Real-Time PCR System. The primers used for RT-qPCR are listed in Table S6 . Relative changes in gene expression were measured using the double delta Ct (ΔΔCt) method with gatB_Yqey as the reference gene [ 18 – 20 ]. Analysis of the supernatant of the culture medium To further confirm the role of BA3317 in sporulation, we exchanged the medium of A16R with 3317 grown in DSM at T0.5. After centrifugation for 6500 pm and 10min, the mediums were sterilized by filtration, and the medium was exchanged to for growth 120 hours to determine the rate of sporulation. We used ammonium sulfate fractionation to track the active components for the formation of spores. The supernatant culture medium of A16R (800 ml, DSM) was collected at T 0.5 after sterilized by filtration. Following the solid ammonium sulfate fractionation table, we use different saturation of the ammonium sulfate to precipitate the supernatant. The precipitate was separated by centrifugation, and the rate of sporulation was measured by dissolving it in water to add into the medium. In vitro promoter pull-down assay. A fragment consisting of the BA_3317 promoter region (500p) was amplified from B. anthracis A16R genomic DNA using the biotinylated primer set BA_3317P-F/BA_3317P-R and purified using a Gel Extraction Kit (CoWin Biosciences, Beijing, China). Cytoplasmic extracts were harvested from wild-type A16R cells grown in DSM at time points T- 2 , T 3 , and T 8 . The biotinylated BA_3317 promoter fragment was immobilized on streptavidin-coated Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) and incubated with various total protein extracts, as per the manufacturer’s instructions. After washing three times, the Dynabeads were eluted with buffer according to the manufacturer’s protocol. Dynabeads lacking the immobilized BA_3317 promoter fragment were used as a negative control, and are referred to here as “empty” magnetic beads. The eluted protein samples were analyzed by LC-MS/MS as described below. Electrophoretic mobility shift assay. His-tagged SpoIIID and SpoVG and GST-tagged GerE proteins were purified from E. coli BL21(DE3) as described above. Corresponding DNA fragments were obtained by PCR from strain A16R genomic DNA using specific BA_3317P-F/R primers (Table S2 ) labeled with a fluorescent 5′-end 6-FAM modification and confirmed as authentic by DNA sequencing. The FAM-labeled probes were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and then quantified using a NanoDrop 2000C spectrophotometer (Thermo, USA). EMSAs were performed in a 20-µl reaction volume containing 50 ng of probe and various concentrations of purified proteins in a reaction buffer consisting of 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 2.5 mM MgCl 2 , 0.2 mM DTT, 2 µg of salmon sperm DNA, and 10% glycerol. Following incubation for 30 min at 25°C, reaction mixtures were loaded into 2% Tris-boric acid EDTA (TBE) gels buffered with 0.5× TBE, and then scanned using ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA). Results Deletion of BA_3317 decreases sporulation efficiency Based on unpublished germination proteomic profiles, we identified one downregulated protein, designated BA_3317 in the Bacillus anthracis A16R. This open reading frame of BA_3317 is predicted to encode an amino acid permease. Using the online tool available at http://smart.embl-heidelberg.de/ , we performed a structural prediction of BA_3317[ 21 ]. Conserved domain analysis indicated that BA_3317 possesses 12 transmembrane regions, a semialdehyde dehydrogenase (semialdehyde_dh) domain, and an AgrB domain (Fig. 1 A). Detailed results of the bioinformatics analysis are provided in Table 1 . Proteins of the semialdehyde dehydrogenase family participate in arginine biosynthesis and in the synthesis of several amino acids derived from aspartate [ 22 ]. AgrB, a transmembrane protein, is involved in the transport and maturation of the staphylococcal quorum-sensing (QS) system [ 11 , 12 ]. Additionally, in Enterococcus faecalis OG1RF, the agr-like gene fsrB has been implicated in the proteolytic processing of the precursor of a QS signal molecule, which regulates the expression of the virulence genes gelatinase ( gelE ) and serine protease ( sprE ) [ 23 ]. Therefore, we hypothesized that BA_3317 might function in amino acid utilization and in the regulation of sporulation and virulence in B. anthracis . Supporting this notion, preliminary data suggest a potential role for BA_3317 in amino acid transport (Fig. S1 and Table S1 , Supplementary Materials). However, virulence assays using the DBA/2 mouse model demonstrated that BA_3317 does not affect the virulence of B. anthracis (Fig. 1 B). Table 1 The bioinformatics analysis of protein BA_3317 Name Start End E-value Reason G3P_acyltransf 19 167 85400 threshold Bac_rhodopsin 35 310 11100 threshold Semialdhyde_dh 41 128 31400 threshold VKc 87 167 5050 threshold AgrB 88 273 26400 threshold PDZ 98 171 1070 threshold PSN 118 294 330 threshold HOLI 124 285 2050 threshold low complexity 125 140 N/A overlap TLC 147 389 2030 threshold Col_cuticle_N 149 199 17500 threshold low complexity 149 169 N/A overlap EXOIII 233 399 869 threshold 7TM_GPCR_Srsx 287 420 41400 threshold acidPPc 319 402 1300 threshold Following the bioinformatics analysis, we next assessed the sporulation efficiency of BA_3317 mutants. Initial growth curve analysis in Difco sporulation medium (DSM) revealed no differences among the three strains, a pattern consistent with that observed in BHI medium (Fig. S1 , Supplementary Materials). The spore-forming ability of the BA_3317-null mutant was quantified by calculating the ratio of heat-resistant spores to total viable cells at 120 hours post-inoculation. As shown in Fig. 1 C, the ΔBA_3317 mutant produced a significantly lower proportion of spores (22 ± 3%) compared to the wild-type A16R strain (93 ± 2%) (P ≤ 0.001). Complementation of the mutant (strain HΔBA_3317) partially restored sporulation, yielding 76.3 ± 7% spores (P ≤ 0.001 versus the mutant), a level similar to that of the wild type. To examine the process of sporulation, cultures of each strain were stained at T 14 and T 38 (where T n represents n hours after T 0 , the end of the exponential growth phase) using malachite green (specific for dormant spores) and safranin O (specific for vegetative cells), followed by optical microscopy (Fig. 1 D). At T 14 , spores were detected in cultures of A16R (61.3 ± 8%) and HΔBA_3317 (32.7 ± 5%) but were absent in the ΔBA_3317 culture. By T 38 , the proportion of spores increased in both A16R (88.3 ± 3%) and HΔBA_3317 (67.3 ± 10%) cultures, while remaining low in the ΔBA_3317 mutant (18.7 ± 4%). Quantitative analysis of micrographs and corresponding statistics are provided in Fig. 1 E. Collectively, these results demonstrate that BA_3317 is involved in regulating spore formation in B. anthracis . Mutant ΔBA_3317 strain was partly arrested on asymmetric division To further delineate the role of BA_3317 in sporulation, we analyzed wild-type A16R, the ΔBA_3317 mutant, and the complemented HΔBA_3317 strain following staining with the membrane-specific dye FM4-64 and laser scanning confocal microscopy. As indicated by gray arrows in Fig. 2 A and 2 B, red fluorescence imaging revealed that A16R, ΔBA_3317, and HΔBA_3317 formed an asymmetric septum by the T 2 phase at proportions of ~ 8%, ~ 1.5%, and ~ 8.3%, respectively. By this time point, however, ~ 22% of wild-type cells and ~ 6.3% of HΔBA_3317 cells had undergone engulfment (yellow arrows). At T 4 , red fluorescence images showed that ~ 65.6% of A16R cells had completed engulfment. In contrast, ~ 44.3% of HΔBA_3317 cells were engulfed at this time point, while ~ 23% remained in asymmetric division; only 3.6% of ΔBA_3317 cells had completed engulfment, with ~ 17% arrested at asymmetric cell division. By T 8 in DSM, red fluorescence imaging demonstrated that ~ 18% of ΔBA_3317 cells were at the engulfment stage, whereas ~ 14.3% still underwent asymmetric division. Bright-field imaging showed that A16R and HΔBA_3317 cultures contained ~ 21.3% and ~ 15% prespore cells, respectively, at T 8 (green arrows, Fig. 2 A), whereas prespores were not detected in ΔBA_3317 cultures. Collectively, these results indicate that fewer ΔBA_3317 cells initiate sporulation, and the sporulation process is partially blocked and delayed in this mutant. Localization of the BA_3317 protein To determine the subcellular localization of BA_3317 and better understand its role during sporulation, we performed fluorescence microscopy on sporulating cells and dormant spores. A16R harboring pBE2-BA_3317- gfp (Fig. 3 A) was cultured in DSM at 37°C. Live cells were stained with the membrane-specific dye FM4-64, followed by observation via laser scanning confocal microscopy; red fluorescence denoted the bacterial cell membrane, and green fluorescence indicated the localization of the BA_3317-GFP fusion protein. Dormant spores and de-coated spores were similarly analyzed using the same protocol. At T 0 , BA_3317-GFP was located on the cell membrane (Fig. 3 B). At T 2 , the BA_3317-GFP was located both on the asymmetric septum and the cell membrane. At T 4 , BA_3317-GFP was located both on the outline of the forespores and the cell membrane. At T 8 period, BA_3317-GFP was also observed on the prespores. In the purified spores, BA_3317-GFP could also be observed as a green circle on the spore surface by inverted fluorescence microscopy (Fig. 3 C). In the de-coated spores with the removal of the spore cell coat, the green fluorescence signal of BA_3317-GFP disappeared (Fig. 3 C). These results confirmed that BA_3317 localizes on the bacterial cell membrane in early stationary stage and within the forespore during asymmetric division. In the late stage of spore development, BA_3317 was localized on the surface of dormant spores, with no BA_3317-specific GFP signal detected on the matured spore outer layer after de-coating. BA_3317 affects spore formation as a transporter of the sporualtion signal molecules AgrB is involved in transport and maturation of the staphylococcal QS pheromone [ 24 ]. To investigate whether BA_3317 functions as a transporter in the quorum-sensing (QS) system to modulate sporulation, culture supernatant was harvested from wild-type A16R and ΔBA_3317 cultures at 0.5 h post-exponential growth ( T 0.5 ), filter-sterilized, and reciprocally exchanged between the two strains (Fig. 4 A). Cultures were further incubated at 37°C for 120 h, after which sporulation efficiency was quantified. As shown in Fig. 4 B, A16R exhibited a modest reduction in sporulation efficiency following medium exchange (60.1 ± 6.3%) compared to the non-exchanged control (88.6 ± 9.2%). In contrast, ΔBA_3317 displayed a significant increase in sporulation efficiency after receiving wild-type culture supernatant (64.7 ± 3.4%) relative to its non-exchanged counterpart (25.1 ± 5.0%). These findings indicate that ΔBA_3317 fails to secrete sufficient sporulation-promoting factors, and uptake of these factors from wild-type culture supernatant partially rescues its compromised sporulation phenotype. To corroborate these results, cultures of all four experimental groups (A16R non-exchanged, A16R exchanged, ΔBA_3317 non-exchanged, ΔBA_3317 exchanged) were stained at T 110 with malachite green (dormant spore-specific) and safranin O (vegetative cell-specific), followed by bright-field microscopy to visualize and quantify spores. As shown in Fig. 4 C, the number and proportion of spores in exchanged A16R cultures were lower than those in non-exchanged A16R, while exchanged ΔBA_3317 cultures showed elevated spore abundance compared to non-exchanged ΔBA_3317. Collectively, these data demonstrate that sporulation-promoting signaling components are less abundant in ΔBA_3317 culture supernatant than in wild-type culture supernatant, and that ΔBA_3317 acquires these critical factors from the exchanged medium. This raises the possibility that BA_3317 may function in the secretion of factors essential for efficient sporulation in Bacillus anthracis . To identify the sporulation-promoting factors that are deficient in the ΔBA_3317 mutant supernatant, we employed ammonium sulfate fractional precipitation to track and enrich the active components (Fig. 4 D). Culture supernatant (800 mL) from wild-type A16R cells at T 0.5 was collected and subjected to stepwise ammonium sulfate precipitation. Based on a standard fractionation scheme, proteinaceous material was precipitated at 60%, 80%, and 100% saturation. Each precipitate was resuspended, and an aliquot equivalent to 1/8 of the original supernatant volume was added to 100 mL cultures of ΔBA_3317 at T 0 . Sporulation efficiency was quantified after 120 h of incubation at 37°C. As shown in Fig. 4 E, only the addition of the 100%-saturation fraction significantly restored sporulation in the ΔBA_3317 mutant, increasing the spore formation rate from 21.7 ± 4% to 62.8 ± 4.5%. The 60% and 80% fractions did not produce a statistically significant improvement. These results indicate that the sporulation-promoting activity secreted by wild-type cells precipitates at high ammonium sulfate concentration, consistent with the properties of a small, soluble molecule. This further supports a role for BA_3317 in the export of small molecule signals required for efficient spore development. BA_3317 modulates sporulation efficiency and PlcR-PapR QS system by transporting for signaling molecules secretion To identify the key genes through which BA_3317 modulates sporulation, we isolated total RNA from bacteria at T 0.5 of sporulation (0.5 h post-exponential growth) for transcriptomic analysis. The analysis revealed 735 upregulated and 835 downregulated genes (Fig. S2 , Supplementary Materials). Gene ontology (GO) and KEGG enrichment analysis of these differentially expressed genes (DEGs) was performed at http://david.ncifcrf.gov/home.jsp/ . The top terms (high score and P-value ≤ 0.1) from the enrichment analysis were selected for imaging. Enrichment analysis showed enrichment primarily in a biological process: sporulation (Fig. 5 A and Table S2 in Supplementary Materials). σ F controls early forespore-specific sporulation gene expression, and SpoIIE is involved in in early sporulation events asymmetric division [ 5 – 7 , 25 , 26 ]. The sporulation genes spoIID , spoIIM , and spoIIP are initially needed for prespore engulfing [ 27 ]. These key sporulation-related genes among the DEGs are shown in Fig. 5 B, the transcriptional levels of spoIIE , spoIIP , spoIIM , and σ F were significantly lower in the ΔBA_3317 mutant than in the wild-type strain A16R, and partially restored in the complemented strain HΔBA_3317. We further performed RT-qPCR to compare the transcriptional levels of these four genes across the three strains, and the results confirmed a trend consistent with that of the transcriptomic analysis (Fig. 5 C). While SpoIIE is known to regulate asymmetric division during sporulation in Bacillus subtilis and plays a critical role in Bacillus anthracis sporulation [ 28 ], its transcriptional and translational levels directly reflect the status of sporulation. Building on transcriptomic and qPCR data, we employed a blue-white screening assay with X-gal (120µg/mL) as the substrate to assess spoIIE promoter activity. Our results showed that spoIIE promoter activity was significantly lower in the ΔBA_3317 mutant than in the wild-type A16R strain in LB after 24 h (Fig. 5 D and 5 E, Fig. S3 A in Supplementary Materials). These findings suggest that measurement of spoIIE promoter activity can serve as a reporter for sporulation-promoting factors in cell-free supernatants. Using a proximity-dependent diffusion assay, we observed that the spoIIE promoter was activated in ΔBA_3317 mutant cells located adjacent to wild-type A16R colonies, as indicated by the hydrolysis of X-gal to yield a blue product (Fig. 5 F). In contrast, ΔBA_3317 cells positioned farther away from A16R showed no detectable promoter activity. This result confirms that the supernatant from A16R cultures contains a diffusible factor that can restore early sporulation signaling in the mutant. Together, these findings demonstrate that the ΔBA_3317 mutant is deficient in producing a medium-diffusible component required for sporulation, and that supplementation with this component is sufficient to reactivate the key early sporulation regulator SpoIIE and initiate sporulation. Together, these findings further confirm that BA_3317 functions as a secretory transporter for QS signaling molecules to modulate sporulation. Notably, deletion of BA_3317 did not affect extracellular protease activity (Fig. S3 B in Supplementary Data). To further dissect its role in QS signaling, we ectopically activated the plcR-papR QS system by introducing an exogenous plasmid in B. anthracis (Fig. 5 G). Given that the hemolytic activity was excessively weak (Fig. S3 C in Supplementary Data)[ 29 ], we subsequently employed lecithinase for activity assessment using egg yolk agar (#HB0262-1, Hopebio, China). No lecithinase activity was observed for the ΔBA_3317 ( plcR-papR ) clone, whereas clear halos formed around both the A16R ( plcR-papR ) clone and the ΔBA_3317 clone supplemented with the PapR7 peptide following 16 h of culture (Fig. 5 H and 5 I). Our results indicated that ΔBA_3317 harboring plcR-papR exhibited markedly reduced PlcR activity, which was restored to varying degrees by exogenous supplementation with the PapR heptapeptide (DVPFEY, 50µM) from B. anthracis and the PapR heptapeptide (KDLPFEY, 50µM) from B. cereus [ 30 , 31 ]. These results collectively demonstrate that BA_3317 acts as a transporter for signaling molecules secretion, thereby influencing both sporulation and PlcR-dependent physiological processes, such as lecithinase activity, in B. anthracis . BA_3317 acts as a membrane protein regulated by SpoVG and GerE Fluorescence localization studies confirmed the membrane association of BA_3317 (Fig. 3 ). To investigate the transcriptional regulation of BA_3317, we performed an in vitro promoter pull-down assay using Dynabeads to capture proteins that interact with the BA_3317 promoter region. Proteins bound to the beads were identified by LC‑MS/MS, yielding approximately 120 candidate binding proteins (Table S3 , Supplementary Materials). After comparing binding profiles against an “empty” magnetic bead control and considering known sporulation regulators in Bacillus subtilis, we identified specific interactors at distinct time points: SpoVG was enriched in the T- 2 and T 3 samples, while GerE and SpoIIID were detected in the T 8 sample (Table 2 ). Table 2 BA_3317 promoter-binding proteins. Accession Description T − 2 T 3 T 8 BA_3858 hup-3 DNA-binding protein HU √ √ √ BA_0047 spoVG regulatory protein SpoVG √ √ BA_1012 BA_1012 3'-5' exoribonuclease YhaM √ √ √ BA_2377 hup-2 DNA-binding protein HU √ √ √ BA_3982 rpsP 30S ribosomal protein S16 √ √ BA_5395 uvrA excinuclease ABC subunit A √ BA_0134 rpmJ 50S ribosomal protein L36 √ √ BA_4547 rpsT 30S ribosomal protein S20 √ BA_0123 rpsN 30S ribosomal protein S14 √ BA_4724 gerE germination protein GerE √ BA_5521 spoIIID stage III sporulation protein D √ To validate the interaction between these candidate proteins and the BA_3317 promoter, we expressed GST-tagged GerE, His-tagged SpoVG, and His-tagged SpoIIID in Escherichia coli and purified them via affinity chromatography. The Electrophoretic mobility shift assays (EMSAs) were performed to assess the binding capacity of GerE, SpoVG, and SpoIIID to a 532-bp fragment of the BA_3317 promoter. Incubation with increasing concentrations of each purified protein resulted in slower-migrating probe-protein complexes, indicating binding. As shown in Fig. 6 A and 6 B, we confirmed that GerE and SpoVG specifically recognize and bind to sequences within the BA_3317 promoter fragment (original blots are provided in Supplemental Fig. S4 ). In contrast, no significant binding was detected between His-tagged SpoIIID and the BA_3317 promoter (Fig. S5 ), with original blots included in Supplementary Fig. S6 . While a conserved GerE recognition sequence has been characterized in B. subtilis , no conserved SpoVG-binding motif is known in this organism. Consistent with this, a GerE consensus sequence (TPuGGPy; Pu = purine, Py = pyrimidine) was identified within the first 60 bp of the BA_3317 promoter (TAGGC; Fig. 6 C)[ 32 ]. To determine whether GerE and SpoVG regulate the transcription of BA_3317, we constructed a PBA_3317-lacZ transcriptional fusion and transformed it into the wild-type A16R strain, as well as the Δ spoVG and Δ gerE mutant strains. In B. anthracis , the sporulation phenotype of the gerE mutant was largely consistent with that observed in B. subtilis . The spoVG mutant exhibited a pronounced sporulation defect [ 33 ]. β-Galactosidase activity assays showed that BA_3317 promoter activity was markedly elevated in the Δ spoVG mutant relative to wild-type A16R during T − 2 to T 1 . In contrast, BA_3317 promoter activity was significantly reduced in the the Δ gerE mutant from T 3 onward, compared with the wild-type control (Fig. 6 D). These results demonstrate that BA_3317 promoter activity is negatively regulated by SpoVG prior to asymmetric cell division ( T − 2 to T 1 ) and positively regulated by GerE in late sporulation ( T 3 onward). BA_3317 mediates Quorum-Sensing signal export to initiate sporulation in B. anthracis Integrating our current findings with established knowledge from the model organism B. subtilis , we propose an integrated model for the role of BA_3317 in B. anthracis sporulation (Fig. 7 ). Orthologs of the sporulation signal transduction phosphorelay are present in members of the Bacillus cereus group, and sporulation is initiated in B. anthracis upon phosphorylation of Spo0A via a multi-component phosphorelay system [ 34 ]. Prior to asymmetric division, forespore-specific σ F is synthesized under the control of phosphorylated Spo0A (Spo0A ~ P) [ 35 ]. SpoIID, SpoIIM, and SpoIIP are required for forespore engulfment [ 1 , 36 ]. σ E directly controls the transcription of sporulation genes spoIID, spoIIM , and spoIIP [ 37 ], suggesting indirect regulation by σ F . Deletion of BA_3317 impairs the transport secretion of quorum-sensing (QS) signaling proteins or peptides, thereby abrogating their extracellular maturation, re-importation, and subsequent Spo0A phosphorylation. This, in turn, leads to decreased levels of key sporulation regulators (SpoIIE, σ F ) and structural proteins (SpoIIM, SpoIIP), ultimately reducing sporulation efficiency. Notably, sporulation is a complex, multi-gene process, and additional factors contributing to the observed phenotype warrant further investigation to fully delineate their roles in mediating the sporulation defect of the ΔBA_3317 mutant. The membrane protein BA_3317 functions as a transporter of sporulation-related signal molecules. Its expression is temporally regulated: SpoVG acts as a repressor prior to asymmetric division ( T − 2 to T 1 ), whereas GerE functions as a positive regulator during late sporulation (from T 3 onward). Ultimately, BA_3317 is incorporated as a structural component of the mature spore in Bacillus anthracis . Consistent with its structural role, the protein is degraded during spore germination and was correspondingly identified among downregulated proteins in germination proteomic profiles. Discussion Sporulation is a pivotal developmental process that enables B. anthracis to survive adverse environments and maintain pathogenicity. In this study, we systematically characterized the function of BA_3317, a previously uncharacterized membrane protein with a QS-related AgrB domain, and identified its essential role as a signal exporter in mediating B. anthracis sporulation. Our findings integrate BA_3317 into the complex regulatory network of B. anthracis sporulation, revealing a novel link between QS signal transport and sporulation initiation. Elsewhere, the agrB null mutants of Clostridium perfringens, C. acetobutylicum , and C. sporogenes also spores formation defects, suggesting that the phenotype is controlled by QS [ 38 – 40 ]. The similarities between the S. aureus and Bacillus group were highlighted by a study that used the machinery from the S. aureus peptide-based agr QS system to engineer a synthetic QS system in B. megaterium s [ 41 ]. BA_3317 contains an AgrB domain, which was identified as an S. aureus QS system member [ 11 , 12 ]. Analysis of the published B. anthracis genomes show that they contain homologs of the staphylococcal agr quorum-sensing system (Table S4 , Supplementary Materials). The decreased sporulation efficiency phenotype of the ΔBA_3317 mutant was restored to some extent by medium exchange between ΔBA_3317 and A16R and the addition of supernatants from the A16R following precipitation by fractionated ammonium sulfate at T 0.5 . The most salient finding is that BA_3317 acts as a signal transporter essential for the asymmetric cell division stage-a critical early checkpoint of sporulation. Asymmetric cell division is tightly controlled by a cascade of transcriptional regulators and signaling molecules, and any disruption at this stage directly blocks subsequent sporulation events[ 42 ]. We further demonstrated that BA_3317 modulates the transcription of spoIIE , a key sporulation-specific gene essential for asymmetric division [ 43 ]. Similarly, the B. anthracis spoIIE mutant was delayed in polar division and failed to activate σ F in the forespore and σ E in the mother cell. Notably, the spoIIE promoter in ΔBA_3317 cells were only activated when adjacent to wild-type colonies, implying that BA_3317 mediates the export of a diffusible sporulation signal that is required for spoIIE expression. This observation aligns with secretion exchange experiments showing that loss of BA_3317 impairs the secretion of sporulation-essential factors, collectively supporting the hypothesis that BA_3317 functions as a sporulation signal transporter. The functional link between BA_3317 and the plcR-papR QS system further reinforces its role in QS signal transport. Our results indicates that BA_3317 is required for the outward transport of the PapR peptide: without BA_3317, the PapR peptide cannot be secreted to activate downstream QS signaling, leading to the loss of lecithinase activity. Despite these significant findings, several limitations of this study should be acknowledged. First, the specific sporulation signal exported by BA_3317 remain to be identified; Second, many other genes also contribute to the complicated sporulation process and should be examined in future studies to determine their roles in the sporulation phenotype. However, based on our current findings and what is already known in the model species B. subtilis , we could propose a multiple-factor model for the role played by BA_3317 in sporulation (Fig. 7 ). The absence of BA_3317 may block the secretion of sporulation signal molecules, thereby preventing its extracellular maturation and re-importation and Spo0A phosphorylation subsequently. This would in turn result in reduced levels of the key SpoIIE, σ F , SpoIIP, and SpoIIM sporulation proteins, thereby affecting sporulation efficiency. As a critical mediator of sporulation initiation via QS signal export, BA_3317 represents a novel potential target for anti-anthrax interventions. Inhibiting BA_3317 function could block the export of QS signals, disrupt sporulation, and thereby reduce the environmental persistence and infectivity of B. anthracis . In conclusion, our study identifies BA_3317 as a novel sporulation signal exporter that is critical for B. anthracis sporulation. BA_3317 mediates the export of QS signals (including the PapR peptide), regulates the transcription of key sporulation genes (e.g., spoIIE ), and is tightly regulated by the canonical sporulation regulators SpoVG and GerE. These findings advance our understanding of the species-specific molecular mechanisms linking QS and sporulation in B. anthracis and provide a novel target for anthrax prevention and control. Future studies focusing on the direct transport activity of BA_3317 and its interacting partners will further deepen our knowledge of this regulatory pathway. Declarations Supplementary Materials Fig. S1-S6; Table S1-S6. Acknowledgments We gratefully acknowledge financial support for National Natural Science Foundation of China (grant numbers: 82102412,82172317) and funded by the State Key Laboratory of Pathogen and Biosecurity (Academy of Military Medical Science, SKLPBS2419). Author contributions Y. L., D. W., X. L. and H. W. investigation; Y. L., D. W., J. J. and M. F. methodology; Y. L., D. W., and M. C. formal analysis; Y. L., D. W., X. L. and H. W. conceptualization; C. P., Y. G. and S. Y. visualization; L. Z. validation; Y. L. and D. W. writing–original draft; X. L. and H. W. writing–review and editing; M.C, M. G., Y. L. and D. W. data curation; X. L. and H. W. supervision; Y. L. and D. W. funding acquisition; H. W. project administration. Conflicts of Interest The authors declare no conflicts of interest. Data availability Original microscopic images are available upon request. All data generated or analyzed during this study are included in this published article and its supplementary files, or available upon request. Source data are provided with this article. Ethics approval The animal experiment was approved by the Animal Care and Use Committee of the Academy of Military Sciences (IACUC-DWZX-2021-037). Consent to participate Not applicable. Consent for publication Not applicable. 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Marchand N, Collins CH: Synthetic Quorum Sensing and Cell–Cell Communication in Gram-Positive Bacillus megaterium . Acs Synthetic Biology 2015, 5 (7):597. Barák I, Muchová K, Labajová N: Asymmetric cell division during Bacillus subtilis sporulation . Future Microbiol 2019, 14 :353-363. Ramírez-Guadiana FH, Brogan AP, Yu Y, Midonet C, Sher JW, Schmid EW, Roney IJ, Rudner DZ: Identification of sporulation genes in Bacillus anthracis highlights similarities and significant differences with Bacillus subtilis . PLoS Biol 2025, 23 (12):e3003521. Additional Declarations No competing interests reported. Supplementary Files TableS1aminoacidcomponentmedium.xlsx TableS4QSsystemsinB.anthracishomologous.xlsx TableS5andTableS6.doc TableS3Invitropromoterpulldownproteinslist.xlsx TableS2mRNAtranscriptomicanalysis.xlsx RevisedFig.S1S6.doc 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-8551494","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":581345215,"identity":"21597cd8-80bd-42ce-b447-962837f64665","order_by":0,"name":"Yufei 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Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiankai","middleName":"","lastName":"Liu","suffix":""},{"id":581345252,"identity":"b5bc1ce8-21f1-4c11-9768-c5c75adef646","order_by":11,"name":"Hengliang Wang","email":"","orcid":"","institution":"Academy of Military Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hengliang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-01-08 13:54:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8551494/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8551494/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101342403,"identity":"19e887ce-3e80-4f06-9718-66e209b117ff","added_by":"auto","created_at":"2026-01-28 16:32:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1177332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeletion of BA_3317 decreases sporulation efficiency \u003c/strong\u003e(A) Domain organization of BA_3317 based on bioinformatic analysis. Blue vertical bars, transmembrane domains; An orange pentagon, the semialdehyde_dh domain; A red ellipse, AgrB domain; (B) Loss of\u0026nbsp; BA_3317 does not affect B. anthracis virulence in mice. There was no significantly difference between the survival curves for DBA_2 mice challenged with ΔBA_3317 spores (red line; n = 10), wild-type spores (blue line; n = 10) and HΔBA_3317 spores(green line, n=10) when the spores were administered intraperitonally (~1×10\u003csup\u003e6\u003c/sup\u003e spores). (C) Sporulation efficiency of A16R, ΔBA_3317, and HΔBA_3317 were defined as the ratio of the number of spores to the total number of viable cells at 120 h post-inoculation. (D) The three strains were stained with malachite green and safranin O at time points T\u003csub\u003e14\u003c/sub\u003e and T\u003csub\u003e38\u003c/sub\u003e. Spores stain green whereas vegetative cells stain red. T\u003csub\u003en\u003c/sub\u003e, n hours after T\u003csub\u003e0\u003c/sub\u003e (the end of the exponential growth phase). Scale bar, 20 μm. (E) The percentage of spores is defined as the ratio of the number of spores to the total number of viable cells in micrographs at T\u003csub\u003e14\u003c/sub\u003e and T\u003csub\u003e38\u003c/sub\u003e post-inoculation. Values represent the means of at least three independent replicates. The data were analyzed in SPSS (version 19.0) using an χ2 test. Error bars represent standard deviations.*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.\u003c/p\u003e","description":"","filename":"RevisedFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/aedfe3d1f32375fddf8b4b39.png"},{"id":101398290,"identity":"d3822936-a899-44a0-ad95-c430c9cc713e","added_by":"auto","created_at":"2026-01-29 09:40:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3011457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe ΔBA_3317 mutant was partially arrested at the asymmetric division stage by laser scanning confocal microscopy.\u003c/strong\u003e (A) Laser scanning confocal microscopy of wild-type A16R, ΔBA_3317, and HΔBA_3317 cells grown in DSM and stained with FM4-64. Bar, 10 μm. The cell membrane is visible as red fluorescence. The gray, yellow, and green arrows indicate asymmetric cells, engulfed cells, and developing spores, respectively. Samples were collected at the indicated time points (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e, \u003cem\u003eT\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e, and \u003cem\u003eT\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e) and prepared for microscopy as described in Materials and Methods; (B) Pooled data from two replicates of the percentage of asymmetric cells, engulfed cells and developing spores over time in the different strains. In the figure, asy stand for asymmetric cells, eng stand for engulfed cells, pre stand for developing spores.\u003c/p\u003e","description":"","filename":"RevisedFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/6086565b7e9e039901bfccda.png"},{"id":101342406,"identity":"cc47304e-16c7-4c07-b273-1b10de88ffba","added_by":"auto","created_at":"2026-01-28 16:32:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2850306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBA_3317 as a membrane protein. \u003c/strong\u003e(A) Construction of the GFP-tagged protein via gene fusion. The GFP tag was added to the 3ʹ-end of the BA_3317 promoter and open reading frame; (B) Laser scanning confocal micrographs of strain A16R(pBE2-BA_3317-\u003cem\u003egfp\u003c/em\u003e) cultured in DSM at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e, \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e, \u003cem\u003eT\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e, and \u003cem\u003eT\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e. GFP, green fluorescent protein signal. FM4-64, red fluorescent signal. Merge, combined green and red fluorescent signals. Bright, phase-contrast microscopy. The yellow arrows indicate the different locations of the protein during spore development. Bar, 10 μm; (C) Image showing A16R (pBE2-BA_3317-\u003cem\u003egfp\u003c/em\u003e) cells in dormant spore phase and spores after de-coating. Arrow indicates a de-coated spore whose coat has split open. Bar,10 μm.\u003c/p\u003e","description":"","filename":"RevisedFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/120b888a862022acf0985a65.png"},{"id":101398149,"identity":"852c3112-289e-47f4-a97b-1999fcc25c6c","added_by":"auto","created_at":"2026-01-29 09:39:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1913204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of BA_3317 impairs the secretion of factors essential for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. anthracis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sporulation. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003esupernatants from A16R and ΔBA_3317 cells were exchanged at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e. (B) The sporulation efficiency of A16R and ΔBA_3317 is assayed after medium exchange between ΔBA_3317 and A16R at time points \u003cem\u003eT\u003c/em\u003e\u003csub\u003e110\u003c/sub\u003e. (C) The four strains were stained with malachite green and safranin O at time points \u003cem\u003eT\u003c/em\u003e\u003csub\u003e110\u003c/sub\u003e. Spores stain green whereas vegetative cells stain red. Scale bar, 20 μm; (D) Culture supernatant by ammonium sulfate fractional\u003cstrong\u003e \u003c/strong\u003eprecipitation. Supernatant (800 mL) from wild-type A16R cells was harvested at the \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e time point for fractionation; (E) Sporulation efficiency of A16R and ΔBA_3317 is assayed after ΔBA_3317 supplemented with the different saturated precipitated ammonium sulfate at 120 h post-inoculation from A16R culture supernatant. The data were analyzed in SPSS (version 19.0) using a χ2 test. Values are the mean values from three independent experiments and their standard deviations. Error bars represent standard deviations. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.\u003c/p\u003e","description":"","filename":"RevisedFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/7f4a67923c346488652d1ebb.png"},{"id":101397871,"identity":"0ac9d99f-16ea-44bd-8049-c6b86c0be023","added_by":"auto","created_at":"2026-01-29 09:37:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1589435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBA_3317 modulates sporulation by regulating the transcription of sporulation-specific genes\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e spoIIE\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and PapR7 heptapeptide export. \u003c/strong\u003e(A) Enrichment analysis of differentially expressed genes from transcriptomic data of wild-type (A16R), ΔBA_3317, and complemented (HΔBA_3317) strains at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e of sporulation; (B) Heatmap showing mRNA levels of selected sporulation‑specific genes in the three strains from the same transcriptomic dataset; (C) RT‑qPCR validation of sporulation‑gene expression in ΔBA_3317 and HΔBA_3317 relative to A16R at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e. Values are the mean values from three independent experiments and their standard deviations. The data were analyzed using a t-test. **P≤0.01; ***P≤0.001; ****P≤0.0001; (D) Schematic of the \u003cem\u003elacZ\u003c/em\u003e promoter‑fusion reporter construct; (E) Blue‑white screening assay using X‑gal to compare \u003cem\u003espoIIE\u003c/em\u003e promoter activity in A16R (clone 1, P\u003cem\u003espoIIE\u003c/em\u003e‑lacZ) and ΔBA_3317 (clone 2, P\u003cem\u003espoIIE\u003c/em\u003e‑lacZ); (F) Spatial activation of the \u003cem\u003espoIIE\u003c/em\u003e promoter: blue color appears in ΔBA_3317 colonies only when adjacent to wild‑type A16R colonies; (G) Strategy for ectopic activation of the \u003cem\u003eplcR‑papR\u003c/em\u003e quorum‑sensing system in \u003cem\u003eB. anthracis\u003c/em\u003e;\u003cem\u003e \u003c/em\u003e(H) and (I) Lecithinase activity assay after supplementation with either the PapR7 peptide (from \u003cem\u003eB. anthracis\u003c/em\u003e or \u003cem\u003eB. cereus\u003c/em\u003e) or the 100%‑saturated ammonium sulfate precipitate from A16R at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e culture supernatant. Clones: 3, A16R; 4, A16R (\u003cem\u003eplcR‑papR\u003c/em\u003e); 5, ΔBA_3317; 6, ΔBA_3317 (\u003cem\u003eplcR‑papR\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"RevisedFigures5.png","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/3e08b96ce7b1f444bcd2c764.png"},{"id":101342410,"identity":"1e69eba7-9739-4de9-a3ae-d5b79a579b21","added_by":"auto","created_at":"2026-01-28 16:32:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":729833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBA_3317 is directly regulated by SpoVG and GerE. \u003c/strong\u003e(A) EMSA assay of the BA_3317 promoter fragment (532 bp) following incubation with SpoVG. Lane 1, FAM-labeled BA_3317 probe; lanes 2–6, incubation of the probe with increasing concentrations of purified SpoVG; (B) EMSA assay of the BA_3317 promoter fragment (532 bp) following incubation with GerE. Lane 1, FAM-labeled BA_3317 probe incubated with GST protein; lane 2, FAM-labeled BA_3317 probe; lanes 3–7, incubation of the probe with increasing concentrations of purified GerE-GST; (C) Sequence analysis of the BA_3317 promoter region. The GerE consensus sequence and the start codon of BA_3317 are shown by underlining and overlining, respectively. (D) β-Galactosidase activity assays were performed to compare the activities of the BA_3317 promoter in three different strains (A16R, blue line; Δ\u003cem\u003egerE\u003c/em\u003e, red line; Δ\u003cem\u003espoVG\u003c/em\u003e, green line) at the indicated time points after growth in DSM at 37°C. \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e is the end of the exponential phase, and \u003cem\u003eT\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e is n hours after \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e. Each value represents the mean value of at least three independent replicates. Error bars show the standard deviations.\u003c/p\u003e","description":"","filename":"RevisedFigures6.png","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/3bb4f2bf751596defadcb450.png"},{"id":101398214,"identity":"dd552f5a-9334-4a18-8100-82d2bdcffb88","added_by":"auto","created_at":"2026-01-29 09:40:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":148788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model for the involvement of BA_3317 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. anthracis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e sporulation and life cycle. \u003c/strong\u003eGreen and orange dots represent the proteins or peptides precursors and mature proteins or peptides, respectively. BA_3317 localized at the cell membrane. BA_3317 was negatively regulated by SpoVG before asymmetric cell division and positively regulated and GerE after asymmetric cell division.\u003c/p\u003e","description":"","filename":"RevisedFigures7.png","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/d102538b7b539ec7f7c6f6ce.png"},{"id":104401599,"identity":"37207e7c-f425-4ad2-854e-3849463f2daf","added_by":"auto","created_at":"2026-03-11 12:13:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15213356,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/d4aeb5f4-f3a3-4259-ae1d-6f7845475b79.pdf"},{"id":101342404,"identity":"ff55736b-c2ce-4942-bb06-a55ef8858990","added_by":"auto","created_at":"2026-01-28 16:32:01","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10668,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1aminoacidcomponentmedium.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/f79ced939374e8f034bfd4bb.xlsx"},{"id":101342412,"identity":"be0e7776-9cca-4173-aafd-eec1a250fd8d","added_by":"auto","created_at":"2026-01-28 16:32:01","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13338,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4QSsystemsinB.anthracishomologous.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/72e651c2d2cff5f334031fc9.xlsx"},{"id":101398601,"identity":"007cb318-7cf0-4e56-8404-6fa896e1ebad","added_by":"auto","created_at":"2026-01-29 09:42:49","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":101888,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5andTableS6.doc","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/c0194e5292a75c5196fc4b8a.doc"},{"id":101342408,"identity":"560a18e9-20cd-413e-8460-f1939698fb36","added_by":"auto","created_at":"2026-01-28 16:32:01","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":143929,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3Invitropromoterpulldownproteinslist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/2f8461080bd90df8eb65338d.xlsx"},{"id":101342414,"identity":"69974fdb-acd8-402a-9546-7263f03a3dfa","added_by":"auto","created_at":"2026-01-28 16:32:02","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":910606,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2mRNAtranscriptomicanalysis.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/1e0d9870d2ddd1e442886fb1.xlsx"},{"id":101342411,"identity":"ca8b491d-89e3-40bc-af85-a8ebda58afc2","added_by":"auto","created_at":"2026-01-28 16:32:01","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1329152,"visible":true,"origin":"","legend":"","description":"","filename":"RevisedFig.S1S6.doc","url":"https://assets-eu.researchsquare.com/files/rs-8551494/v1/028eb24957b58a529bb8aa3d.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Disruption of Protein BA_3317 Affects Asymmetric Division as an Exporter of Sporulation Signal Molecules in Bacillus anthracis","fulltext":[{"header":"Background","content":"\u003cp\u003eLike other members of the \u003cem\u003eBacillus\u003c/em\u003e genus, \u003cem\u003eBacillus anthracis\u003c/em\u003e initiates sporulation under nutrient-limiting conditions. While the spore serves as the primary infectious agent for anthrax, the specific molecular pathways that regulate its formation in this pathogen are not fully elucidated. The mechanistic framework for \u003cem\u003eB. anthracis\u003c/em\u003e sporulation heavily relies on that established for the model bacterium \u003cem\u003eBacillus subtilis\u003c/em\u003e. Endospore formation is a complex developmental process involving asymmetric cell division, forespore engulfment, and spore morphogenesis in \u003cem\u003eB. subtilis\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Asymmetric cell division is a critical early event that generates two cellular compartments with distinct sizes and developmental fates. This process, essential for establishing sporulation polarity, requires SpoIIE [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and is partly repressed by SpoVG [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The resulting cellular asymmetry directly enables the activation of the forespore-specific sigma factor, σ\u003csup\u003eF\u003c/sup\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which in turn regulates the expression of genes involved in forespore engulfment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. During this engulfment process, the membrane-associated proteins SpoIID, SpoIIM, and SpoIIP are essential for membrane migration, and their expression is indirectly regulated by σ\u003csup\u003eF\u003c/sup\u003e via σ\u003csup\u003eE\u003c/sup\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As an auxiliary transcriptional regulator, SpoIIID exerts both positive and negative control over a broad range of genes within the σ\u003csup\u003eE\u003c/sup\u003e regulon during early mother cell development [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The mother cell-specific sigma factor σ\u003csup\u003eK\u003c/sup\u003e regulates the transcription factor GerE, which controls the expression of numerous spore coat proteins and cell wall hydrolases responsible for mother cell lysis and spore release [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA key interface between environmental cues and cellular response is quorum sensing (QS). Sporulation is initiated when Spo0A, the master regulator of sporulation, is activated via a \u0026ldquo;phosphorelay\u0026rdquo; system governed by autophosphorylating histidine kinases that respond to various environmental stresses. In \u003cem\u003eB. subtilis\u003c/em\u003e, the Rap protein dephosphorylates Spo0F\u0026thinsp;~\u0026thinsp;P, thereby blocking the transfer of ATP-derived phosphate to Spo0A via Spo0B and Spo0F. Notably, Rap activity is inhibited by a QS peptide exported by secretory proteins and reimported via an oligopeptide permease (Opp) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The critical role of QS systems in regulating sporulation in \u003cem\u003eBacillus\u003c/em\u003e spp. is well established: the absence of functional App and Opp transporters leads to a marked reduction in sporulation in \u003cem\u003eB. subtilis\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and modulates the activity of the Spo0A response regulator [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite this, compared with other QS components, little is known about the secretion and maturation of small QS signaling molecules. In the Gram-positive bacterium Staphylococcus aureus, the accessory gene regulator (\u003cem\u003eagr\u003c/em\u003e) operon (\u003cem\u003eagrA\u003c/em\u003e, \u003cem\u003eagrB\u003c/em\u003e, \u003cem\u003eagrC\u003c/em\u003e, \u003cem\u003eagrD\u003c/em\u003e) encodes a canonical QS system [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], where AgrB\u0026mdash;a transmembrane protein\u0026mdash;is essential for the maturation of QS pheromones [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, it remains unclear whether AgrB-like proteins are involved in the import/export of quorum-signaling peptides that regulate endospore formation in \u003cem\u003eB. anthracis\u003c/em\u003e A16R.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the role of the putative BA_3317 protein in \u003cem\u003eB. anthracis\u003c/em\u003e A16R (pXO1⁺, pXO2⁻). The \u003cem\u003eB. anthracis\u003c/em\u003e vaccine strain A16R is derived from strain A16 via UV irradiation[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. BA_3317 is annotated to contain two functional domains (Semialdehyde_dh and AgrB) and is predicted to act as an amino acid permease. We constructed an in-frame deletion mutant of BA_3317 and confirmed that disruption of this locus significantly reduced sporulation efficiency. In addition, the BA_3317 protein likely functions as a sporulation signal molecule secretory transporter protein, suggesting that the impaired sporulation in the ΔBA_3317 mutant is associated with disrupted sporulation signal transduction.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of sporulation efficiency\u003c/h2\u003e \u003cp\u003eThe material and methods of bacterial strains and plasmids used in this study are listed in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e (Supplementary Materials). The oligonucleotides used in this study are listed in Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e (Supplementary Materials). Strains A16R, ΔBA_3317, and HΔBA_3317 were grown in liquid DSM [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] at 37\u0026deg;C for 120 h with vigorous shaking. The number of viable cells in each culture was defined as the total number of colony-forming units, and was determined by serial dilution plate counts on LB agar medium. The number of spores per culture was defined as the number of heat-resistant cells in each culture, and was determined by plating serial dilutions of heat-treated (70℃ for 30 min) cells onto LB agar plates. Sporulation efficiency was defined as the ratio of the number of spores to the number of viable cells. At least three biological replicates were included for each sporulation efficiency assay. The data were analyzed by analysis of Chi-square in SPSS (version 19.0).\u003c/p\u003e \u003cp\u003e \u003cb\u003eVirulence assays.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe DBA_2 mouse was purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. for virulence assay of these strains and housed in the Laboratory Animal Center which was constant ambient temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C) and humidity (55\u0026thinsp;\u0026plusmn;\u0026thinsp;5%). Food, bedding, and water were replaced every four days. Mice received an intraperitoneal injection of bacterial spores (~\u0026thinsp;2 \u0026times; 10⁶ CFU). The animals were monitored at least hourly, with predefined humane endpoints (e.g., 15\u0026ndash;20% body weight loss, severe lethargy) established to trigger prompt euthanasia if necessary, thereby minimizing undue suffering. Euthanasia was performed using the CO₂ inhalation method. All animal experiments were conducted in accordance with the recommendations and approvals of the Animal Care and Use Committee of the Academy of Military Sciences (IACUC-DWZX-2021-037).\u003c/p\u003e \u003cp\u003e \u003cb\u003eLaser scanning confocal microscopy.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFM4-64 (Molecular Probes Inc., Eugene, OR, USA), a lipophilic membrane stain, was dissolved in dimethyl sulfoxide at a final concentration of 100 \u0026micro;M. Aliquots (1 mL) of bacterial cultures were collected at designated time points (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e, \u003cem\u003eT\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e, \u003cem\u003eT\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e, where \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e is the end of the exponential phase and \u003cem\u003eT\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e is n hours after \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e) and centrifuged. Cell pellets were resuspended in 100 \u0026micro;L of PBS and then incubated with an equal volume of FM4-64 (100 \u0026micro;M) for 1 min on ice. The stained cells were observed using a laser scanning confocal microscope (Carl Zeiss, Germany).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLocalization of the BA_3317 protein\u003c/h3\u003e\n\u003cp\u003eTo examine the localization of BA_3317-GFP in sporulating cells and dormant spores, we used fluorescence microscopy to observe the morphology of the various \u003cem\u003eB. anthracis\u003c/em\u003e strains, as described in the laser scanning confocal microscopy analysis. Additionally, spores and de-coated spores were observed using a normal fluorescence inverted microscope (Nikon, Tokyo, Japan) equipped with an Endow GFP filter. Spores were de-coated by treatment for 30 min at 70\u0026deg;C with 0.1 M NaCl, 0.1 M NaOH, 1% sodium dodecyl sulfate (SDS), and 0.1 M dithiothreitol before being washed, as described previously [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eConstruction of promoter fusions\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eConstruction of \u003cem\u003elacZ\u003c/em\u003e promoter fusions\u003c/div\u003e \u003cp\u003eThe \u003cem\u003egerE, spoIIID\u003c/em\u003e, and \u003cem\u003espoVG\u003c/em\u003e deletion strains were constructed as described previously [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To analyze the transcriptional activity of the BA_3317 promoter in \u003cem\u003eB. anthracis\u003c/em\u003e cells, the putative PBA_3317 (500 bp) promoter fragment was PCR amplified from A16R genomic DNA using BA_3317P-F-specific primers (containing a \u003cem\u003eHindIII\u003c/em\u003e restriction site and 15 bp of sequence homologous to the vector) and BA_3317P-R (containing a \u003cem\u003eBamHI\u003c/em\u003e restriction site and 15 bp of sequence homologous to the vector). The P\u003csub\u003eBA_3317\u003c/sub\u003e fragment was then integrated into vector pHT304-18Z, harboring a promoterless \u003cem\u003elacZ\u003c/em\u003e gene, using the CloneEZ PCR Cloning Kit (Genscript Biotech Co., Nanjing, China). The demethylated pHT-PBA_3317-lacZ plasmid was electroporated into various \u003cem\u003eB. anthracis\u003c/em\u003e strains to generate A16R(PBA_3317-\u003cem\u003elacZ\u003c/em\u003e), A16R\u003cem\u003eΔgerE\u003c/em\u003e(PBA_3317-\u003cem\u003elacZ\u003c/em\u003e), and A16R\u003cem\u003eΔspoVG\u003c/em\u003e(PBA_3317-\u003cem\u003elacZ\u003c/em\u003e) strains. Transformants were selected on plates containing erythromycin and confirmed by PCR and sequencing.\u003c/p\u003e \u003cp\u003eTo analyze the transcriptional activity of the \u003cem\u003espoIIE\u003c/em\u003e promoter in \u003cem\u003eB. anthracis\u003c/em\u003e cells, the putative P\u003cem\u003espoIIE\u003c/em\u003e (500 bp) promoter fragment was PCR amplified from A16R genomic DNA using P\u003csub\u003espoIIE\u003c/sub\u003e-F/R primers in Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e. The demethylated pHT-P\u003cem\u003espoIIE\u003c/em\u003e-lacZ plasmid was electroporated into various \u003cem\u003eB. anthracis\u003c/em\u003e strains to generate A16R(P\u003cem\u003espoIIE\u003c/em\u003e-lacZ) and A16RΔBA_3317(P\u003cem\u003espoIIE\u003c/em\u003e -lacZ) strains.\u003c/p\u003e\n\u003ch3\u003eβ-Galactosidase assays\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eB. anthracis\u003c/em\u003e strains containing \u003cem\u003elacZ\u003c/em\u003e transcriptional fusions were grown in liquid DSM at 37\u0026deg;C. Culture samples (1.5 ml) were collected every hour from the post-exponential (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u003cem\u003e-\u003c/em\u003e2\u003c/sub\u003e) phase until post-stationary phase (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e7\u003c/sub\u003e). The β-galactosidase activities of the samples were measured with O-Nitrophenyl β-D-galactopyranoside (ONPG) as the substrate previously described[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and expressed as Miller units per mg of protein. At least three independent cultures were assayed for enzyme activity.\u003c/p\u003e\n\u003ch3\u003eTranscriptic and qRT-PCR analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from \u003cem\u003eB. anthracis\u003c/em\u003e A16R, ΔBA_3317 and HΔBA_3317 cells cultured in DSM at time points \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e. Sequencing data were aligned to \u003cem\u003eBacillus anthracis\u003c/em\u003e Ames Ancestor reference genome (assembly: AE017334, AE017336). The qRT-PCR was performed to identify differences in the expression of genes between the wild-type and mutant strains. Genomic DNA contamination was removed using the HiFiScript gDNA Removal Kit. Prior to reverse transcription, total RNA was subjected to PCR using primers specific for the 16S rRNA and 23S rRNA genes to exclude the possibility of genomic DNA contamination. Double-stranded cDNA was generated using a cDNA synthesis kit, and qRT-PCR analysis was performed with UltraSYBR Mix (CoWin Biosciences, Beijing, China) and the BioRad (Hercules, CA, USA) CFX96 Connect Real-Time PCR System. The primers used for RT-qPCR are listed in Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e. Relative changes in gene expression were measured using the double delta Ct (ΔΔCt) method with \u003cem\u003egatB_Yqey\u003c/em\u003e as the reference gene [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the supernatant of the culture medium\u003c/h2\u003e \u003cp\u003eTo further confirm the role of BA3317 in sporulation, we exchanged the medium of A16R with 3317 grown in DSM at T0.5. After centrifugation for 6500 pm and 10min, the mediums were sterilized by filtration, and the medium was exchanged to for growth 120 hours to determine the rate of sporulation.\u003c/p\u003e \u003cp\u003eWe used ammonium sulfate fractionation to track the active components for the formation of spores. The supernatant culture medium of A16R (800 ml, DSM) was collected at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e after sterilized by filtration. Following the solid ammonium sulfate fractionation table, we use different saturation of the ammonium sulfate to precipitate the supernatant. The precipitate was separated by centrifugation, and the rate of sporulation was measured by dissolving it in water to add into the medium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro promoter pull-down assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA fragment consisting of the BA_3317 promoter region (500p) was amplified from B. anthracis A16R genomic DNA using the biotinylated primer set BA_3317P-F/BA_3317P-R and purified using a Gel Extraction Kit (CoWin Biosciences, Beijing, China). Cytoplasmic extracts were harvested from wild-type A16R cells grown in DSM at time points \u003cem\u003eT-\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e, \u003cem\u003eT\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e, and \u003cem\u003eT\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e. The biotinylated BA_3317 promoter fragment was immobilized on streptavidin-coated Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) and incubated with various total protein extracts, as per the manufacturer\u0026rsquo;s instructions. After washing three times, the Dynabeads were eluted with buffer according to the manufacturer\u0026rsquo;s protocol. Dynabeads lacking the immobilized BA_3317 promoter fragment were used as a negative control, and are referred to here as \u0026ldquo;empty\u0026rdquo; magnetic beads. The eluted protein samples were analyzed by LC-MS/MS as described below.\u003c/p\u003e \u003cp\u003e \u003cb\u003eElectrophoretic mobility shift assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHis-tagged SpoIIID and SpoVG and GST-tagged GerE proteins were purified from \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) as described above. Corresponding DNA fragments were obtained by PCR from strain A16R genomic DNA using specific BA_3317P-F/R primers (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) labeled with a fluorescent 5\u0026prime;-end 6-FAM modification and confirmed as authentic by DNA sequencing. The FAM-labeled probes were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and then quantified using a NanoDrop 2000C spectrophotometer (Thermo, USA). EMSAs were performed in a 20-\u0026micro;l reaction volume containing 50 ng of probe and various concentrations of purified proteins in a reaction buffer consisting of 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 2.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.2 mM DTT, 2 \u0026micro;g of salmon sperm DNA, and 10% glycerol. Following incubation for 30 min at 25\u0026deg;C, reaction mixtures were loaded into 2% Tris-boric acid EDTA (TBE) gels buffered with 0.5\u0026times; TBE, and then scanned using ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDeletion of BA_3317 decreases sporulation efficiency\u003c/h2\u003e \u003cp\u003eBased on unpublished germination proteomic profiles, we identified one downregulated protein, designated BA_3317 in the \u003cem\u003eBacillus anthracis\u003c/em\u003e A16R. This open reading frame of BA_3317 is predicted to encode an amino acid permease. Using the online tool available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, we performed a structural prediction of BA_3317[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Conserved domain analysis indicated that BA_3317 possesses 12 transmembrane regions, a semialdehyde dehydrogenase (semialdehyde_dh) domain, and an AgrB domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Detailed results of the bioinformatics analysis are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Proteins of the semialdehyde dehydrogenase family participate in arginine biosynthesis and in the synthesis of several amino acids derived from aspartate [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. AgrB, a transmembrane protein, is involved in the transport and maturation of the staphylococcal quorum-sensing (QS) system [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Additionally, in Enterococcus faecalis OG1RF, the agr-like gene \u003cem\u003efsrB\u003c/em\u003e has been implicated in the proteolytic processing of the precursor of a QS signal molecule, which regulates the expression of the virulence genes gelatinase (\u003cem\u003egelE\u003c/em\u003e) and serine protease (\u003cem\u003esprE\u003c/em\u003e) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, we hypothesized that BA_3317 might function in amino acid utilization and in the regulation of sporulation and virulence in \u003cem\u003eB. anthracis\u003c/em\u003e. Supporting this notion, preliminary data suggest a potential role for BA_3317 in amino acid transport (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Supplementary Materials). However, virulence assays using the DBA/2 mouse model demonstrated that BA_3317 does not affect the virulence of \u003cem\u003eB. anthracis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \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\u003eThe bioinformatics analysis of protein BA_3317\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStart\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eE-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReason\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG3P_acyltransf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBac_rhodopsin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e310\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSemialdhyde_dh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVKc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgrB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e273\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePSN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHOLI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elow complexity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eoverlap\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCol_cuticle_N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003elow complexity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eoverlap\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEXOIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e869\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7TM_GPCR_Srsx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e420\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eacidPPc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e319\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethreshold\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\u003eFollowing the bioinformatics analysis, we next assessed the sporulation efficiency of BA_3317 mutants. Initial growth curve analysis in Difco sporulation medium (DSM) revealed no differences among the three strains, a pattern consistent with that observed in BHI medium (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Supplementary Materials). The spore-forming ability of the BA_3317-null mutant was quantified by calculating the ratio of heat-resistant spores to total viable cells at 120 hours post-inoculation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the ΔBA_3317 mutant produced a significantly lower proportion of spores (22\u0026thinsp;\u0026plusmn;\u0026thinsp;3%) compared to the wild-type A16R strain (93\u0026thinsp;\u0026plusmn;\u0026thinsp;2%) (P\u0026thinsp;\u0026le;\u0026thinsp;0.001). Complementation of the mutant (strain HΔBA_3317) partially restored sporulation, yielding 76.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7% spores (P\u0026thinsp;\u0026le;\u0026thinsp;0.001 versus the mutant), a level similar to that of the wild type.\u003c/p\u003e \u003cp\u003eTo examine the process of sporulation, cultures of each strain were stained at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e14\u003c/sub\u003e and \u003cem\u003eT\u003c/em\u003e\u003csub\u003e38\u003c/sub\u003e (where \u003cem\u003eT\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e represents n hours after \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e, the end of the exponential growth phase) using malachite green (specific for dormant spores) and safranin O (specific for vegetative cells), followed by optical microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). At \u003cem\u003eT\u003c/em\u003e\u003csub\u003e14\u003c/sub\u003e, spores were detected in cultures of A16R (61.3\u0026thinsp;\u0026plusmn;\u0026thinsp;8%) and HΔBA_3317 (32.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5%) but were absent in the ΔBA_3317 culture. By \u003cem\u003eT\u003c/em\u003e\u003csub\u003e38\u003c/sub\u003e, the proportion of spores increased in both A16R (88.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3%) and HΔBA_3317 (67.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10%) cultures, while remaining low in the ΔBA_3317 mutant (18.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4%). Quantitative analysis of micrographs and corresponding statistics are provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. Collectively, these results demonstrate that BA_3317 is involved in regulating spore formation in \u003cem\u003eB. anthracis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMutant ΔBA_3317 strain was partly arrested on asymmetric division\u003c/h2\u003e \u003cp\u003eTo further delineate the role of BA_3317 in sporulation, we analyzed wild-type A16R, the ΔBA_3317 mutant, and the complemented HΔBA_3317 strain following staining with the membrane-specific dye FM4-64 and laser scanning confocal microscopy. As indicated by gray arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, red fluorescence imaging revealed that A16R, ΔBA_3317, and HΔBA_3317 formed an asymmetric septum by the \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e phase at proportions of ~\u0026thinsp;8%, ~\u0026thinsp;1.5%, and ~\u0026thinsp;8.3%, respectively. By this time point, however, ~\u0026thinsp;22% of wild-type cells and ~\u0026thinsp;6.3% of HΔBA_3317 cells had undergone engulfment (yellow arrows). At \u003cem\u003eT\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e, red fluorescence images showed that ~\u0026thinsp;65.6% of A16R cells had completed engulfment. In contrast, ~\u0026thinsp;44.3% of HΔBA_3317 cells were engulfed at this time point, while\u0026thinsp;~\u0026thinsp;23% remained in asymmetric division; only 3.6% of ΔBA_3317 cells had completed engulfment, with ~\u0026thinsp;17% arrested at asymmetric cell division. By \u003cem\u003eT\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e in DSM, red fluorescence imaging demonstrated that ~\u0026thinsp;18% of ΔBA_3317 cells were at the engulfment stage, whereas ~\u0026thinsp;14.3% still underwent asymmetric division. Bright-field imaging showed that A16R and HΔBA_3317 cultures contained\u0026thinsp;~\u0026thinsp;21.3% and ~\u0026thinsp;15% prespore cells, respectively, at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e (green arrows, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), whereas prespores were not detected in ΔBA_3317 cultures. Collectively, these results indicate that fewer ΔBA_3317 cells initiate sporulation, and the sporulation process is partially blocked and delayed in this mutant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLocalization of the BA_3317 protein\u003c/h2\u003e \u003cp\u003eTo determine the subcellular localization of BA_3317 and better understand its role during sporulation, we performed fluorescence microscopy on sporulating cells and dormant spores. A16R harboring pBE2-BA_3317-\u003cem\u003egfp\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) was cultured in DSM at 37\u0026deg;C. Live cells were stained with the membrane-specific dye FM4-64, followed by observation via laser scanning confocal microscopy; red fluorescence denoted the bacterial cell membrane, and green fluorescence indicated the localization of the BA_3317-GFP fusion protein. Dormant spores and de-coated spores were similarly analyzed using the same protocol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e, BA_3317-GFP was located on the cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At \u003cem\u003eT\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e, the BA_3317-GFP was located both on the asymmetric septum and the cell membrane. At \u003cem\u003eT\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e, BA_3317-GFP was located both on the outline of the forespores and the cell membrane. At \u003cem\u003eT\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e period, BA_3317-GFP was also observed on the prespores. In the purified spores, BA_3317-GFP could also be observed as a green circle on the spore surface by inverted fluorescence microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In the de-coated spores with the removal of the spore cell coat, the green fluorescence signal of BA_3317-GFP disappeared (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results confirmed that BA_3317 localizes on the bacterial cell membrane in early stationary stage and within the forespore during asymmetric division. In the late stage of spore development, BA_3317 was localized on the surface of dormant spores, with no BA_3317-specific GFP signal detected on the matured spore outer layer after de-coating.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBA_3317 affects spore formation as a transporter of the sporualtion signal molecules\u003c/h2\u003e \u003cp\u003eAgrB is involved in transport and maturation of the staphylococcal QS pheromone [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To investigate whether BA_3317 functions as a transporter in the quorum-sensing (QS) system to modulate sporulation, culture supernatant was harvested from wild-type A16R and ΔBA_3317 cultures at 0.5 h post-exponential growth (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e), filter-sterilized, and reciprocally exchanged between the two strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Cultures were further incubated at 37\u0026deg;C for 120 h, after which sporulation efficiency was quantified. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, A16R exhibited a modest reduction in sporulation efficiency following medium exchange (60.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3%) compared to the non-exchanged control (88.6\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2%). In contrast, ΔBA_3317 displayed a significant increase in sporulation efficiency after receiving wild-type culture supernatant (64.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4%) relative to its non-exchanged counterpart (25.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0%). These findings indicate that ΔBA_3317 fails to secrete sufficient sporulation-promoting factors, and uptake of these factors from wild-type culture supernatant partially rescues its compromised sporulation phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo corroborate these results, cultures of all four experimental groups (A16R non-exchanged, A16R exchanged, ΔBA_3317 non-exchanged, ΔBA_3317 exchanged) were stained at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e110\u003c/sub\u003e with malachite green (dormant spore-specific) and safranin O (vegetative cell-specific), followed by bright-field microscopy to visualize and quantify spores. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, the number and proportion of spores in exchanged A16R cultures were lower than those in non-exchanged A16R, while exchanged ΔBA_3317 cultures showed elevated spore abundance compared to non-exchanged ΔBA_3317.\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrate that sporulation-promoting signaling components are less abundant in ΔBA_3317 culture supernatant than in wild-type culture supernatant, and that ΔBA_3317 acquires these critical factors from the exchanged medium. This raises the possibility that BA_3317 may function in the secretion of factors essential for efficient sporulation in \u003cem\u003eBacillus anthracis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo identify the sporulation-promoting factors that are deficient in the ΔBA_3317 mutant supernatant, we employed ammonium sulfate fractional precipitation to track and enrich the active components (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Culture supernatant (800 mL) from wild-type A16R cells at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e was collected and subjected to stepwise ammonium sulfate precipitation. Based on a standard fractionation scheme, proteinaceous material was precipitated at 60%, 80%, and 100% saturation. Each precipitate was resuspended, and an aliquot equivalent to 1/8 of the original supernatant volume was added to 100 mL cultures of ΔBA_3317 at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e. Sporulation efficiency was quantified after 120 h of incubation at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, only the addition of the 100%-saturation fraction significantly restored sporulation in the ΔBA_3317 mutant, increasing the spore formation rate from 21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4% to 62.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5%. The 60% and 80% fractions did not produce a statistically significant improvement. These results indicate that the sporulation-promoting activity secreted by wild-type cells precipitates at high ammonium sulfate concentration, consistent with the properties of a small, soluble molecule. This further supports a role for BA_3317 in the export of small molecule signals required for efficient spore development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBA_3317 modulates sporulation efficiency and PlcR-PapR QS system by transporting for signaling molecules secretion\u003c/h2\u003e \u003cp\u003eTo identify the key genes through which BA_3317 modulates sporulation, we isolated total RNA from bacteria at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e of sporulation (0.5 h post-exponential growth) for transcriptomic analysis. The analysis revealed 735 upregulated and 835 downregulated genes (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Supplementary Materials). Gene ontology (GO) and KEGG enrichment analysis of these differentially expressed genes (DEGs) was performed at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://david.ncifcrf.gov/home.jsp/\u003c/span\u003e\u003cspan address=\"http://david.ncifcrf.gov/home.jsp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The top terms (high score and P-value\u0026thinsp;\u0026le;\u0026thinsp;0.1) from the enrichment analysis were selected for imaging. Enrichment analysis showed enrichment primarily in a biological process: sporulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e in Supplementary Materials). σ\u003csup\u003eF\u003c/sup\u003e controls early forespore-specific sporulation gene expression, and SpoIIE is involved in in early sporulation events asymmetric division [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The sporulation genes \u003cem\u003espoIID\u003c/em\u003e, \u003cem\u003espoIIM\u003c/em\u003e, and \u003cem\u003espoIIP\u003c/em\u003e are initially needed for prespore engulfing [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These key sporulation-related genes among the DEGs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the transcriptional levels of \u003cem\u003espoIIE\u003c/em\u003e, \u003cem\u003espoIIP\u003c/em\u003e, \u003cem\u003espoIIM\u003c/em\u003e, and σ\u003csup\u003eF\u003c/sup\u003e were significantly lower in the ΔBA_3317 mutant than in the wild-type strain A16R, and partially restored in the complemented strain HΔBA_3317. We further performed RT-qPCR to compare the transcriptional levels of these four genes across the three strains, and the results confirmed a trend consistent with that of the transcriptomic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile SpoIIE is known to regulate asymmetric division during sporulation in \u003cem\u003eBacillus subtilis\u003c/em\u003e and plays a critical role in \u003cem\u003eBacillus anthracis\u003c/em\u003e sporulation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], its transcriptional and translational levels directly reflect the status of sporulation. Building on transcriptomic and qPCR data, we employed a blue-white screening assay with X-gal (120\u0026micro;g/mL) as the substrate to assess \u003cem\u003espoIIE\u003c/em\u003e promoter activity. Our results showed that \u003cem\u003espoIIE\u003c/em\u003e promoter activity was significantly lower in the ΔBA_3317 mutant than in the wild-type A16R strain in LB after 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA in Supplementary Materials). These findings suggest that measurement of \u003cem\u003espoIIE\u003c/em\u003e promoter activity can serve as a reporter for sporulation-promoting factors in cell-free supernatants. Using a proximity-dependent diffusion assay, we observed that the \u003cem\u003espoIIE\u003c/em\u003e promoter was activated in ΔBA_3317 mutant cells located adjacent to wild-type A16R colonies, as indicated by the hydrolysis of X-gal to yield a blue product (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In contrast, ΔBA_3317 cells positioned farther away from A16R showed no detectable promoter activity. This result confirms that the supernatant from A16R cultures contains a diffusible factor that can restore early sporulation signaling in the mutant. Together, these findings demonstrate that the ΔBA_3317 mutant is deficient in producing a medium-diffusible component required for sporulation, and that supplementation with this component is sufficient to reactivate the key early sporulation regulator SpoIIE and initiate sporulation. Together, these findings further confirm that BA_3317 functions as a secretory transporter for QS signaling molecules to modulate sporulation.\u003c/p\u003e \u003cp\u003eNotably, deletion of BA_3317 did not affect extracellular protease activity (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB in Supplementary Data). To further dissect its role in QS signaling, we ectopically activated the \u003cem\u003eplcR-papR\u003c/em\u003e QS system by introducing an exogenous plasmid in \u003cem\u003eB. anthracis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Given that the hemolytic activity was excessively weak (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC in Supplementary Data)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], we subsequently employed lecithinase for activity assessment using egg yolk agar (#HB0262-1, Hopebio, China). No lecithinase activity was observed for the ΔBA_3317 (\u003cem\u003eplcR-papR\u003c/em\u003e) clone, whereas clear halos formed around both the A16R (\u003cem\u003eplcR-papR\u003c/em\u003e) clone and the ΔBA_3317 clone supplemented with the PapR7 peptide following 16 h of culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eH and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). Our results indicated that ΔBA_3317 harboring \u003cem\u003eplcR-papR\u003c/em\u003e exhibited markedly reduced PlcR activity, which was restored to varying degrees by exogenous supplementation with the PapR heptapeptide (DVPFEY, 50\u0026micro;M) from \u003cem\u003eB. anthracis\u003c/em\u003e and the PapR heptapeptide (KDLPFEY, 50\u0026micro;M) from \u003cem\u003eB. cereus\u003c/em\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These results collectively demonstrate that BA_3317 acts as a transporter for signaling molecules secretion, thereby influencing both sporulation and PlcR-dependent physiological processes, such as lecithinase activity, in \u003cem\u003eB. anthracis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBA_3317 acts as a membrane protein regulated by SpoVG and GerE\u003c/h2\u003e \u003cp\u003eFluorescence localization studies confirmed the membrane association of BA_3317 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). To investigate the transcriptional regulation of BA_3317, we performed an in vitro promoter pull-down assay using Dynabeads to capture proteins that interact with the BA_3317 promoter region. Proteins bound to the beads were identified by LC‑MS/MS, yielding approximately 120 candidate binding proteins (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Supplementary Materials). After comparing binding profiles against an \u0026ldquo;empty\u0026rdquo; magnetic bead control and considering known sporulation regulators in Bacillus subtilis, we identified specific interactors at distinct time points: SpoVG was enriched in the \u003cem\u003eT-\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e and \u003cem\u003eT\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e samples, while GerE and SpoIIID were detected in the \u003cem\u003eT\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e sample (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBA_3317 promoter-binding proteins.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" 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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u0026minus;\u0026thinsp;2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eT\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eT\u003c/em\u003e\u003csub\u003e8\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_3858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehup-3 DNA-binding protein HU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_0047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003espoVG regulatory protein SpoVG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_1012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBA_1012 3'-5' exoribonuclease YhaM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_2377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehup-2 DNA-binding protein HU\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_3982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erpsP 30S ribosomal protein S16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_5395\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003euvrA excinuclease ABC subunit A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_0134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erpmJ 50S ribosomal protein L36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_4547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erpsT 30S ribosomal protein S20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_0123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erpsN 30S ribosomal protein S14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_4724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003egerE germination protein GerE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBA_5521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003espoIIID stage III sporulation protein D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e\u0026radic;\u003c/b\u003e\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\u003eTo validate the interaction between these candidate proteins and the BA_3317 promoter, we expressed GST-tagged GerE, His-tagged SpoVG, and His-tagged SpoIIID in \u003cem\u003eEscherichia coli\u003c/em\u003e and purified them via affinity chromatography. The Electrophoretic mobility shift assays (EMSAs) were performed to assess the binding capacity of GerE, SpoVG, and SpoIIID to a 532-bp fragment of the BA_3317 promoter. Incubation with increasing concentrations of each purified protein resulted in slower-migrating probe-protein complexes, indicating binding. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, we confirmed that GerE and SpoVG specifically recognize and bind to sequences within the BA_3317 promoter fragment (original blots are provided in Supplemental Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). In contrast, no significant binding was detected between His-tagged SpoIIID and the BA_3317 promoter (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), with original blots included in Supplementary Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e. While a conserved GerE recognition sequence has been characterized in \u003cem\u003eB. subtilis\u003c/em\u003e, no conserved SpoVG-binding motif is known in this organism. Consistent with this, a GerE consensus sequence (TPuGGPy; Pu\u0026thinsp;=\u0026thinsp;purine, Py\u0026thinsp;=\u0026thinsp;pyrimidine) was identified within the first 60 bp of the BA_3317 promoter (TAGGC; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eC)[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether GerE and SpoVG regulate the transcription of BA_3317, we constructed a PBA_3317-lacZ transcriptional fusion and transformed it into the wild-type A16R strain, as well as the Δ\u003cem\u003espoVG\u003c/em\u003e and Δ\u003cem\u003egerE\u003c/em\u003e mutant strains. In \u003cem\u003eB. anthracis\u003c/em\u003e, the sporulation phenotype of the \u003cem\u003egerE\u003c/em\u003e mutant was largely consistent with that observed in \u003cem\u003eB. subtilis\u003c/em\u003e. The \u003cem\u003espoVG\u003c/em\u003e mutant exhibited a pronounced sporulation defect [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. β-Galactosidase activity assays showed that BA_3317 promoter activity was markedly elevated in the Δ\u003cem\u003espoVG\u003c/em\u003e mutant relative to wild-type A16R during \u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u0026minus;\u0026thinsp;2\u003c/sub\u003e to \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e. In contrast, BA_3317 promoter activity was significantly reduced in the the Δ\u003cem\u003egerE\u003c/em\u003e mutant from \u003cem\u003eT\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e onward, compared with the wild-type control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These results demonstrate that BA_3317 promoter activity is negatively regulated by SpoVG prior to asymmetric cell division (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u0026minus;\u0026thinsp;2\u003c/sub\u003e to \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e) and positively regulated by GerE in late sporulation (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e onward).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBA_3317 mediates Quorum-Sensing signal export to initiate sporulation in\u003c/b\u003e \u003cb\u003eB. anthracis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIntegrating our current findings with established knowledge from the model organism \u003cem\u003eB. subtilis\u003c/em\u003e, we propose an integrated model for the role of BA_3317 in \u003cem\u003eB. anthracis\u003c/em\u003e sporulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Orthologs of the sporulation signal transduction phosphorelay are present in members of the \u003cem\u003eBacillus cereus\u003c/em\u003e group, and sporulation is initiated in \u003cem\u003eB. anthracis\u003c/em\u003e upon phosphorylation of Spo0A via a multi-component phosphorelay system [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Prior to asymmetric division, forespore-specific σ\u003csup\u003eF\u003c/sup\u003e is synthesized under the control of phosphorylated Spo0A (Spo0A\u0026thinsp;~\u0026thinsp;P) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. SpoIID, SpoIIM, and SpoIIP are required for forespore engulfment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. σ\u003csup\u003eE\u003c/sup\u003e directly controls the transcription of sporulation genes \u003cem\u003espoIID, spoIIM\u003c/em\u003e, and \u003cem\u003espoIIP\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], suggesting indirect regulation by σ\u003csup\u003eF\u003c/sup\u003e. Deletion of BA_3317 impairs the transport secretion of quorum-sensing (QS) signaling proteins or peptides, thereby abrogating their extracellular maturation, re-importation, and subsequent Spo0A phosphorylation. This, in turn, leads to decreased levels of key sporulation regulators (SpoIIE, σ\u003csup\u003eF\u003c/sup\u003e) and structural proteins (SpoIIM, SpoIIP), ultimately reducing sporulation efficiency. Notably, sporulation is a complex, multi-gene process, and additional factors contributing to the observed phenotype warrant further investigation to fully delineate their roles in mediating the sporulation defect of the ΔBA_3317 mutant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe membrane protein BA_3317 functions as a transporter of sporulation-related signal molecules. Its expression is temporally regulated: SpoVG acts as a repressor prior to asymmetric division (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e\u0026minus;\u0026thinsp;2\u003c/sub\u003e to \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e), whereas GerE functions as a positive regulator during late sporulation (from \u003cem\u003eT\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e onward). Ultimately, BA_3317 is incorporated as a structural component of the mature spore in \u003cem\u003eBacillus anthracis\u003c/em\u003e. Consistent with its structural role, the protein is degraded during spore germination and was correspondingly identified among downregulated proteins in germination proteomic profiles.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSporulation is a pivotal developmental process that enables \u003cem\u003eB. anthracis\u003c/em\u003e to survive adverse environments and maintain pathogenicity. In this study, we systematically characterized the function of BA_3317, a previously uncharacterized membrane protein with a QS-related AgrB domain, and identified its essential role as a signal exporter in mediating \u003cem\u003eB. anthracis\u003c/em\u003e sporulation. Our findings integrate BA_3317 into the complex regulatory network of \u003cem\u003eB. anthracis\u003c/em\u003e sporulation, revealing a novel link between QS signal transport and sporulation initiation.\u003c/p\u003e \u003cp\u003eElsewhere, the \u003cem\u003eagrB\u003c/em\u003e null mutants of \u003cem\u003eClostridium perfringens, C. acetobutylicum\u003c/em\u003e, and \u003cem\u003eC. sporogenes\u003c/em\u003e also spores formation defects, suggesting that the phenotype is controlled by QS [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The similarities between the \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e group were highlighted by a study that used the machinery from the \u003cem\u003eS. aureus\u003c/em\u003e peptide-based \u003cem\u003eagr\u003c/em\u003e QS system to engineer a synthetic QS system in \u003cem\u003eB. megaterium\u003c/em\u003es [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. BA_3317 contains an AgrB domain, which was identified as an \u003cem\u003eS. aureus\u003c/em\u003e QS system member [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Analysis of the published \u003cem\u003eB. anthracis\u003c/em\u003e genomes show that they contain homologs of the \u003cem\u003estaphylococcal agr\u003c/em\u003e quorum-sensing system (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e, Supplementary Materials). The decreased sporulation efficiency phenotype of the ΔBA_3317 mutant was restored to some extent by medium exchange between ΔBA_3317 and A16R and the addition of supernatants from the A16R following precipitation by fractionated ammonium sulfate at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe most salient finding is that BA_3317 acts as a signal transporter essential for the asymmetric cell division stage-a critical early checkpoint of sporulation. Asymmetric cell division is tightly controlled by a cascade of transcriptional regulators and signaling molecules, and any disruption at this stage directly blocks subsequent sporulation events[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. We further demonstrated that BA_3317 modulates the transcription of \u003cem\u003espoIIE\u003c/em\u003e, a key sporulation-specific gene essential for asymmetric division [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Similarly, the B. \u003cem\u003eanthracis spoIIE\u003c/em\u003e mutant was delayed in polar division and failed to activate σ\u003csup\u003eF\u003c/sup\u003e in the forespore and σ\u003csup\u003eE\u003c/sup\u003e in the mother cell. Notably, the \u003cem\u003espoIIE\u003c/em\u003e promoter in ΔBA_3317 cells were only activated when adjacent to wild-type colonies, implying that BA_3317 mediates the export of a diffusible sporulation signal that is required for \u003cem\u003espoIIE\u003c/em\u003e expression. This observation aligns with secretion exchange experiments showing that loss of BA_3317 impairs the secretion of sporulation-essential factors, collectively supporting the hypothesis that BA_3317 functions as a sporulation signal transporter.\u003c/p\u003e \u003cp\u003eThe functional link between BA_3317 and the \u003cem\u003eplcR-papR\u003c/em\u003e QS system further reinforces its role in QS signal transport. Our results indicates that BA_3317 is required for the outward transport of the PapR peptide: without BA_3317, the PapR peptide cannot be secreted to activate downstream QS signaling, leading to the loss of lecithinase activity.\u003c/p\u003e \u003cp\u003eDespite these significant findings, several limitations of this study should be acknowledged. First, the specific sporulation signal exported by BA_3317 remain to be identified; Second, many other genes also contribute to the complicated sporulation process and should be examined in future studies to determine their roles in the sporulation phenotype. However, based on our current findings and what is already known in the model species \u003cem\u003eB. subtilis\u003c/em\u003e, we could propose a multiple-factor model for the role played by BA_3317 in sporulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The absence of BA_3317 may block the secretion of sporulation signal molecules, thereby preventing its extracellular maturation and re-importation and Spo0A phosphorylation subsequently. This would in turn result in reduced levels of the key SpoIIE, σ\u003csup\u003eF\u003c/sup\u003e, SpoIIP, and SpoIIM sporulation proteins, thereby affecting sporulation efficiency. As a critical mediator of sporulation initiation via QS signal export, BA_3317 represents a novel potential target for anti-anthrax interventions. Inhibiting BA_3317 function could block the export of QS signals, disrupt sporulation, and thereby reduce the environmental persistence and infectivity of \u003cem\u003eB. anthracis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, our study identifies BA_3317 as a novel sporulation signal exporter that is critical for \u003cem\u003eB. anthracis\u003c/em\u003e sporulation. BA_3317 mediates the export of QS signals (including the PapR peptide), regulates the transcription of key sporulation genes (e.g., \u003cem\u003espoIIE\u003c/em\u003e), and is tightly regulated by the canonical sporulation regulators SpoVG and GerE. These findings advance our understanding of the species-specific molecular mechanisms linking QS and sporulation in \u003cem\u003eB. anthracis\u003c/em\u003e and provide a novel target for anthrax prevention and control. Future studies focusing on the direct transport activity of BA_3317 and its interacting partners will further deepen our knowledge of this regulatory pathway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. S1-S6; Table S1-S6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge financial support for National Natural Science Foundation of China (grant numbers: 82102412,82172317) and funded by the State Key Laboratory of Pathogen and Biosecurity (Academy of Military Medical Science, SKLPBS2419).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. L., D. W., X. L. and H. W. investigation; Y. L., D. W., J. J. and M. F. methodology; Y. L., D. W., and M. C. formal analysis; Y. L., D. W., X. L. and H. W. conceptualization; C. P., Y. G. and S. Y. visualization; L. Z. validation; Y. L. and D. W. writing\u0026ndash;original draft; X. L. and H. W. writing\u0026ndash;review and editing; M.C, M. G., Y. L. and D. W. data curation; X. L. and H. W. supervision; Y. L. and D. W. funding acquisition; H. W. project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; The authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOriginal microscopic images are available upon request. All data generated or analyzed during this study are included in this published article and its supplementary files, or available upon request. Source data are provided with this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiment\u0026nbsp;was\u0026nbsp;approved by the Animal Care and Use Committee of the Academy of Military Sciences (IACUC-DWZX-2021-037).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eErrington J: \u003cstrong\u003eRegulation of endospore formation in Bacillus subtilis\u003c/strong\u003e. \u003cem\u003eNature Reviews Microbiology \u003c/em\u003e2003, \u003cstrong\u003e1\u003c/strong\u003e(2):117.\u003c/li\u003e\n\u003cli\u003eDehghani B, Rodrigues CDA: \u003cstrong\u003eSpoIIQ-dependent 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sporulation\u003c/strong\u003e. \u003cem\u003eFuture Microbiol \u003c/em\u003e2019, \u003cstrong\u003e14\u003c/strong\u003e:353-363.\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez-Guadiana FH, Brogan AP, Yu Y, Midonet C, Sher JW, Schmid EW, Roney IJ, Rudner DZ: \u003cstrong\u003eIdentification of sporulation genes in Bacillus anthracis highlights similarities and significant differences with Bacillus subtilis\u003c/strong\u003e. \u003cem\u003ePLoS Biol \u003c/em\u003e2025, \u003cstrong\u003e23\u003c/strong\u003e(12):e3003521.\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":"Bacillus anthracis, transporter, sporulation, asymmetric cell division, spoIIE, Quorum-sensing","lastPublishedDoi":"10.21203/rs.3.rs-8551494/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8551494/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBacillus anthracis\u003c/em\u003e forms dormant spores that constitute the primary infectious agent of anthrax. BA_3317, a membrane protein harboring a quorum-sensing (QS)-related AgrB domain, is essential for sporulation in \u003cem\u003eB. anthracis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe constructed an in-frame deletion mutant of BA_3317 in \u003cem\u003eB. anthracis\u003c/em\u003e vaccine strain A16R. Sporulation efficiency was quantified, and mutant morphology was observed via confocal microscopy. To investigate the role of BA_3317 in spore germination, we performed secretion exchange experiments between A16R and ΔBA_3317 at \u003cem\u003eT\u003c/em\u003e\u003csub\u003e0.5\u003c/sub\u003e, analyzed the transcriptional activity of \u003cem\u003espoIIE\u003c/em\u003e, and determined lecithinase activity after activating the \u003cem\u003eplcR-papR\u003c/em\u003e QS system. Additionally, we identified the upstream regulators of BA_3317 using in vitro promoter pull-down assays.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDeletion of BA_3317 severely reduced sporulation efficiency and ΔBA_3317 mutant was partially arrested at the asymmetric cell division stage. Cells secretion exchange experiments and spatial reporter assays revealed that BA_3317 exports a signal molecule required for sporulation, and its loss downregulated key sporulation gene \u003cem\u003espoIIE\u003c/em\u003e. The mutant also lacked lecithinase activity via the ectopically activated the \u003cem\u003eplcR-papR\u003c/em\u003e QS system, which was restored by adding the PapR heptapeptide, indicating BA_3317 mediates peptide signal transport. BA_3317 expression is repressed by SpoVG prior to asymmetric division and positively regulated by GerE during late sporulation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings identify BA_3317 as a critical regulator of \u003cem\u003eB. anthracis\u003c/em\u003e sporulation that functions as an exporter of sporulation signaling molecules. This study advances understanding of species-specific sporulation mechanisms in \u003cem\u003eB. anthracis\u003c/em\u003e and provides a potential target for anthrax prevention and control.\u003c/p\u003e","manuscriptTitle":"Disruption of Protein BA_3317 Affects Asymmetric Division as an Exporter of Sporulation Signal Molecules in Bacillus anthracis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 16:31:53","doi":"10.21203/rs.3.rs-8551494/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":"3dcd9246-7f57-41d6-8e24-ee2efd99a92e","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-04T12:27:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 16:31:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8551494","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8551494","identity":"rs-8551494","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}