Journal of Bacteriology最新文献

{"title":"DNA repair is essential for <i>Vibrio cholerae</i> growth on thiosulfate-citrate-bile salts-sucrose (TCBS) medium.","authors":"Alex J Wessel, Drew T T Johnson, Christopher M Waters","doi":"10.1128/jb.00004-25","DOIUrl":"10.1128/jb.00004-25","url":null,"abstract":"<p><p>Thiosulfate-citrate-bile salts-sucrose (TCBS) agar is a selective and differential media for the enrichment of pathogenic <i>Vibrios</i>. We observed that an exonuclease VII (<i>exoVII</i>) mutant of <i>Vibrio cholerae</i> failed to grow on TCBS agar, suggesting that DNA repair mutant strains may be hampered for growth in this selective media. Examination of the selective components of TCBS revealed that bile acids were primarily responsible for the toxicity of the <i>exoVII</i> mutant. Suppressor mutations in DNA gyrase restored growth of the <i>exoVII</i> mutants on TCBS, suggesting that TCBS inhibits DNA gyrase similar to the antibiotic ciprofloxacin. To better understand what factors are important for <i>V. cholerae</i> to grow on TCBS, we generated a randomly barcoded TnSeq (RB-TnSeq) library in <i>V. cholerae</i> and have used it to uncover a range of DNA repair mutants that also fail to grow on TCBS agar. The results of this study suggest that TCBS agar causes DNA damage to <i>V. cholerae</i> similarly to the mechanism of action of fluoroquinolones, and overcoming this DNA damage is critical for <i>Vibrio</i> growth on this selective medium.IMPORTANCETCBS is often used to diagnose cholera infection. We found that many mutant <i>V. cholerae</i> strains are attenuated for growth on TCBS agar, meaning they could remain undetected using this culture-dependent method. Hypermutator strains with defects in DNA repair pathways might be especially inhibited by TCBS. In addition, <i>V. cholerae</i> grown successively on TCBS agar develops resistance to ciprofloxacin.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0000425"},"PeriodicalIF":2.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143709751","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of a novel phage depolymerase against ST11 K64 carbapenem-resistant <i>Klebsiella pneumoniae</i> and its therapeutic potential.","authors":"Peini Yang, Bin Shan, Xing Hu, Li Xue, Guibo Song, Pingan He, Xu Yang","doi":"10.1128/jb.00387-24","DOIUrl":"https://doi.org/10.1128/jb.00387-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Carbapenem-resistant &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; (CRKP) is a clinical pathogen with a high mortality rate, and its clinical management and infection control have become a serious challenge. Phage-encoded depolymerase cleaves the capsular polysaccharide, a major virulence factor of &lt;i&gt;K. pneumoniae&lt;/i&gt;. This study aimed to identify a phage depolymerase targeting ST11 K64 CRKP, evaluate its antimicrobial activity and therapeutic efficacy, and provide new alternative therapeutic strategies for K64 CRKP. Phages were screened from untreated hospital sewage using clinically isolated CRKP as the host bacterium. The host range, efficiency of plaque formation, optimal multiplicity of infection, adsorption efficiency, and one-step growth curve of phage vB_KpnP_IME1309 were determined by the double-layer agar plate culture method. The morphology of the phage was observed by transmission electron microscopy. Phage nucleic acids were extracted for whole-genome sequencing, and the phage-encoded depolymerase gene ORF37 was amplified by polymerase chain reaction. Next, a recombinant plasmid was constructed to induce depolymerase expression, which was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. &lt;i&gt;In vitro&lt;/i&gt; bactericidal activity was determined using a combined serum assay, and the anti-&lt;i&gt;K&lt;/i&gt;. &lt;i&gt;pneumoniae&lt;/i&gt; biofilm effect of depolymerase was determined by crystal violet staining. Finally, a &lt;i&gt;Galleria mellonella&lt;/i&gt; larvae infection model was established to investigate the therapeutic effect of depolymerase on larvae &lt;i&gt;in vivo&lt;/i&gt;. Here, we isolated and characterized a phage vB_KpnP_IME1309 targeting ST11 K64 CRKP, which featured a latent period of 20 min and a burst size of approximately 290 plaque-forming units/cell. It contained 41 predicted open reading frames, of which ORF37 encoded depolymerase. The expressed and purified depolymerase Dep37 cleaved only ST11 K64 CRKP and formed a translucent halo on the agar plate. Dep37 increased the susceptibility of &lt;i&gt;K. pneumoniae&lt;/i&gt; B1 to serum killing, inhibited CRKP biofilm formation, and degraded mature biofilms. The combination of Dep37 and kanamycin was significantly more effective in treating CRKP biofilms compared to either Dep37 or kanamycin alone. An injection of Dep37 at 5 min and 2 h after the CRKP infection of &lt;i&gt;Galleria mellonella&lt;/i&gt; larvae increased their survival rates by up to 73% and 53%, respectively. Depolymerase Dep37 may be used as a potential method for capsule typing of &lt;i&gt;K. pneumoniae&lt;/i&gt;, showing great promise for the development of novel alternative therapeutic strategies against ST11 K64 CRKP.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;A novel phage vB_KpnP_IME1309 targeting ST11 K64 carbapenem-resistant &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; (CRKP) was isolated and characterized. The ORF37 encoding depolymerase gene of phage vB_KpnP_IME1309 was successfully expressed and purified. Depolymerase increases the susceptibility of CRKP to serum killing, inhibits ","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0038724"},"PeriodicalIF":2.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143709844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The <i>Streptococcus pyogenes</i> mannose phosphotransferase system (Man-PTS) influences antimicrobial activity and niche-specific nasopharyngeal infection.","authors":"Amanda C Marple, Blake A Shannon, Aanchal Rishi, Lana Estafanos, Brent D Armstrong, Veronica Guariglia-Oropeza, Stephen W Tuffs, John K McCormick","doi":"10.1128/jb.00492-24","DOIUrl":"https://doi.org/10.1128/jb.00492-24","url":null,"abstract":"<p><p><i>Streptococcus pyogenes</i> is a human-adapted pathogen that can cause multiple diseases, including pharyngitis and skin infections. Although this bacterium produces many virulence factors, how <i>S. pyogenes</i> competes with the host microbiota is not well understood. Here, we detected antimicrobial activity from <i>S. pyogenes</i> MGAS8232 that prevented the growth of <i>Micrococcus luteus</i>. This activity was produced when cells were grown in 5% CO₂ in M17 media supplemented with galactose; however, the addition of alternative sugars coupled with genome sequencing experiments revealed that the antimicrobial phenotype was not related to classical bacteriocins. To further determine genes involved in the production of this activity, a transposon mutant library in <i>S. pyogenes</i> MGAS8232 identified the mannose phosphotransferase system (Man-PTS), a major sugar transporter, as important for the antimicrobial phenotype. Loss-of-function transposon mutants linked to the antimicrobial activity were identified to also be involved in alternative sugar utilization, and additionally, the Man-PTS was further identified from an inadvertent secondary mutation in a bacteriocin operon mutant. Sugar utilization in the Man-PTS mutants demonstrated that galactose, mannose, and N-acetylglucosamine utilization was impaired. RNA-seq experiments in high and low glucose concentrations further characterized the Man-PTS as a glucose transporter; however, transcriptional regulators or virulence factors were not affected with the loss of the Man-PTS. Deletion of Man-PTS demonstrated defects in a mouse model of nasopharyngeal infection but not skin infection. This work suggests that the ability of <i>S. pyogenes</i> to utilize alternative sugars presented by glycans may play a role in acute infection and interactions with the endogenous microbial population existing in the nasopharynx.IMPORTANCE<i>Streptococcus pyogenes</i> is responsible for over 500,000 deaths per year primarily due to invasive infections and post-infection sequelae, although the most common manifestations include pharyngitis and impetigo. <i>S. pyogenes</i> can adapt to its environment through alternative sugar metabolism. Here, we identified an antimicrobial phenotype that was not bacteriocin-related but a by-product of alternative sugar metabolism. The mannose phosphotransferase system was involved in the production of the antimicrobial and was also important for <i>S. pyogenes</i> to utilize alternative sugars and establish nasopharyngeal infection but not skin infection. Overall, this study identified potential strategies used by <i>S. pyogenes</i> for interactions with the endogenous microbiota and further elucidated the importance of sugar metabolism in acute upper respiratory tract infection.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0049224"},"PeriodicalIF":2.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143709846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Calprotectin elicits aberrant iron starvation responses in <i>Pseudomonas aeruginosa</i> under anaerobic conditions.","authors":"Jacob M Weiner, Wei Hao Lee, Elizabeth M Nolan, Amanda G Oglesby","doi":"10.1128/jb.00029-25","DOIUrl":"10.1128/jb.00029-25","url":null,"abstract":"<p><p><i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that uses several mechanisms to survive in the iron-limiting host environment. The innate immune protein calprotectin (CP) sequesters ferrous iron [Fe(II)], among other divalent transition metal ions, to limit its availability to pathogens. CP levels are increased in individuals with cystic fibrosis (CF), a hereditary disease that leads to chronic pulmonary infection by <i>P. aeruginosa</i>. We previously showed that aerobic CP treatment of <i>P. aeruginosa</i> induces a multi-metal starvation response that alters expression of several virulence properties. However, the CF lung is a hypoxic environment due to the growth of <i>P. aeruginosa</i> in dense biofilms. Here, we report that anaerobic CP treatment of <i>P. aeruginosa</i> induces many processes associated with an aerobic iron starvation response, including decreased phenazine production and increased expression of the PrrF small regulatory RNAs (sRNAs). However, the iron starvation response elicited by CP in anaerobic conditions shows characteristics that are distinct from responses observed in aerobic growth, including a lack of siderophore production and increased induction of genes for the FeoAB Fe(II) and Phu heme uptake systems. Also distinct from aerobic conditions, CP treatment induces expression of genes for predicted manganese transporters MntH1 and MntH2 during anaerobic growth while eliciting a less robust zinc starvation response compared to aerobic conditions. Induction of <i>mntH2</i> is dependent on the PrrF sRNAs, suggesting a novel example of metal regulatory cross-talk. Thus, anaerobic CP treatment results in a multi-metal starvation response with key distinctions from aerobic conditions, revealing differences in <i>P. aeruginosa</i> metal homeostasis during anaerobic growth.IMPORTANCEIron is critical for most microbial pathogens, and the innate immune system sequesters this metal to limit microbial growth. Pathogens must overcome iron sequestration to survive during infection. For many pathogens, iron homeostasis has primarily been studied in aerobic conditions. Nevertheless, some host environments are hypoxic, including chronic lung infection sites in individuals with cystic fibrosis (CF). Here, we use the innate immune protein calprotectin, which sequesters divalent metal ions including Fe(II), to study the anaerobic iron starvation response of a common CF lung pathogen, <i>Pseudomonas aeruginosa</i>. We report several distinctions of this response during anaerobiosis, highlighting the importance of carefully considering the host environment when investigating the role of nutritional immunity in host-pathogen interactions.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0002925"},"PeriodicalIF":2.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143709632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A SecA-associated protease modulates the extent of surface display of staphylococcal protein A.","authors":"Muhammad S Azam, Amany M Ibrahim, Owen Leddy, So-Young Oh, Olaf Schneewind, Dominique Missiakas","doi":"10.1128/jb.00522-24","DOIUrl":"10.1128/jb.00522-24","url":null,"abstract":"<p><p>In bacteria, signal peptides direct pre-proteins to the SecYEG secretion channel and are typically cleaved by signal peptidases during translocation across the membrane. In gram-positive bacteria, such as <i>Staphylococcus aureus</i>, some signal peptides have a pre-translocation function. Staphylococcal protein A (SpA) carries such an atypical signal sequence, with a YSIRK/GXXS motif that directs its precursor into the cross-wall of dividing cells for subsequent anchoring by sortase A. Here, we report that PepV-a member of the M20 peptidase family which has been described as a manganese-dependent dipeptidase <i>in vitro</i>-may influence the surface display of precursors with a YSIRK/GXXS motif. SpA deposition into cross-walls was increased in Δ<i>pepV</i> bacteria. Yet, in the absence of <i>pepV</i>, neither the kinetics of signal sequence processing nor the final product of the sorting reaction was altered. In pull-down experiments, PepV was identified as a ligand of SecA. When purified PepV was incubated with SpA precursors, this interaction triggered self-cleavage of the enzyme, an unexpected activity exacerbated by the presence of a chelating agent. In agreement with this finding, a pulse-chase experiment revealed that the half-life of PepV is extended in bacteria lacking <i>spa</i>. Collectively, these data reveal a mutually inhibitory relationship between SpA precursors and PepV, the net result suggesting that while PepV may reduce the surface display of SpA, SpA precursors destabilize PepV possibly to overcome such inhibition.</p><p><strong>Importance: </strong>The \"signal hypothesis\" proposed that N-terminal sequences of secretory proteins contain targeting cues directing nascent polypeptides to the endoplasmic reticulum. This concept was later confirmed as broadly applicable, even to prokaryotes with a single membrane. In gram-positive bacteria, signal sequences bearing the YSIRK/GXXS motif are necessary and sufficient to direct precursors to septal membranes. However, trans-acting factors involved in this spatially restricted targeting remain largely unknown. Here, we identify a member of the M20 metalloprotease family as a potential contributor to the septal surface display of proteins containing YSIRK/GXXS signal peptides.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0052224"},"PeriodicalIF":2.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143709677","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Mycobacterium tuberculosis</i> FtsB and PerM interact via a C-terminal helix in FtsB to modulate cell division.","authors":"João Ramalheira Ferreira, Ruilan Xu, Zach Hensel","doi":"10.1128/jb.00444-24","DOIUrl":"https://doi.org/10.1128/jb.00444-24","url":null,"abstract":"<p><p>Latent infection by <i>Mycobacterium tuberculosis</i> (Mtb) impedes effective tuberculosis therapy and eradication. The protein PerM is essential for chronic Mtb infections in mice and acts via the divisome protein FtsB to modulate cell division. Using transgenic co-expression in <i>Escherichia coli</i>, we studied the Mtb PerM-FtsB interaction in isolation from other Mtb proteins, engineering PerM to enhance expression in the <i>E. coli</i> membrane. Using fluorescence microscopy in <i>E. coli</i>, we observed that the previously reported PerM-dependent instability of Mtb FtsB required a segment of FtsB predicted to bind cell-division proteins FtsL and FtsQ. Furthermore, we found that the stability of membrane-localized PerM hinged on its interaction with a conserved, C-terminal helix in FtsB. We also observed that removing this helix disrupted PerM-FtsB interaction using single-molecule tracking. Molecular dynamics results supported the observation that FtsB stabilized PerM and suggested that interactions at the PerM-FtsB interface differ from our initial structure prediction in a way that is consistent with PerM sequence conservation. Although narrowly conserved, the PerM-FtsB interaction emerges as a potential therapeutic target for persistent infections by disrupting the regulation of cell division. Integrating protein structure prediction, molecular dynamics, and single-molecule microscopy, our approach is primed to screen potential inhibitors of the PerM-FtsB interaction and can be straightforwardly adapted to explore other putative interactions.IMPORTANCEOur research reveals significant insights into the dynamic interaction between the proteins PerM and FtsB within <i>Mycobacterium tuberculosis</i>, contributing to our understanding of bacterial cell division mechanisms crucial for infection persistence. By combining innovative fluorescence microscopy and molecular dynamics, we established that the stability of these proteins is interdependent; molecular dynamics placing PerM-FtsB in the context of the mycobacterial divisome shows how disrupting PerM-FtsB interactions can plausibly impact bacterial cell wall synthesis. These findings highlight the PerM-FtsB interface as a promising target for novel therapeutics aimed at combating persistent bacterial infections. Importantly, our approach can be adapted for similar studies in other bacterial systems, suggesting broad implications for microbial biology and antibiotic development.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0044424"},"PeriodicalIF":2.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143709660","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interplay of virulence factors and signaling molecules: albumin and calcium-mediated biofilm regulation in <i>Bordetella bronchiseptica</i>.","authors":"Sabrina Laura Mugni, Nicolás Ambrosis, George A O Toole, Federico Sisti, Julieta Fernández","doi":"10.1128/jb.00445-24","DOIUrl":"https://doi.org/10.1128/jb.00445-24","url":null,"abstract":"<p><p><i>Bordetella bronchiseptica,</i> a respiratory pathogen capable of infecting various mammals, including humans, is associated with chronic infections. <i>B. bronchiseptica</i> can form biofilm-like structures <i>in vivo</i>, providing tolerance against environmental stresses. Recent studies have highlighted the role of cyclic diguanylate monophosphate (c-di-GMP) in this process <i>in vitro</i>: elevated c-di-GMP levels stimulate biofilm formation, whereas phosphodiesterase (PDE) activation reduces biofilms. Respiratory secretions, which contain albumin and calcium at higher concentrations than standard growth media, promote an increase in the amount and extracellular localization of the adenylate cyclase toxin (ACT), an important virulence factor of <i>Bordetella spp</i>. Secreted ACT, present in the extracellular medium or attached to the outer membrane, inhibits biofilm formation. Based on these observations, we hypothesized that serum albumin and calcium together inhibit biofilm formation and explored the potential role of c-di-GMP in this process. Our findings suggest that serum albumin and calcium inhibit <i>B. bronchiseptica</i> biofilm formation through two potentially independent mechanisms: one involving ACT secretion and another promoting c-di-GMP degradation. In the presence of albumin and calcium, intracellular levels of c-di-GMP were reduced, and specific PDEs appear to be involved in this process. In addition, albumin and calcium stimulated the secretion of the adhesin BrtA. This study contributes to the understanding of the mechanisms governing <i>B. bronchiseptica</i> biofilm formation and its modulation by host factors.IMPORTANCE<i>Bordetella bronchiseptica</i>, a respiratory pathogen capable of infecting various mammals, forms biofilms that enhance its ability to withstand environmental stresses. This study reveals that host-derived factors, specifically serum albumin and calcium, inhibit biofilm formation through two independent mechanisms: increasing adenylate cyclase toxin secretion and promoting the degradation of cyclic diguanylate monophosphate (c-di-GMP), a key biofilm regulator. These findings provide insights into how host conditions influence <i>B. bronchiseptica</i> biofilm dynamics, shedding light on the complex interactions between pathogen and host that contribute to infection persistence. Understanding these mechanisms may inform strategies to mitigate chronic infections caused by <i>B. bronchiseptica</i>.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0044524"},"PeriodicalIF":2.7,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143709845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A platform supporting generation and isolation of random transposon mutants in <i>Chlamydia trachomatis</i>.","authors":"Caroline Hawk, Nur Hamdzah, Zoe Dimond, Kenneth A Fields","doi":"10.1128/jb.00500-24","DOIUrl":"10.1128/jb.00500-24","url":null,"abstract":"<p><p><i>Chlamydia</i> species represent a paradigm for understanding successful obligate intracellular parasitism. Despite limited genetic malleability, development of genetic tools has facilitated the elucidation of molecular mechanisms governing infectivity. Random mutagenesis approaches provide one of the most powerful strategies available to accomplish untargeted elucidation of gene function. Unfortunately, initial progress in transposon-mediated mutagenesis of <i>Chlamydia</i> has been challenging. To increase efficiency, we developed a plasmid-based system that couples conditional plasmid maintenance with a previously described strategy leveraging inducible expression of the <i>Himar1</i>-derived C9 transposase. Our pOri-Tn(Q) construct was maintained in <i>Chlamydia trachomatis</i> cultivated with antibiotics but was rapidly cured in the absence of antibiotic selection. pOri-Tn(Q) supported transposition events when transposase expression was induced during infection. Induction was accompanied by loss of the plasmid backbone when penicillin G was used to select for only the transposable element. C9 induction during iterative passaging was used to increase the overall insertion frequency and accumulate an expanded pool of transposon mutants. The approach supported isolation of individual mutant strains from the mixed pool, and whole-genome sequencing confirmed that the recovered strains harbored single insertions.IMPORTANCE<i>Chlamydia trachomatis</i> is a prevalent human pathogen exerting a tremendous negative impact on human health. A complete understanding of how these bacteria create and maintain an intracellular niche and avoid/subvert host defense mechanisms to cause disease is lacking. The utility of transposon-mediated, random mutagenesis in supporting forward genetic studies is well established in a multitude of genetically tractable systems. This study reports the development of a plasmid-based system capable of generating mutant pools and supporting subsequent isolation of individual transposon mutants. This step is an important advance in providing a mechanism capable of supporting downstream studies interrogating chlamydial biology.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0050024"},"PeriodicalIF":2.7,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11925237/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143414280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Chlamydia trachomatis</i>: a model for intracellular bacterial parasitism.","authors":"Erin P Smith, Raphael H Valdivia","doi":"10.1128/jb.00361-24","DOIUrl":"10.1128/jb.00361-24","url":null,"abstract":"<p><p><i>Chlamydia</i> comprises a diverse group of obligate intracellular bacteria that cause infections in animals, including humans. These organisms share fascinating biology, including distinct developmental stages, non-canonical cell surface structures, and adaptations to intracellular parasitism. <i>Chlamydia trachomatis</i> is of particular interest due to its significant clinical importance, causing both ocular and sexually transmitted infections. The strain L2/434/Bu, responsible for lymphogranuloma venereum, is the most common strain used to study chlamydial molecular and cell biology because it grows readily in cell culture and is amenable to genetic manipulation. Indeed, this strain has enabled researchers to tackle fundamental questions about the molecular mechanisms underlying <i>Chlamydia's</i> developmental transitions and biphasic lifecycle and cellular adaptations to obligate intracellular parasitism, including characterizing numerous conserved virulence genes and defining immune responses. However, L2/434/Bu is not representative of <i>C. trachomatis</i> strains that cause urogenital infections in humans, limiting its utility in addressing questions of host tropism and immune evasion in reproductive organs. Recent research efforts are shifting toward understanding the unique attributes of more clinically relevant <i>C. trachomatis</i> genovars.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0036124"},"PeriodicalIF":2.7,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11925236/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143458087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rho-dependent termination and RNase E-mediated cleavage: dual pathways for RNA 3' end processing in polycistronic mRNA.","authors":"Heung Jin Jeon, Monford Paul Abishek N, Xun Wang, Heon M Lim","doi":"10.1128/jb.00437-24","DOIUrl":"10.1128/jb.00437-24","url":null,"abstract":"<p><p>\"Pre-full-length\" transcripts are produced at the end of the polycistronic galactose (<i>gal</i>) operon, 5' <i>galE-galT-galK-galM 3'</i>, via Rho-dependent transcription termination (RDT) and -independent transcription termination. The 3' end of the full-length <i>galETKM</i> mRNA is acquired by exonucleolytic processing of the 3'-OH ends of the pre-full-length transcripts. However, the <i>gal</i> operon produces an mRNA termed <i>galE</i> whose 3' end forms approximately 120 nucleotides downstream of the <i>galE</i> stop codon, within the subsequent gene, <i>galT</i>, thereby establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3' end of <i>galE</i> mRNA. We discovered that the 3' ends of pre-galE mRNA are produced in the middle of <i>galT</i> as a result of the combination of two separate molecular processes-one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3' ends of pre-<i>galE</i> mRNA undergo exonucleolytic processing to the 3' end of <i>galE</i> mRNA observed <i>in vivo</i>. A hairpin structure containing an 8 bp stem and a 4-nucleotide loop, located 5-10 nucleotides upstream of the 3' ends of <i>galE</i> mRNA, blocks exoribonuclease digestion and renders transcript stability. These findings demonstrate that RNase E-contrary to its general role in mRNA degradation-produces RNA 3' ends that regulate polarity in gene expression.IMPORTANCEThis study reports the findings of two molecular mechanisms that generate the 3' ends of pre-<i>galE</i> mRNA in the <i>gal</i> operon, viz., Rho-dependent transcription termination and RNase E-mediated cleavage. These 3' ends are subsequently processed to produce stable <i>galE</i> mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3' end of <i>galE</i> mRNA within <i>galT</i> in contrast to the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0043724"},"PeriodicalIF":2.7,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11925234/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143515652","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}