Journal of Bacteriology最新文献

{"title":"In memoriam: Pierre Cornelis (1949-2023).","authors":"Jean-Paul Pirnay, Françoise Van Bambeke, Christine Baysse, Miguel Cámara, Sylvie Chevalier, Jean François Collet, Aurélie Crabbé, Jozef Dingemans, Alain Dufour, Linda Eeckhaudt, Alain Filloux, Olivier Lesouhaitier, Qing Wei, Daniel De Vos","doi":"10.1128/jb.00179-24","DOIUrl":"10.1128/jb.00179-24","url":null,"abstract":"","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":"206 7","pages":"e0017924"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11270859/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141758838","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":"An insight into the role of branched-chain α-keto acid dehydrogenase (BKD) complex in branched-chain fatty acid biosynthesis and virulence of <i>Listeria monocytogenes</i>.","authors":"Q M Monzur Kader Chowdhury, Shamima Islam, Lakshmi Narayanan, Seto C Ogunleye, Shangshang Wang, Dinh Thu, Nancy E Freitag, Mark L Lawrence, Hossam Abdelhamed","doi":"10.1128/jb.00033-24","DOIUrl":"10.1128/jb.00033-24","url":null,"abstract":"<p><p><i>Listeria monocytogenes</i> is a foodborne bacterial pathogen that causes listeriosis. Positive regulatory factor A (PrfA) is a pleiotropic master activator of virulence genes of <i>L. monocytogenes</i> that becomes active upon the entry of the bacterium into the cytosol of infected cells. <i>L. monocytogenes</i> can survive and multiply at low temperatures; this is accomplished through the maintenance of appropriate membrane fluidity via branched-chain fatty acid (BCFA) synthesis. Branched-chain α-keto acid dehydrogenase (BKD), which is composed of four polypeptides encoded by <i>lpd</i>, <i>bkdA1</i>, <i>bkdA2</i>, and <i>bkdB</i>, is known to play a vital role in BCFA biosynthesis. Here, we constructed BKD-deficient <i>Listeria</i> strains by in-frame deletion of <i>lpd</i>, <i>bkdA1</i>, <i>bkdA2</i>, and <i>bkdB</i> genes. To determine the role in <i>in vivo</i> and <i>in vitro</i>, mouse model challenges, plaque assay in murine L2 fibroblast, and intracellular replication in J744A.1 macrophage were conducted. BKD-deficient strains exhibited defects in BCFA composition, virulence, and PrfA-regulon function within the host cells. Transcriptomics analysis revealed that the transcript level of the PrfA-regulon was lower in Δ<i>bkdA1</i> strain than those in the wild-type. This study demonstrates that <i>L. monocytogenes</i> strains lacking BKD complex components were defective in PrfA-regulon function, and full activation of wild-type <i>prfA</i> may not occur within host cells in the absence of BKD. Further study will investigate the consequences of BKD deletion on PrfA function through altering BCFA catabolism.IMPORTANCE<i>Listeria monocytogenes</i> is the causative agent of listeriosis, a disease with a high mortality rate. In this study, we have shown that the deletion of BKD can impact the function of PrfA and the PrfA-regulon. The production of virulence proteins within host cells is necessary for <i>L. monocytogenes</i> to promote its intracellular survival and is likely dependent on membrane integrity. We thus report a link between <i>L. monocytogenes</i> membrane integrity and the function of PrfA. This knowledge will increase our understanding of <i>L. monocytogenes</i> pathogenesis, which may provide insight into the development of antimicrobial agents.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0003324"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11270904/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141426955","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":"DarA-the central processing unit for the integration of osmotic with potassium and amino acid homeostasis in <i>Bacillus subtilis</i>.","authors":"Robert Warneke, Christina Herzberg, Martin Weiß, Thorben Schramm, Dietrich Hertel, Hannes Link, Jörg Stülke","doi":"10.1128/jb.00190-24","DOIUrl":"10.1128/jb.00190-24","url":null,"abstract":"<p><p>Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes including osmolyte uptake, cell wall homeostasis, as well as antibiotic and heat resistance. This study investigates the role of the c-di-AMP receptor protein DarA in the osmotic stress response in <i>Bacillus subtilis</i>. Through a series of experiments, we demonstrate that DarA plays a central role in the cellular response to osmotic fluctuations. Our findings show that DarA becomes essential under extreme potassium limitation as well as upon salt stress, highlighting its significance in mediating osmotic stress adaptation. Suppressor screens with <i>darA</i> mutants reveal compensatory mechanisms involving the accumulation of osmoprotectants, particularly potassium and citrulline. Mutations affecting various metabolic pathways, including the citric acid cycle as well as glutamate and arginine biosynthesis, indicate a complex interplay between the osmotic stress response and metabolic regulation. In addition, the growth defects of the <i>darA</i> mutant during potassium starvation and salt stress in a strain lacking the high-affinity potassium uptake systems KimA and KtrAB can be rescued by increased affinity of the remaining potassium channel KtrCD or by increased expression of <i>ktrD,</i> thus resulting in increased potassium uptake. Finally, the <i>darA</i> mutant can respond to salt stress by the increased expression of MleN , which can export sodium ions.IMPORTANCEEnvironmental bacteria are exposed to rapidly changing osmotic conditions making an effective adaptation to these changes crucial for the survival of the cells. In Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in this adaptation by controlling (i) the influx of physiologically compatible organic osmolytes and (ii) the biosynthesis of such osmolytes. In several bacteria, cyclic di-adenosine monophosphate (c-di-AMP) can bind to a signal transduction protein, called DarA, in <i>Bacillus subtilis</i>. So far, no function for DarA has been discovered in any organism. We have identified osmotically challenging conditions that make DarA essential and have identified suppressor mutations that help the bacteria to adapt to those conditions. Our results indicate that DarA is a central component in the integration of osmotic stress with the synthesis of compatible amino acid osmolytes and with the homeostasis of potassium, the first response to osmotic stress.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0019024"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11270874/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141237576","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":"ComI inhibits transformation in <i>Bacillus subtilis</i> by selectively killing competent cells.","authors":"Dominique R Smith, Daniel B Kearns, Briana M Burton","doi":"10.1128/jb.00413-23","DOIUrl":"10.1128/jb.00413-23","url":null,"abstract":"<p><p>Many bacteria build elaborate molecular machines to import DNA via natural competence, yet this activity is often not identified until strains have been handled and domesticated in laboratory settings. For example, one of the best studied Gram-positive model organisms, <i>Bacillus subtilis,</i> has a poorly transformable ancestor. Transformation in the ancestral strain is inhibited by a transmembrane peptide, ComI, which is encoded on an extrachromosomal plasmid. Although ComI was shown to be necessary and sufficient to inhibit transformation when produced at high levels under an inducible promoter, the mechanism by which ComI inhibits transformation is unknown. Here, we examine the native regulation and mechanism of transformation inhibition by ComI. We find that under native regulation, ComI expression is restricted in the absence of the plasmid. In the presence of the plasmid, we find that ComI is expressed at higher levels in cells that are differentiating into a competent state. The subcellular localization of ComI, however, does not depend on any other competence proteins, and permeabilization activity is concentration-dependent. Time-lapse microscopy reveals that competent cells producing ComI are first permeabilized and then die. Based on these observations, we propose a new model for the mechanism of ComI in which response to competence activation leads to selective elimination of the competent subpopulation.</p><p><strong>Importance: </strong>Natural transformation mechanisms have been studied across several bacterial systems, but few examples of inhibition exist. This work investigates the mechanism of action of a plasmid-encoded transmembrane inhibitor of natural transformation. The data reveal that the peptide can cause cell permeabilization. Permeabilization is synergistic with entry of <i>Bacillus subtilis</i> into the \"competent\" state, such that cells with the ability to be transformed are preferentially killed. These findings reveal a self-preservation mechanism coupled to the physiological state of the cells that ensures that the population can maintain an unaltered plasmid and its predicted prophage.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0041323"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11270867/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141317425","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":"A sequence invariable region in TcdB2 is required for toxin escape from <i>Clostridioides difficile</i>.","authors":"Megan L Kempher, Tyler M Shadid, Jason L Larabee, Jimmy D Ballard","doi":"10.1128/jb.00096-24","DOIUrl":"10.1128/jb.00096-24","url":null,"abstract":"<p><p>Sequence differences among the subtypes of <i>Clostridioides difficile</i> toxin TcdB (2,366 amino acids) are broadly distributed across the entire protein, with the notable exception of 76 residues at the protein's carboxy terminus. This sequence invariable region (SIR) is identical at the DNA and protein level among the TcdB variants, suggesting this string of amino acids has undergone selective pressure to prevent alterations. The functional role of the SIR domain in TcdB has not been determined. Analysis of a recombinantly constructed TcdB mutant lacking the SIR domain did not identify changes in TcdB's enzymatic or cytopathic activities. To further assess the SIR region, we constructed a <i>C. difficile</i> strain with the final 228 bp deleted from the <i>tcdB</i> gene, resulting in the production of a truncated form of TcdB lacking the SIR (TcdB2_∆2291-2366). Using a combination of approaches, we found in the absence of the SIR sequence TcdB2_∆2291-2366 retained cytotoxic activity but was not secreted from <i>C. difficile</i>. TcdB2_∆2291-2366 was not released from the cell under autolytic conditions, indicating the SIR is involved in a more discrete step in toxin escape from the bacterium. Fractionation experiments combined with antibody detection found that TcdB2_∆2291-2366 accumulates at the cell membrane but is unable to complete steps in secretion beyond this point. These data suggest conservation of the SIR domain across variants of TcdB could be influenced by the sequence's role in efficient escape of the toxin from <i>C. difficile</i>.</p><p><strong>Importance: </strong><i>Clostridioides difficile</i> is a leading cause of antibiotic associated disease in the United States. The primary virulence factors produced by <i>C. difficile</i> are two large glucosylating toxins TcdA and TcdB. To date, several sequence variants of TcdB have been identified that differ in various functional properties. Here, we identified a highly conserved region among TcdB subtypes that is required for release of the toxin from <i>C. difficile</i>. This study reveals a putative role for the longest stretch of invariable sequence among TcdB subtypes and provides new details regarding toxin release into the extracellular environment. Improving our understanding of the functional roles of the conserved regions of TcdB variants aids in the development of new, broadly applicable strategies to treat CDI.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0009624"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11323933/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141419292","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":"Guarding the walls: the multifaceted roles of Bce modules in cell envelope stress sensing and antimicrobial resistance.","authors":"Natasha L George, Ellen C Bennett, Benjamin J Orlando","doi":"10.1128/jb.00123-24","DOIUrl":"10.1128/jb.00123-24","url":null,"abstract":"<p><p>Bacteria have developed diverse strategies for defending their cell envelopes from external threats. In Firmicutes, one widespread strategy is to use Bce modules-membrane protein complexes that unite a peptide-detoxifying ABC transporter with a stress response coordinating two-component system. These modules provide specific, front-line defense for a wide variety of antimicrobial peptides and small molecule antibiotics as well as coordinate responses for heat, acid, and oxidative stress. Because of these abilities, Bce modules play important roles in virulence and the development of antibiotic resistance in a variety of pathogens, including <i>Staphylococcus</i>, <i>Streptococcus</i>, and <i>Enterococcus</i> species. Despite their importance, Bce modules are still poorly understood, with scattered functional data in only a small number of species. In this review, we will discuss Bce module structure in light of recent cryo-electron microscopy structures of the <i>B. subtilis</i> BceABRS module and explore the common threads and variations-on-a-theme in Bce module mechanisms across species. We also highlight the many remaining questions about Bce module function. Understanding these multifunctional membrane complexes will enhance our understanding of bacterial stress sensing and may point toward new therapeutic targets for highly resistant pathogens.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0012324"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11270860/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141310749","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":"An expanded genetic toolkit for inducible expression and targeted gene silencing in <i>Rickettsia parkeri</i>.","authors":"Jon McGinn, Annie Wen, Desmond L Edwards, David M Brinkley, Rebecca L Lamason","doi":"10.1128/jb.00091-24","DOIUrl":"10.1128/jb.00091-24","url":null,"abstract":"<p><p>Pathogenic species within the <i>Rickettsia</i> genus are transmitted to humans through arthropod vectors and cause a spectrum of diseases ranging from mild to life-threatening. Despite rickettsiae posing an emerging global health risk, the genetic requirements of their infectious life cycles remain poorly understood. A major hurdle toward building this understanding has been the lack of efficient tools for genetic manipulation, owing to the technical difficulties associated with their obligate intracellular nature. To this end, we implemented the Tet-On system to enable conditional gene expression in <i>Rickettsia parkeri</i>. Using Tet-On, we show inducible expression of antibiotic resistance and a fluorescent reporter. We further used this inducible promoter to screen the ability of <i>R. parkeri</i> to express four variants of the catalytically dead Cas9 (dCas9). We demonstrate that all four dCas9 variants can be expressed in <i>R. parkeri</i> and used for CRISPR interference (CRISPRi)-mediated targeted gene knockdown. We show targeted knockdown of an antibiotic resistance gene as well as the endogenous virulence factor <i>sca2</i>. Altogether, we have developed systems for inducible gene expression and CRISPRi-mediated gene knockdown for the first time in rickettsiae, laying the groundwork for more scalable, targeted mechanistic investigations into their infectious life cycles.IMPORTANCEThe spotted fever group of <i>Rickettsia</i> contains vector-borne pathogenic bacteria that are neglected and emerging threats to public health. Due to the obligate intracellular nature of rickettsiae, the development of tools for genetic manipulation has been stunted, and the molecular and genetic underpinnings of their infectious lifecycle remain poorly understood. Here, we expand the genetic toolkit by introducing systems for conditional gene expression and CRISPR interference (CRISPRi)-mediated gene knockdown. These systems allow for relatively easy manipulation of rickettsial gene expression. We demonstrate the effectiveness of these tools by disrupting the intracellular life cycle using CRISPRi to deplete the <i>sca2</i> virulence factor. These tools will be crucial for building a more comprehensive and detailed understanding of rickettsial biology and pathogenesis.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0009124"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11270864/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141261550","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":"Identification of a copper-responsive small molecule inhibitor of uropathogenic <i>Escherichia coli</i>.","authors":"Braden S Hanson, Amanuel Hailemariam, Yongjian Yang, Faras Mohamed, George L Donati, Dwight Baker, James Sacchettini, James J Cai, Sargurunathan Subashchandrabose","doi":"10.1128/jb.00112-24","DOIUrl":"10.1128/jb.00112-24","url":null,"abstract":"<p><p>Urinary tract infections (UTIs) are a major global health problem and are caused predominantly by uropathogenic <i>Escherichia coli</i> (UPEC). UTIs are a leading cause of prescription antimicrobial use. Incessant increase in antimicrobial resistance in UPEC and other uropathogens poses a serious threat to the current treatment practices. Copper is an effector of nutritional immunity that impedes the growth of pathogens during infection. We hypothesized that copper would augment the toxicity of select small molecules against bacterial pathogens. We conducted a small molecule screening campaign with a library of 51,098 molecules to detect hits that inhibit a UPEC Δ<i>tolC</i> mutant in a copper-dependent manner. A molecule, denoted as <i>E. coli</i> inhibitor or ECIN, was identified as a copper-responsive inhibitor of wild-type UPEC strains. Our gene expression and metal content analysis results demonstrate that ECIN works in concert with copper to exacerbate Cu toxicity in UPEC. ECIN has a broad spectrum of activity against pathogens of medical and veterinary significance including <i>Acinetobacter baumannii, Pseudomonas aeruginosa</i>, and methicillin-resistant <i>Staphylococcus aureus</i>. Subinhibitory levels of ECIN eliminate UPEC biofilm formation. Transcriptome analysis of UPEC treated with ECIN reveals induction of multiple stress response systems. Furthermore, we demonstrate that L-cysteine rescues the growth of UPEC exposed to ECIN. In summary, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC.IMPORTANCEUrinary tract infection (UTI) is a ubiquitous infectious condition affecting millions of people annually. Uropathogenic <i>Escherichia coli</i> (UPEC) is the predominant etiological agent of UTI. However, UTIs are becoming increasingly difficult to resolve with antimicrobials due to increased antimicrobial resistance in UPEC and other uropathogens. Here, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. In addition to <i>E. coli</i>, this small molecule also inhibits pathogens of medical and veterinary significance including <i>Acinetobacter baumannii, Pseudomonas aeruginosa,</i> and methicillin-resistant <i>Staphylococcus aureus</i>.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0011224"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11270900/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141296058","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":"Non-interchangeable functions of efflux transporters of <i>Pseudomonas aeruginosa</i> in survival under infection-associated stress.","authors":"Justyna W Adamiak, Laiba Ajmal, Helen I Zgurskaya","doi":"10.1128/jb.00054-24","DOIUrl":"10.1128/jb.00054-24","url":null,"abstract":"<p><p><i>Pseudomonas aeruginosa</i> is a challenging opportunistic pathogen due to its intrinsic and acquired mechanisms of antibiotic resistance. A large repertoire of efflux transporters actively expels antibiotics, toxins, and metabolites from cells and enables growth of <i>P. aeruginosa</i> in diverse environments. In this study, we analyzed the roles of representative efflux pumps from the Resistance-Nodulation-Division (RND), Major Facilitator Superfamily (MFS), and Small Multidrug Resistance (SMR) families of proteins in the susceptibility of <i>P. aeruginosa</i> to antibiotics and bacterial growth under stresses imposed by human hosts during bacterial infections: an elevated temperature, osmotic stress, low iron, bile salts, and acidic pH. We selected five RND pumps MexAB-OprM, MexEF-OprN, MexCD-OprJ, MuxABC-OpmB, and TriABC-OpmH that differ in their substrate specificities and expression profiles, two MFS efflux pumps PA3136-3137 and PA5158-5160 renamed here into MfsAB and MfsCD-OpmG, respectively, and an SMR efflux transporter PA1540-1541 (MdtJI). We found that the most promiscuous RND pumps such as MexEF-OprN and MexAB-OprM are integrated into diverse survival mechanisms and enable <i>P. aeruginosa</i> growth under various stresses. MuxABC-OpmB and TriABC-OpmH pumps with narrower substrate spectra are beneficial only in the presence of the iron chelator 2,2'-dipyridyl and bile salts, respectively. MFS pumps do not contribute to antibiotic efflux but play orthogonal roles in acidic pH, low iron, and in the presence of bile salts. In contrast, MdtJI protects against polycationic antibiotics but does not contribute to survival under stress. Thus, efflux pumps play specific, non-interchangeable functions in <i>P. aeruginosa</i> cell physiology and bacterial survival under stresses.</p><p><strong>Importance: </strong>The role of multidrug efflux pumps in the intrinsic and clinical levels of antibiotic resistance in <i>Pseudomonas aeruginosa</i> and other gram-negative bacteria is well-established. Their functions in bacterial physiology, however, remain unclear. The <i>P. aeruginosa</i> genome comprises an arsenal of efflux pumps from different protein families, the substrate specificities of which are typically assessed by measuring their impact on susceptibility to antibiotics. In this study, we analyzed how deletions and overproductions of efflux pumps affect <i>P. aeruginosa</i> growth under human-infection-induced stresses. Our results show that the physiological functions of multidrug efflux pumps are non-redundant and essential for the survival of this important human pathogen under stress.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0005424"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11323973/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141317426","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":"Comprehensive analysis of insertion sequences within rRNA genes of CPR bacteria and biochemical characterization of a homing endonuclease encoded by these sequences.","authors":"Megumi Tsurumaki, Asako Sato, Motofumi Saito, Akio Kanai","doi":"10.1128/jb.00074-24","DOIUrl":"10.1128/jb.00074-24","url":null,"abstract":"<p><p>The Candidate Phyla Radiation (CPR) represents an extensive bacterial clade comprising primarily uncultured lineages and is distinguished from other bacteria by a significant prevalence of insertion sequences (ISs) within their rRNA genes. However, our understanding of the taxonomic distribution and characteristics of these ISs remains limited. In this study, we used a comprehensive approach to systematically determine the nature of the rRNA ISs in CPR bacteria. The analysis of hundreds of rRNA gene sequences across 65 CPR phyla revealed that ISs are present in 48% of 16S rRNA genes and 82% of 23S rRNA genes, indicating a broad distribution across the CPR clade, with exceptions in the 16S and 23S rRNA genes of <i>Candidatus</i> (<i>Ca</i>.) Saccharibacteria and the 16S rRNA genes of <i>Ca</i>. Peregrinibacteria. Over half the ISs display a group-I-intron-like structure, whereas specific 16S rRNA gene ISs display features reminiscent of group II introns. The ISs frequently encode proteins with homing endonuclease (HE) domains, centered around the LAGLIDADG motif. The LAGLIDADG HE (LHE) proteins encoded by the rRNA ISs of CPR bacteria predominantly have a single-domain structure, deviating from the usual single- or double-domain configuration observed in typical prokaryotic LHEs. Experimental analysis of one LHE protein, I-ShaI from <i>Ca</i>. Shapirobacteria, confirmed that its endonuclease activity targets the DNA sequence of its insertion site, and chemical cross-linking experiments demonstrated its capacity to form homodimers. These results provide robust evidence supporting the hypothesis that the explosive proliferation of rRNA ISs in CPR bacteria was facilitated by mechanisms involving LHEs.</p><p><strong>Importance: </strong>Insertion sequences (ISs) in rRNA genes are relatively limited and infrequent in most bacterial phyla. With a comprehensive bioinformatic analysis, we show that in CPR bacteria, these ISs occur in 48% of 16S rRNA genes and 82% of 23S rRNA genes. We also report the systematic and biochemical characterization of the LAGLIDADG homing endonucleases (LHEs) encoded by these ISs in the first such analysis of the CPR bacteria. This study significantly extends our understanding of the phylogenetic positions of rRNA ISs within CPR bacteria and the biochemical features of their LHEs.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0007424"},"PeriodicalIF":2.7,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11270868/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141296057","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}