Journal of Bacteriology最新文献

{"title":"Inhibitory proteins of <i>Bacillus subtilis</i> interact within the membrane to block intramembrane protease activity.","authors":"Saikat Mandal, Alanah Soriano, Caroline Erpelding, Jackson Ruffner, Eric Smith, Benjamin J Orlando, Lee Kroos","doi":"10.1128/jb.00186-25","DOIUrl":"https://doi.org/10.1128/jb.00186-25","url":null,"abstract":"<p><p>Intramembrane proteases (IPs) are crucial for diverse signaling pathways, including some that regulate the virulence of pathogenic bacteria. A better understanding of mechanisms controlling IP activity is necessary to guide therapeutic development. <i>Bacillus subtilis</i> SpoIVFB is an IP with two natural inhibitory proteins, BofA and SpoIVFA. These proteins form a complex with SpoIVFB and prevent it from cleaving Pro-σ^K during endosporulation. We investigated proximity between BofA and SpoIVFA in the SpoIVFB inhibition complex using <i>in vivo</i> disulfide crosslinking in <i>Escherichia coli</i>. We discovered that two parts of the BofA C-terminal region are proximal to the SpoIVFA transmembrane segment (TMS). Our results support predictions that the BofA C-terminal region adopts an unusual structure within the membrane and interacts with the SpoIVFA TMS to block SpoIVFB cleavage of Pro-σ^K. Endospore-forming <i>Bacilli</i>, including several pathogenic species, encode BofA and SpoIVFA orthologs, which likely interact in a similar fashion to inhibit their SpoIVFB ortholog. It may be possible to control SpoIVFB activity and hence endosporulation with modulators that target the interaction between BofA and SpoIVFA.IMPORTANCERegulated intramembrane proteolysis (RIP) pathways govern important processes in all three domains of life. A key component of RIP pathways is an intramembrane protease (IP), which cleaves one or more substrates within a membrane. Developing modulators of IPs has been challenging, particularly for metallo-IPs. Bacterial metallo-IPs function in RIP pathways that control the virulence of many pathogens. SpoIVFB is a metallo-IP necessary for endosporulation of <i>Bacillus subtilis</i>. Endosporulation enhances the survival of pathogenic <i>Bacilli</i>, which encode orthologs of SpoIVFB and its natural inhibitory proteins BofA and SpoIVFA. Here, we present the first experimental evidence for contacts between BofA and SpoIVFA within the membrane-embedded SpoIVFB inhibition complex, providing foundations for a deeper understanding of mechanisms controlling metallo-IP activity.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0018625"},"PeriodicalIF":3.0,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145251243","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":"Segregation of sister chromosomes during the shape change of developing <i>Myxococcus xanthus</i> cells.","authors":"Y Hoang, Yann S Dufour, Lee Kroos","doi":"10.1128/jb.00328-25","DOIUrl":"https://doi.org/10.1128/jb.00328-25","url":null,"abstract":"<p><p>Chromosome organization is critical for the maintenance of genetic integrity. Most studies of bacterial nucleoids have focused on growing rod-shaped organisms. Studying nucleoid dynamics during <i>Myxococcus xanthus</i> development offers the unique opportunity to investigate the localization of two sister chromosomes as rod-shaped cells transition into round spores. During starvation-induced multicellular development, DNA replication is required for <i>M. xanthus</i> rods to transition into spores with two copies of the chromosome. Here, we report novel approaches using confocal fluorescence microscopy to observe the chromosome number and arrangement, and nucleoid localization in developing cells <i>in situ</i>. We discovered that sister chromosomes are present in some rods and transitioning cells (TCs) early in development. The arrangement of the two chromosomes in developing cells was novel compared to predivisional growing cells studied previously. We observed segregated nucleoids in ~40% of TCs and spores. The majority of TCs contained a crescent-shaped nucleoid along one side, perhaps due to ongoing chromosome segregation, whereas most spores appeared to have undergone nucleoid decondensation. During unicellular glycerol-induced sporulation of <i>M. xanthus</i>, we observed segregated nucleoids in only ~10%-20% of TCs and spores. In addition, early in starvation-induced development, we discovered a subpopulation of cells that may be spheroplasts destined for lysis, which is the fate of most cells under these conditions. Chromosome segregation in developing <i>M. xanthus</i> may be a bet-hedging strategy to increase survival under different conditions and/or an evolutionary remnant of ancestral events that included cell division to produce spores with one copy of the chromosome.IMPORTANCEThe cell cycle normally involves DNA replication, chromosome segregation, and cell division. During starvation-induced <i>Myxococcus xanthus</i> development, DNA replication is necessary for progression to spore formation, which occurs without cell division, resulting in spores with two copies of the chromosome. The organization of sister chromosomes during the morphological change of rod-shaped cells into round spores was unknown. We discovered that the two nucleoids often segregate during the transition from rods to spores. Mature spores contained decondensed nucleoids. Our observations raise important questions about the mechanism of chromosome segregation during <i>M. xanthus</i> development and the reason for its existence. We also discovered a subpopulation of developing cells with characteristics suggesting they are spheroplasts on the verge of cell death.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0032825"},"PeriodicalIF":3.0,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145149212","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":"Adaptation to cinnamaldehyde shapes <i>Pseudomonas aeruginosa</i> resistance to major antibiotics.","authors":"Eline Dubois, Susie Gaillot, Benoît Valot, Maxime Bour, Jean-Michel Brunel, Lison Schmidlin, Patrick Plésiat, Catherine Llanes","doi":"10.1128/jb.00180-25","DOIUrl":"https://doi.org/10.1128/jb.00180-25","url":null,"abstract":"<p><p>In France, the use of essential oils to treat bacterial infections is common, with approximately 40% of cystic fibrosis patients regularly using these natural products to control infections caused by <i>Pseudomonas aeruginosa</i> often in combination with their antibiotic treatments. Previous research has demonstrated that prolonged exposure of <i>P. aeruginosa</i> PA14 to cinnamaldehyde (CNA), the main component of cinnamon essential oil, can select for mutants resistant to β-lactams through overproduction of the MexAB-OprM efflux pump, some of which are also hypersusceptible to aminoglycosides and colistin. We showed here that this hypersusceptibility is not due to an efflux defect, as the deletion of MexXY(OprM)-the specific efflux pump for aminoglycosides-still results in decreased minimum inhibitory concentrations of aminoglycosides. Genome sequencing of hypersusceptible mutants revealed mutations in the ATP synthase operon or its promoter (<i>atpIBEFHAGDC</i>). Surprisingly, although mutations in the <i>atp</i> operon reduced bacterial growth and ATP production, they are not uncommon in clinical strains. We found that ATP synthase alterations modified the respiratory chain and led to inner membrane hyperpolarization, likely enhancing positively charged antibiotic (aminoglycosides and colistin) uptake and susceptibility to these molecules. In addition, the modified respiratory chain increased the proton motive force, allowing the overproduction of the MexAB-OpM efflux pump, which protects bacteria from CNA and from the clinically relevant β-lactam antibiotics. Altogether, these results indicate a trade-off between CNA resistance and aminoglycoside/colistin susceptibility, a reaction that may question the survival of <i>P. aeruginosa</i> in the lung of CF patients possibly submitted to these therapeutic molecules.<b>IMPORTANCE</b>In France, essential oils are widely used by cystic fibrosis patients (40%), often alongside antibiotic therapies, to help control <i>Pseudomonas aeruginosa</i> infections. Cinnamaldehyde from cinnamon essential oil appears to select for <i>P. aeruginosa</i> mutants that are resistant to β-lactam antibiotics due to the overproduction of the MexAB-OprM efflux pump and hypersusceptible to aminoglycosides and colistin. This increased susceptibility is associated with mutations in ATP synthase, which elevate the proton motive force (PMF) and facilitate both (i) increased uptake of positively charged antibiotics (aminoglycosides, colistin) and (ii) more efficient efflux of β-lactams via MexAB-OpM. Thus, the use of cinnamaldehyde may drive a trade-off in <i>P. aeruginosa</i> between β-lactam resistance and aminoglycosides/polymyxins susceptibility, potentially compromising bacterial persistence in the lung of patients.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0018025"},"PeriodicalIF":3.0,"publicationDate":"2025-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145175404","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":"Mechanisms of 10-hydroxyoctadecanoic acid resistance in <i>Streptococcus pneumoniae</i>.","authors":"Cydney N Johnson, Matthew W Frank, Chrispin Chaguza, Brendan T Morrow, Qidong Jia, Christopher D Radka, Jason W Rosch","doi":"10.1128/jb.00223-25","DOIUrl":"10.1128/jb.00223-25","url":null,"abstract":"<p><p>Profiles of human nasal colonization consistently demonstrate that <i>Staphylococcus aureus</i> and <i>Streptococcus pneumoniae</i> can co-exist in the nasopharynx. Several studies have demonstrated the antagonist relationship between the two organisms via several molecular mechanisms, including competition for nutrients as well as via direct killing by hydrogen peroxide. During nasal colonization, the pneumococcus is in direct contact with the fatty acid <i>h</i>18:0, which is released into the extracellular environment by <i>S. aureus</i>. We report that <i>h</i>18:0 is specifically toxic to the pneumococcus among the pathogenic streptococci, providing a unique mechanism for interspecies competition during colonization. Exposure of cells to <i>h</i>18:0 revealed that <i>S. pneumoniae</i> could rapidly adapt to and overcome the observed toxicity. Whole-genome analysis revealed the mechanism underlying this resistance being linked to a truncation of a glycosyltransferase in the capsule biosynthesis locus and a genomic inversion in the phase variation locus, leading to altered cell surface charge and membrane lipid composition. These physiological differences in the resistant isolates may aid in repelling toxic, charged fatty acids such as <i>h</i>18:0 from the cell membrane.IMPORTANCEThe pneumococcus and <i>S. aureus</i> are two of the most well-characterized residents of the human nasopharynx; yet much remains unknown regarding how the two bacteria interact. Here, we describe the potential of <i>S. aureus</i>-produced <i>h</i>18:0, whose function and biological impact are still being described, to act as an interspecies competition molecule against <i>S. pneumoniae</i>, and how the pneumococcus can adapt to overcome its toxicity.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0022325"},"PeriodicalIF":3.0,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145085926","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 type IV pilus steering committee: how Pil-Chp controls directional motility.","authors":"Kaitlin D Yarrington, Dominique H Limoli","doi":"10.1128/jb.00396-24","DOIUrl":"https://doi.org/10.1128/jb.00396-24","url":null,"abstract":"<p><p>Many microbial species live on surfaces and employ various strategies for initiation of and survival within a surface-attached community. One such strategy implemented by many bacterial species is to move across surfaces using grappling hook-like appendages called type IV pili (TFP) which extend, attach to the surface, and retract to pull the cell body forward. In the bacterium <i>Pseudomonas aeruginosa</i>, TFP motility, or twitching, is controlled by the Pil-Chp system. <i>P. aeruginosa</i> uses this system to traverse surfaces and gather information about the local chemical and physical environment. The Pil-Chp system shares many similarities to the well-studied flagellar chemotaxis system (Che), which biases locomotion of swimming cells up or down gradients of chemical stimuli. However, many important differences have been described, while others await discovery. Some of these differences have even led to speculation that chemotaxis may not be a primary role for Pil-Chp. Thus, recent studies have focused on addressing whether <i>P. aeruginosa</i> uses chemotaxis to bias the direction of motility on a surface, and if so, what role does Pil-Chp play in this process? In this review, we focus on current progress in the field toward gaining insight into these questions.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0039624"},"PeriodicalIF":3.0,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145086064","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":"Beyond plasmid addiction: the role of toxin-antitoxin systems in the selfish behavior of mobile genetic elements.","authors":"Bradd Mendoza-Guido, Keilor Rojas-Jimenez","doi":"10.1128/jb.00232-25","DOIUrl":"https://doi.org/10.1128/jb.00232-25","url":null,"abstract":"<p><p>Toxin-antitoxin (TA) systems were initially described as \"addiction\" modules that promote plasmid maintenance through a post-segregational killing (PSK) mechanism. In this process, the cells are forced to retain plasmids to avoid death caused by the longer half-life of the toxin compared to the antitoxin. However, TA systems have since been widely identified across a broad range of mobile genetic elements (MGEs), suggesting that TA systems support the maintenance of these MGEs within bacterial hosts and contribute to the exclusion of competing MGEs such as plasmids and phages. This perspective highlights their broader role beyond plasmid addiction, functioning as key components in safeguarding MGE persistence and enhancing MGE fitness. Therefore, the concept of \"plasmid addiction\" should be reconsidered as a subset of a more comprehensive phenomenon referred to as \"MGE selfishness,\" which more accurately captures the widespread distribution and conserved, self-serving functions of TA systems across diverse MGEs. Additionally, TA systems facilitate the establishment of MGEs as \"molecular symbionts\" within bacterial cells. While initially considered parasitic, the relationships can evolve to provide mutual benefits for both the MGE and the host. From a gene-centered evolutionary perspective, the proposed molecular symbiosis may progress to a point where most of the MGE's original content is lost, leaving only essential genes that are retained and functionally co-opted by the host. Further studies should investigate the role of TA systems in MGEs beyond plasmids, as well as their evolutionary trajectories toward specialized functions that may influence the adaptation and evolution of key bacterial groups, including pathogens.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0023225"},"PeriodicalIF":3.0,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145085972","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":"Molecular interplay between peptidoglycan integrity and outer membrane asymmetry in maintaining cell envelope homeostasis.","authors":"Sinjini Nandy, Arshya F Tehrani, Augusto C Hunt-Serracin, Jacob Biboy, Christine Pybus, Waldemar Vollmer, Joseph M Boll","doi":"10.1128/jb.00331-25","DOIUrl":"10.1128/jb.00331-25","url":null,"abstract":"<p><p>The bacterial cell envelope is a critical interface with the environment, particularly in Gram-negative species where the outer membrane (OM) and peptidoglycan (PG) layers coordinate to maintain structural integrity and resist turgor. Although this coordination is essential for survival, the molecular mechanisms linking OM and PG homeostasis remain poorly understood. LD-transpeptidases (LDTs) are enzymes that crosslink peptides in PG and incorporate d-amino acids, but their physiological roles are not fully defined. Here, we characterize the activity of the LDT enzyme LdtJ in <i>Acinetobacter baumannii</i> and investigate the consequences of its deletion. Loss of LdtJ disrupts cell morphology, downregulates PG precursor genes (e.g., <i>dadA</i> and <i>alr</i>), and activates the stringent response, including elevated ppGpp levels and <i>dksA</i> upregulation. These defects are fully suppressed in a ∆<i>ldtJ</i> ∆<i>mla</i> double mutant, implicating the OM lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the ∆<i>ldtJ</i> mutant are reversed in the double mutant, highlighting a functional interplay between PG remodeling and OM lipid asymmetry. Our findings suggest that LdtJ contributes to envelope integrity not only through PG modification but also by influencing broader regulatory and metabolic networks.IMPORTANCE<i>Acinetobacter baumannii</i> is a leading cause of hospital-acquired infections and is highly resistant to antibiotics. Its survival relies on the integrity of the cell envelope, composed of the peptidoglycan (PG) layer and outer membrane (OM). While LD-transpeptidases are traditionally known for reinforcing PG structure through non-canonical crosslinking, our findings reveal that the LdtJ enzyme also plays a critical role in regulating cellular metabolism and stress responses. Deletion of <i>ldtJ</i> results in pronounced growth defects and abnormal cell morphology-phenotypes that are fully suppressed by disrupting the OM lipid asymmetry transport system, Mla. This genetic interaction uncovers a previously unrecognized link between PG remodeling and OM lipid homeostasis. These insights deepen our understanding of envelope coordination in Gram-negative bacteria.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0033125"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145080695","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":"Colorectal cancer-associated <i>Streptococcus gallolyticus</i>: a hidden diversity expose.","authors":"Bruno Périchon, Thomas Cokelaer, Wooi Keong Teh, Laurence du Merle, Laurence Ma, Marie Touchon, Alexandra Doloy, Claire Poyart, Michael Givskov, Patrick Trieu-Cuot, Shaynoor Dramsi","doi":"10.1128/jb.00230-25","DOIUrl":"10.1128/jb.00230-25","url":null,"abstract":"<p><p><i>Streptococcus gallolyticus</i> subsp. <i>gallolyticus</i> (<i>SGG</i>) is a bacterial pathogen implicated in bacteremia and endocarditis and is often associated with colon tumors in elderly individuals. The development of colorectal cancer (CRC) has been linked to intestinal dysbiosis, characterized by increased proportions of <i>SGG</i> and other intestinal microbes. In this study, we present the complete nucleotide sequence of five novel clinical isolates of <i>SGG</i> associated with colorectal cancer, revealing unexpected genetic diversity. Sequencing an additional 30 <i>SGG</i> clinical isolates provided a more comprehensive description of this genetic diversity. We did not identify a pathogenicity island specific to CRC-associated <i>SGG</i> isolates. Most of these human-derived <i>SGG</i> isolates exhibit resistance to multiple antibiotics. Our findings also offer additional insights into multilocus sequence typing (MLST), capsular loci, and pilus organization. Analysis of the repertoire of surface proteins reveals high potential for binding and foraging complex polysaccharides. Finally, comparative genomics with the phylogenetically closest non-pathogenic subspecies <i>S. gallolyticus</i> subsp. <i>macedonicus</i> confirmed that <i>SGG</i> pathogenicity-associated factors mostly rely on a large repertoire of surface proteins involved in host colonization, presence of C5a peptidase to avoid innate immunity, bile salt hydrolase to persist in the gut, and of specific bacteriocin and type VII-dependent effectors to colonize the host colon. Additionally, the presence of extracellular polysaccharides in <i>SGG</i> probably helps the bacterium survive in harsher conditions.IMPORTANCE<i>Streptococcus gallolyticus</i> subsp. <i>gallolyticus</i> (<i>SGG</i>) was the first intestinal bacterium associated with colorectal cancer. It is now widely accepted that colonic microbiota dysbiosis contributes to oncogenesis, with a higher relative abundance of several potentially pro-carcinogenic bacteria. For example, the oncogenic role of <i>Escherichia coli pks</i>+ and enterotoxinogenic <i>Bacteroides fragilis</i> in colorectal cancer has been well established, identifying the role of genetic loci encoding toxins. Through the sequencing and analysis of 11 clinical <i>SGG</i> isolates from CRC patients and comparisons with non-CRC isolates, we uncovered a significant diversity among CRC-associated strains. Our findings suggest that <i>SGG</i> association with CRC is complex and is not linked to a specific strain or pathogenicity island, thus highlighting the opportunistic and versatile nature of <i>SGG</i>.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0023025"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12445087/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855244","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":"AphA-dependent c-di-GMP production in <i>Vibrio parahaemolyticus</i> is mediated by direct regulation of <i>eapA</i> transcription encoding an EAL domain-containing protein.","authors":"Nan Zhang, Wu Xu, Xue Li, Miaomiao Zhang, Xi Luo, Bin Ni, Renfei Lu, Yiquan Zhang","doi":"10.1128/jb.00104-25","DOIUrl":"https://doi.org/10.1128/jb.00104-25","url":null,"abstract":"<p><p><i>Vibrio parahaemolyticus,</i> a major seafood-borne pathogen, employs quorum sensing (QS) and c-di-GMP to regulate virulence, motility, and biofilm formation. While the master QS regulator AphA promotes c-di-GMP accumulation at low cell density (LCD), the underlying mechanism remained unclear. Here, we show that AphA drives net c-di-GMP accumulation by elevating c-di-GMP production while also activating the transcription of <i>eapA</i> (<i>vp0376</i>), encoding an EAL domain-containing phosphodiesterase, revealing a complex regulatory node. RNA sequencing revealed that AphA regulates 1,542 genes, including 23 potentially linked to c-di-GMP metabolism. Among these, <i>eapA</i> exhibited the strongest predicted AphA-binding motif. Experimental validation confirmed AphA binds the <i>eapA</i> promoter to activate its transcription. The expression of <i>eapA</i> peaked at LCD and decreased with increasing cell density. Deletion of <i>eapA</i> elevated c-di-GMP levels at LCD, enhanced biofilm formation, and impaired swimming motility, while <i>aphA</i> deletion reduced c-di-GMP. The <i>aphA</i>- and <i>eapA</i> double mutant exhibited c-di-GMP and biofilm phenotypes resembling the <i>eapA</i> mutant, placing EapA downstream of AphA. Transcriptional analysis showed <i>eapA</i> deletion upregulated exopolysaccharide biosynthesis genes while downregulating polar flagellar genes, aligning with c-di-GMP-mediated biofilm-motility trade-offs. Our findings establish a direct AphA-EapA-c-di-GMP pathway that critically regulates the biofilm-motility switch in <i>V. parahaemolyticus</i>, revealing how QS integrates with second-messenger signaling to optimize environmental adaptation.IMPORTANCE<i>Vibrio parahaemolyticus</i> (<i>V. parahaemolyticus</i>) poses significant threats to human health and aquaculture, yet the mechanisms linking QS to c-di-GMP signaling remain poorly understood. This work uncovers AphA as a pivotal regulator that directly activates <i>eapA</i>, an EAL domain phosphodiesterase (PDE), to elevate c-di-GMP levels at low cell density (LCD). We identify EapA as the LCD-specific PDE that degrades c-di-GMP and is directly activated by AphA. Deletion of <i>eapA</i> elevates c-di-GMP levels, enhancing biofilm formation while suppressing swimming motility; these phenotypes are epistatic to AphA. The discovery of the AphA-<i>eapA</i>-c-di-GMP axis provides novel insights into how QS integrates with second messengers to optimize bacterial fitness. This study underscores the complexity of c-di-GMP metabolism and highlights AphA's dual role as a global transcriptional regulator, bridging gaps in our understanding of bacterial signaling networks.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0010425"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145080738","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":"Structure-guided engineering of an aromatic ring-hydroxylating dioxygenase for broad-spectrum phthalate degradation.","authors":"Jai Krishna Mahto, Ishani Mishra, Kuldeep Jangid, Pravindra Kumar","doi":"10.1128/jb.00221-25","DOIUrl":"10.1128/jb.00221-25","url":null,"abstract":"<p><p>Phthalates such as isophthalate, phthalate, and terephthalate are widespread environmental pollutants with significant health and ecological impacts. <i>Comamonas testosteroni</i> KF1 initiates isophthalate degradation through a specialized two-component enzyme system composed of isophthalate dioxygenase (IPDO) and its cognate reductase, isophthalate dioxygenase reductase. Despite its environmental significance, the lack of structural insights into IPDO has hindered efforts to rationally redesign, optimize, and harness its chemistry. Here, we report the first crystal structures of substrate-free IPDO and its complex with isophthalate, revealing unique structural features that underpin its substrate specificity. Unlike related oxygenases, phthalate dioxygenase (α₃α₃) and terephthalate dioxygenase (α₃β₃), IPDO adopts a trimer (α₃) architecture, with distinct active site residues tailored to isophthalate binding. The comparative structural analysis identified steric and electrostatic constraints-particularly involving residue V178-that preclude the binding of ortho- or para-substituted substrates. Leveraging these structural insights, we engineered IPDO variants with broadened substrate specificity. Notably, the V178A and F249H substitutions enabled the enzyme to degrade three regioisomers of phthalate (phthalate, isophthalate, and terephthalate) without diminishing its native activity against isophthalate. The catalytic turnover (<i>k</i>_cat) of the V178A/F249H double mutant was found to be 4.8 ± 0.3, 4.9 ± 0.2, and 4.0 ± 0.2 s^-1 for isophthalate, terephthalate, and phthalate, respectively, demonstrating comparable catalytic efficiency for all three substrates. Overall, this work advances our understanding of the molecular mechanisms involved in isophthalate dihydroxylation and elucidates a rational engineering approach to expand the catalytic repertoire of IPDO for biotechnological and environmental applications.IMPORTANCEPhthalate pollution poses a major environmental concern due to its widespread use as plasticizers and its persistence in ecosystems. Microbial degradation of phthalates offers a sustainable solution for mitigating this contamination. Among the key enzymes involved, aromatic-ring-hydroxylating dioxygenases initiate the first critical step in phthalate breakdown. However, most known enzymes exhibit narrow substrate specificity, limiting their utility for degrading diverse phthalate isomers such as isophthalate, phthalate, and terephthalate. This research addresses a critical gap by elucidating the structural basis of substrate specificity in isophthalate dioxygenase and applying rational engineering to expand its catalytic range. By generating enzyme variants capable of degrading all three phthalate regioisomers, this work provides a blueprint for designing versatile biocatalysts tailored for pollutant detoxification.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0022125"},"PeriodicalIF":3.0,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12445078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144821500","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}