Xingru Chen, Emily Perez, Eleanor C Scheeres, Rosemary Northcote, Aretha Fiebig, Andrew J Olive, Sean Crosson
The bacterial cell envelope is essential for viability and host interaction. In the intracellular pathogen Brucella ovis, the orphan HWE-family histidine kinase PhyK has been implicated in processes that influence cell envelope homeostasis, yet its function remains largely uncharacterized. We show that deletion of phyK (∆phyK) disrupts cell size control, increases resistance to anionic detergents, enhances sensitivity to cationic envelope disruptors, and triggers broad transcriptional changes, including reduced expression of aerobic respiration genes and increased expression of genes involved in transport and lipid metabolism. This transcriptional profile mirrors that of wild-type B. ovis exposed to an anionic detergent, indicating that loss of PhyK function primes cells to resist this stress. Despite its altered cell envelope properties, the ∆phyK mutant exhibits no fitness defect in ex vivo macrophage infection models. However, it elicits a significantly reduced pro-inflammatory cytokine response in activated murine macrophages compared to the wild-type strain. We further show that purified PhyK can form multiple stable oligomeric species in solution, reflecting the structural plasticity observed in other HWE-family kinases and likely contributing to its signaling function in vivo. Our results establish PhyK as a key regulator of B. ovis cell envelope properties that can modulate host immune interactions.
{"title":"An HWE-Family Histidine Kinase Modulates Brucella Cell Envelope Properties and Host Innate Immune Response.","authors":"Xingru Chen, Emily Perez, Eleanor C Scheeres, Rosemary Northcote, Aretha Fiebig, Andrew J Olive, Sean Crosson","doi":"10.1111/mmi.70006","DOIUrl":"10.1111/mmi.70006","url":null,"abstract":"<p><p>The bacterial cell envelope is essential for viability and host interaction. In the intracellular pathogen Brucella ovis, the orphan HWE-family histidine kinase PhyK has been implicated in processes that influence cell envelope homeostasis, yet its function remains largely uncharacterized. We show that deletion of phyK (∆phyK) disrupts cell size control, increases resistance to anionic detergents, enhances sensitivity to cationic envelope disruptors, and triggers broad transcriptional changes, including reduced expression of aerobic respiration genes and increased expression of genes involved in transport and lipid metabolism. This transcriptional profile mirrors that of wild-type B. ovis exposed to an anionic detergent, indicating that loss of PhyK function primes cells to resist this stress. Despite its altered cell envelope properties, the ∆phyK mutant exhibits no fitness defect in ex vivo macrophage infection models. However, it elicits a significantly reduced pro-inflammatory cytokine response in activated murine macrophages compared to the wild-type strain. We further show that purified PhyK can form multiple stable oligomeric species in solution, reflecting the structural plasticity observed in other HWE-family kinases and likely contributing to its signaling function in vivo. Our results establish PhyK as a key regulator of B. ovis cell envelope properties that can modulate host immune interactions.</p>","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":" ","pages":""},"PeriodicalIF":2.6,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12354254/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144506872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Natalia de Miguel, María Carolina Touz, Manuela Blasco Pedreros, Gabriel Luna Pizarro
Infections caused by single‐celled protozoan parasites affect countless individuals around the world, particularly in low‐ and middle‐income countries. Despite the significant impact on global health, available therapeutic intervention strategies are still limited. Gaining insight into the mechanisms of infection is essential for developing new vaccines and drug therapies. While host–parasite interactions are well‐recognized as critical factors in disease, recent evidence highlights the growing importance of communication among parasites. Various studies indicate that protozoans can interact and communicate with one another, facilitating coordinated behaviors and the emergence of specialized roles within their populations. These interactions help optimize survival strategies and strengthen their response to infections. This review explores how communication among parasites influences social activities, physiological processes, and disease progression.
{"title":"Understanding Cell Communication Among Protozoan Parasites","authors":"Natalia de Miguel, María Carolina Touz, Manuela Blasco Pedreros, Gabriel Luna Pizarro","doi":"10.1111/mmi.70005","DOIUrl":"https://doi.org/10.1111/mmi.70005","url":null,"abstract":"Infections caused by single‐celled protozoan parasites affect countless individuals around the world, particularly in low‐ and middle‐income countries. Despite the significant impact on global health, available therapeutic intervention strategies are still limited. Gaining insight into the mechanisms of infection is essential for developing new vaccines and drug therapies. While host–parasite interactions are well‐recognized as critical factors in disease, recent evidence highlights the growing importance of communication among parasites. Various studies indicate that protozoans can interact and communicate with one another, facilitating coordinated behaviors and the emergence of specialized roles within their populations. These interactions help optimize survival strategies and strengthen their response to infections. This review explores how communication among parasites influences social activities, physiological processes, and disease progression.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"22 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144500547","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Adrian Jinich, Anisha Zaveri, Michael A. DeJesus, Amanda Spencer, Ricardo Almada‐Monter, Emanuel Flores‐Bautista, Clare M. Smith, Christopher M. Sassetti, Jeremy M. Rock, Sabine Ehrt, Dirk Schnappinger, Thomas R. Ioerger, Kyu Y. Rhee
Characterizing genetic essentiality across various conditions is fundamental for understanding gene function. Transposon sequencing (TnSeq) is a powerful technique to generate genome‐wide essentiality profiles in bacteria and has been extensively applied to Mycobacterium tuberculosis (Mtb). Dozens of TnSeq screens have yielded valuable insights into the biology of Mtb in vitro, inside macrophages, and in model host organisms. Despite their value, these Mtb TnSeq profiles have not been standardized or collated into a single, easily searchable database. This results in significant challenges when attempting to query and compare these resources, limiting our ability to obtain a comprehensive and consistent understanding of genetic conditional essentiality in Mtb. We address this problem by building a central repository of publicly available Mtb TnSeq screens, the Mtb transposon sequencing database (MtbTnDB). The MtbTnDB is a living resource that encompasses to date ≈150 standardized TnSeq screens, enabling open access to data, visualizations, and functional predictions through an interactive web app (www.mtbtndb.app). We conduct several statistical analyses on the complete database, such as demonstrating that (i) genes in the same genomic neighborhood have similar TnSeq profiles, and (ii) clusters of genes with similar TnSeq profiles are enriched for genes from similar functional categories. We further analyze the performance of machine learning models trained on TnSeq profiles to predict the functional annotation of orphan genes in Mtb. By facilitating the comparison of TnSeq screens across conditions, the MtbTnDB will accelerate the exploration of conditional genetic essentiality, provide insights into the functional organization of Mtb genes, and help predict gene function in this important human pathogen.
{"title":"The Mycobacterium tuberculosis Transposon Sequencing Database (MtbTnDB): A Large‐Scale Guide to Genetic Conditional Essentiality","authors":"Adrian Jinich, Anisha Zaveri, Michael A. DeJesus, Amanda Spencer, Ricardo Almada‐Monter, Emanuel Flores‐Bautista, Clare M. Smith, Christopher M. Sassetti, Jeremy M. Rock, Sabine Ehrt, Dirk Schnappinger, Thomas R. Ioerger, Kyu Y. Rhee","doi":"10.1111/mmi.15370","DOIUrl":"https://doi.org/10.1111/mmi.15370","url":null,"abstract":"Characterizing genetic essentiality across various conditions is fundamental for understanding gene function. Transposon sequencing (TnSeq) is a powerful technique to generate genome‐wide essentiality profiles in bacteria and has been extensively applied to <jats:styled-content style=\"fixed-case\"><jats:italic>Mycobacterium tuberculosis</jats:italic></jats:styled-content> (<jats:italic>Mtb</jats:italic>). Dozens of TnSeq screens have yielded valuable insights into the biology of <jats:italic>Mtb</jats:italic> in vitro, inside macrophages, and in model host organisms. Despite their value, these <jats:italic>Mtb</jats:italic> TnSeq profiles have not been standardized or collated into a single, easily searchable database. This results in significant challenges when attempting to query and compare these resources, limiting our ability to obtain a comprehensive and consistent understanding of genetic conditional essentiality in <jats:italic>Mtb</jats:italic>. We address this problem by building a central repository of publicly available <jats:italic>Mtb</jats:italic> TnSeq screens, the <jats:italic>Mtb</jats:italic> transposon sequencing database (MtbTnDB). The MtbTnDB is a living resource that encompasses to date ≈150 standardized TnSeq screens, enabling open access to data, visualizations, and functional predictions through an interactive web app (<jats:ext-link xmlns:xlink=\"http://www.w3.org/1999/xlink\" xlink:href=\"http://www.mtbtndb.app\">www.mtbtndb.app</jats:ext-link>). We conduct several statistical analyses on the complete database, such as demonstrating that (i) genes in the same genomic neighborhood have similar TnSeq profiles, and (ii) clusters of genes with similar TnSeq profiles are enriched for genes from similar functional categories. We further analyze the performance of machine learning models trained on TnSeq profiles to predict the functional annotation of orphan genes in <jats:italic>Mtb</jats:italic>. By facilitating the comparison of TnSeq screens across conditions, the MtbTnDB will accelerate the exploration of conditional genetic essentiality, provide insights into the functional organization of <jats:italic>Mtb</jats:italic> genes, and help predict gene function in this important human pathogen.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"14 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144311389","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anshu Chauhan, Hans Carolus, Dimitrios Sofras, Mohit Kumar, Praveen Kumar, Remya Nair, Aswathy Narayanan, Kusum Yadav, Basharat Ali, Vladislav Biriukov, Amandeep Saini, Ian Leaves, Rudy Vergauwen, Celia Lobo Romero, Dhara Malavia-Jones, Ashutosh Singh, Atanu Banerjee, Shivaprakash M. Rudramurthy, Arunaloke Chakrabarti, Alok K. Mondal, Naseem A. Gaur, Kaustuv Sanyal, Jeffrey M. Rybak, Toni Gabaldón, Patrick Van Dijck, Neil A. R. Gow, Rajendra Prasad
Clinical isolates of Candida auris show a high prevalence of resistance to Amphotericin B (AmB)—an uncommon trait in most Candida species. Alterations in ergosterol biosynthesis can contribute to acquired AmB resistance in C. auris laboratory strains but are rarely seen in clinical isolates. In this study, we experimentally evolved two drug-susceptible Clade II isolates of C. auris to develop AmB resistance. The evolved strains displayed a four to eight fold increase in MIC50 compared to the parental cells. We analyzed changes in their karyotype, genome, lipidome, and transcriptome associated with this acquired resistance. In one lineage, AOX2 was upregulated, and its deletion reversed the AmB resistance phenotype. The aox2Δ mutant also failed to evolve AmB resistance under experimental conditions. In the same lineage, restoring the UPC2S332Rand RTG3S101T mutations to the wild-type allele restored AmB susceptibility. In another lineage, the ergosterol and sphingolipid pathways were observed to play a critical role, and upregulation of the ERG genes elevated the total sterol content, while significant downregulation of HSX11 (glucosylceramide synthase) resulted in lower levels of glucosylceramides. To our knowledge, this study is the first to show that AmB resistance in C. auris can be acquired through mechanisms both dependent on or independent of sterol content modulation, highlighting Aox2 and Upc2 as key regulators of amphotericin resistance.
{"title":"Multi-Omics Analysis of Experimentally Evolved Candida auris Isolates Reveals Modulation of Sterols, Sphingolipids, and Oxidative Stress in Acquired Amphotericin B Resistance","authors":"Anshu Chauhan, Hans Carolus, Dimitrios Sofras, Mohit Kumar, Praveen Kumar, Remya Nair, Aswathy Narayanan, Kusum Yadav, Basharat Ali, Vladislav Biriukov, Amandeep Saini, Ian Leaves, Rudy Vergauwen, Celia Lobo Romero, Dhara Malavia-Jones, Ashutosh Singh, Atanu Banerjee, Shivaprakash M. Rudramurthy, Arunaloke Chakrabarti, Alok K. Mondal, Naseem A. Gaur, Kaustuv Sanyal, Jeffrey M. Rybak, Toni Gabaldón, Patrick Van Dijck, Neil A. R. Gow, Rajendra Prasad","doi":"10.1111/mmi.15379","DOIUrl":"https://doi.org/10.1111/mmi.15379","url":null,"abstract":"Clinical isolates of <i>Candida auris</i> show a high prevalence of resistance to Amphotericin B (AmB)—an uncommon trait in most <i>Candida</i> species. Alterations in ergosterol biosynthesis can contribute to acquired AmB resistance in <i>C. auris</i> laboratory strains but are rarely seen in clinical isolates. In this study, we experimentally evolved two drug-susceptible Clade II isolates of <i>C. auris</i> to develop AmB resistance. The evolved strains displayed a four to eight fold increase in MIC<sub>50</sub> compared to the parental cells. We analyzed changes in their karyotype, genome, lipidome, and transcriptome associated with this acquired resistance. In one lineage, <i>AOX2</i> was upregulated, and its deletion reversed the AmB resistance phenotype. The <i>aox2Δ</i> mutant also failed to evolve AmB resistance under experimental conditions. In the same lineage, restoring the <i>UPC2</i><sup><i>S332R</i></sup> <i>and RTG3</i><sup><i>S101T</i></sup> mutations to the wild-type allele restored AmB susceptibility. In another lineage, the ergosterol and sphingolipid pathways were observed to play a critical role, and upregulation of the ERG genes elevated the total sterol content, while significant downregulation of <i>HSX11</i> (glucosylceramide synthase) resulted in lower levels of glucosylceramides. To our knowledge, this study is the first to show that AmB resistance in <i>C. auris</i> can be acquired through mechanisms both dependent on or independent of sterol content modulation, highlighting Aox2 and Upc2 as key regulators of amphotericin resistance.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"102 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144219516","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Number and arrangement of flagella, the bacterial locomotion organelles, are species-specific and serve as key taxonomic markers. The FlhG ATPase (also: YlxH, FleN), along with FlhF, plays pivotal roles in determining flagellation patterns. In Bacillus subtilis, FlhG and FlhF govern the spatial arrangement of peritrichous flagella. FlhG aids in flagellar assembly by interacting with the flagellar C-ring protein FliY, yet the molecular implications of this interaction have been unclear. Our study reveals that the ATP-dependent FlhG homodimer interacts with the C-terminal domain of GpsB, a cell cycle regulator, which recruits the peptidoglycan synthase PBP1 (also: ponA) to sites of cell wall elongation. A deletion of gpsB leads to dysregulation of the flagellation pattern mimicking the effects of a flhG deletion strain. The finding that GpsB can interact simultaneously with FlhG and PBP1, combined with the observation that GpsB and FliY can simultaneously interact with FlhG, strongly argues for a model in which FlhG confines flagella biosynthesis to regions of active cell wall biosynthesis. Thus, the FlhG-GpsB interaction appears to enable the locally restrained stimulation of the GTPase FlhF, known for its role to localize flagella in various bacterial species.
{"title":"FlhG Cooperates With the Cell Cycle Regulator GpsB to Confine Peritrichous Flagella in B. subtilis.","authors":"Anita Dornes,Patrica Bedrunka,Benjamin Pillet,Dieter Kressler,Thomas Heimerl,Jan Pané-Farré,Gert Bange","doi":"10.1111/mmi.15375","DOIUrl":"https://doi.org/10.1111/mmi.15375","url":null,"abstract":"Number and arrangement of flagella, the bacterial locomotion organelles, are species-specific and serve as key taxonomic markers. The FlhG ATPase (also: YlxH, FleN), along with FlhF, plays pivotal roles in determining flagellation patterns. In Bacillus subtilis, FlhG and FlhF govern the spatial arrangement of peritrichous flagella. FlhG aids in flagellar assembly by interacting with the flagellar C-ring protein FliY, yet the molecular implications of this interaction have been unclear. Our study reveals that the ATP-dependent FlhG homodimer interacts with the C-terminal domain of GpsB, a cell cycle regulator, which recruits the peptidoglycan synthase PBP1 (also: ponA) to sites of cell wall elongation. A deletion of gpsB leads to dysregulation of the flagellation pattern mimicking the effects of a flhG deletion strain. The finding that GpsB can interact simultaneously with FlhG and PBP1, combined with the observation that GpsB and FliY can simultaneously interact with FlhG, strongly argues for a model in which FlhG confines flagella biosynthesis to regions of active cell wall biosynthesis. Thus, the FlhG-GpsB interaction appears to enable the locally restrained stimulation of the GTPase FlhF, known for its role to localize flagella in various bacterial species.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"83 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144087646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Social interactions among bacteria can induce behaviors that affect their fitness and influence how complex communities assemble. Here we report a new socially induced motility behavior that we refer to as baited expansion in Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), a plant pathogenic bacterium. We found Pst DC3000 displayed strongly induced swimming motility towards nearby colonies of Dickeya dianthicola or Escherichia coli. We developed a controlled system to visualize and characterize the development of baited expansion. Our results provide evidence that baited expansion behavior occurs in response to a chemical gradient established and maintained by the bait colony. We also found this behavior correlated with distinct transcriptional profiles and identified molybdenum cofactor (Moco) and a Moco‐utilizing oxidoreductase as crucial factors facilitating the baited expansion behavior.
{"title":"Pseudomonas syringae Socially Induced Swimming Motility Requires the Molybdenum Cofactor","authors":"Zichu Yang, Bryan Swingle","doi":"10.1111/mmi.15378","DOIUrl":"https://doi.org/10.1111/mmi.15378","url":null,"abstract":"Social interactions among bacteria can induce behaviors that affect their fitness and influence how complex communities assemble. Here we report a new socially induced motility behavior that we refer to as baited expansion in <jats:styled-content style=\"fixed-case\"><jats:italic>Pseudomonas syringae</jats:italic></jats:styled-content> pv. tomato DC3000 (<jats:italic>Pst</jats:italic> DC3000), a plant pathogenic bacterium. We found <jats:italic>Pst</jats:italic> DC3000 displayed strongly induced swimming motility towards nearby colonies of <jats:styled-content style=\"fixed-case\"><jats:italic>Dickeya dianthicola</jats:italic></jats:styled-content> or <jats:styled-content style=\"fixed-case\"><jats:italic>Escherichia coli</jats:italic></jats:styled-content>. We developed a controlled system to visualize and characterize the development of baited expansion. Our results provide evidence that baited expansion behavior occurs in response to a chemical gradient established and maintained by the bait colony. We also found this behavior correlated with distinct transcriptional profiles and identified molybdenum cofactor (Moco) and a Moco‐utilizing oxidoreductase as crucial factors facilitating the baited expansion behavior.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"1 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144097134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Iron is vital for most organisms, serving as a cofactor in enzymes, regulatory proteins, and respiratory cytochromes. In Corynebacterium glutamicum, iron and heme homeostasis are tightly interconnected and controlled by the global regulators DtxR and HrrA. While DtxR senses intracellular Fe2+, HrrSA is activated by heme. This study provides the first genome-wide analysis of DtxR and HrrA binding dynamics under varying iron and heme conditions using chromatin affinity purification and sequencing (ChAP-Seq). We revealed 25 novel DtxR targets and 210 previously unrecognized HrrA targets. Among these, metH, encoding homocysteine methyltransferase, and xerC, encoding a tyrosine recombinase, were bound by DtxR exclusively under heme conditions, underscoring condition-dependent variation. Activation of metH by DtxR links iron metabolism to methionine synthesis, potentially relevant for the mitigation of oxidative stress. Beyond novel targets, 16 shared targets between DtxR and HrrA, some with overlapping operator sequences, highlight their interconnected regulons. Strikingly, we demonstrate the significance of weak ChAP-Seq peaks that are often disregarded in global approaches, but feature an impact of the regulator on differential gene expression. These findings emphasize the importance of genome-wide profiling under different conditions to uncover novel targets and shed light on the complexity and dynamic nature of bacterial regulatory networks.
{"title":"Genome-Wide Analysis of DtxR and HrrA Regulons Reveals Novel Targets and a High Level of Interconnectivity Between Iron and Heme Regulatory Networks in Corynebacterium glutamicum","authors":"Aileen Krüger, Ulrike Weber, Julia Frunzke","doi":"10.1111/mmi.15376","DOIUrl":"https://doi.org/10.1111/mmi.15376","url":null,"abstract":"Iron is vital for most organisms, serving as a cofactor in enzymes, regulatory proteins, and respiratory cytochromes. In <i>Corynebacterium glutamicum</i>, iron and heme homeostasis are tightly interconnected and controlled by the global regulators DtxR and HrrA. While DtxR senses intracellular Fe<sup>2+</sup>, HrrSA is activated by heme. This study provides the first genome-wide analysis of DtxR and HrrA binding dynamics under varying iron and heme conditions using chromatin affinity purification and sequencing (ChAP-Seq). We revealed 25 novel DtxR targets and 210 previously unrecognized HrrA targets. Among these, <i>metH,</i> encoding homocysteine methyltransferase, and <i>xerC,</i> encoding a tyrosine recombinase, were bound by DtxR exclusively under heme conditions, underscoring condition-dependent variation. Activation of <i>metH</i> by DtxR links iron metabolism to methionine synthesis, potentially relevant for the mitigation of oxidative stress. Beyond novel targets, 16 shared targets between DtxR and HrrA, some with overlapping operator sequences, highlight their interconnected regulons. Strikingly, we demonstrate the significance of weak ChAP-Seq peaks that are often disregarded in global approaches, but feature an impact of the regulator on differential gene expression. These findings emphasize the importance of genome-wide profiling under different conditions to uncover novel targets and shed light on the complexity and dynamic nature of bacterial regulatory networks.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"43 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144066349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nathan Hill, Lara M. Matulina, Cameron MacIntyre, M. Amine Hassani, Sheila Thomas, Matteo Luban, Isabelle Ward, Amina Abdalla, John M. Leong, Brandon L. Garcia, Jacob E. Lemieux
Lyme disease is a tick-borne spirochetosis with diverse clinical manifestations. Genotypic and phenotypic variation among Borrelia burgdorferi strains correlates with variable manifestations of Lyme disease in humans; this diversity is attributed in part to variation in surface-exposed lipoproteins, which are targets of the human antibody response and contribute to tissue adhesion, immune evasion, and other host interactions. Many B. burgdorferi lipoproteins are encoded as multi-copy gene families, such as the OspE/F-like leader peptide (Elp) protein family, which inhibits classical complement activation by binding complement C1s. To characterize Elp allelic variants, we adapted the Pseudomonas syringae ice nucleation protein (INP) system to present B. burgdorferi lipoproteins on the surface of Escherichia coli. Using this system, we identified interactions with classical complement proteins and mapped binding regions, then validated interactions using recombinant proteins and B. burgdorferi surface display. We also discovered a novel potential interaction between Elp proteins and the mammalian basement membrane protein perlecan, thus revealing a bifunctional nature of Elps. Our findings indicate that Elps have undergone functional diversification while maintaining classical complement inhibition mediated by potent and conserved C1s binding and demonstrate that E. coli surface display offers an efficient, cost-effective, and relatively high-throughput approach to characterize B. burgdorferi lipoproteins.
{"title":"Heterologous Surface Display Reveals Conserved Complement Inhibition and Functional Diversification of Borrelia burgdorferi Elp Proteins","authors":"Nathan Hill, Lara M. Matulina, Cameron MacIntyre, M. Amine Hassani, Sheila Thomas, Matteo Luban, Isabelle Ward, Amina Abdalla, John M. Leong, Brandon L. Garcia, Jacob E. Lemieux","doi":"10.1111/mmi.15369","DOIUrl":"https://doi.org/10.1111/mmi.15369","url":null,"abstract":"Lyme disease is a tick-borne spirochetosis with diverse clinical manifestations. Genotypic and phenotypic variation among <i>Borrelia burgdorferi</i> strains correlates with variable manifestations of Lyme disease in humans; this diversity is attributed in part to variation in surface-exposed lipoproteins, which are targets of the human antibody response and contribute to tissue adhesion, immune evasion, and other host interactions. Many <i>B. burgdorferi</i> lipoproteins are encoded as multi-copy gene families, such as the OspE/F-like leader peptide (Elp) protein family, which inhibits classical complement activation by binding complement C1s. To characterize Elp allelic variants, we adapted the <i>Pseudomonas syringae</i> ice nucleation protein (INP) system to present <i>B. burgdorferi</i> lipoproteins on the surface of <i>Escherichia coli</i>. Using this system, we identified interactions with classical complement proteins and mapped binding regions, then validated interactions using recombinant proteins and <i>B. burgdorferi</i> surface display. We also discovered a novel potential interaction between Elp proteins and the mammalian basement membrane protein perlecan, thus revealing a bifunctional nature of Elps. Our findings indicate that Elps have undergone functional diversification while maintaining classical complement inhibition mediated by potent and conserved C1s binding and demonstrate that <i>E. coli</i> surface display offers an efficient, cost-effective, and relatively high-throughput approach to characterize <i>B. burgdorferi</i> lipoproteins.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"11 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144066647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
All methanogens that can fix nitrogen use molybdenum (Mo) nitrogenase. Some methanogens, including Methanosarcina acetivorans, also contain alternative vanadium- and iron-nitrogenases, encoded by the vnf and anf operons, respectively. These nitrogenases are produced when there is insufficient Mo to support Mo-nitrogenase activity. The factors that control the expression of the alternative nitrogenases in response to Mo availability are unknown in methanogens. Here we show that ModE is the regulator that represses transcription of the vnf and anf operons in M. acetivorans when cells are grown with Mo. CRISPRi repression of modE results in a significant increase in the transcription of the vnf and anf operons as well as the detection of Fe-nitrogenase during nitrogen fixation in the presence of Mo. Gel shift assays with recombinant ModE demonstrated that ModE binds a specific sequence motif upstream of the vnf and anf operons, as well as other genes and operons related to nitrogen fixation and Mo transport. However, purified ModE does not contain Mo, and the addition of Mo does not alter the affinity of ModE for DNA, indicating M. acetivorans ModE may not directly bind Mo. This study shows that ModE is the primary Mo-responsive regulator of alternative nitrogenase expression in M. acetivorans, but other factor(s) are likely involved in directly sensing Mo.
{"title":"ModE Regulates Alternative Nitrogenase Expression in the Methanogen Methanosarcina acetivorans.","authors":"Melissa Chanderban,Daniel J Lessner","doi":"10.1111/mmi.15377","DOIUrl":"https://doi.org/10.1111/mmi.15377","url":null,"abstract":"All methanogens that can fix nitrogen use molybdenum (Mo) nitrogenase. Some methanogens, including Methanosarcina acetivorans, also contain alternative vanadium- and iron-nitrogenases, encoded by the vnf and anf operons, respectively. These nitrogenases are produced when there is insufficient Mo to support Mo-nitrogenase activity. The factors that control the expression of the alternative nitrogenases in response to Mo availability are unknown in methanogens. Here we show that ModE is the regulator that represses transcription of the vnf and anf operons in M. acetivorans when cells are grown with Mo. CRISPRi repression of modE results in a significant increase in the transcription of the vnf and anf operons as well as the detection of Fe-nitrogenase during nitrogen fixation in the presence of Mo. Gel shift assays with recombinant ModE demonstrated that ModE binds a specific sequence motif upstream of the vnf and anf operons, as well as other genes and operons related to nitrogen fixation and Mo transport. However, purified ModE does not contain Mo, and the addition of Mo does not alter the affinity of ModE for DNA, indicating M. acetivorans ModE may not directly bind Mo. This study shows that ModE is the primary Mo-responsive regulator of alternative nitrogenase expression in M. acetivorans, but other factor(s) are likely involved in directly sensing Mo.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"137 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143932897","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cheyenne D. Lee, Arshad Rizvi, Zavier A. Carter, Adrianne N. Edwards, Shonna M. McBride
Clostridioides difficile is an anaerobic enteric pathogen that disseminates in the environment as a dormant spore. For C. difficile and other sporulating bacteria, the initiation of sporulation is a regulated process that prevents spore formation under favorable growth conditions. In Bacillus subtilis, one such mechanism for preventing sporulation is the prokaryotic 5-oxoprolinase, PxpB (KipI), which impedes the activation of the main sporulation kinase. In addition, PxpB functions as part of a complex that detoxifies the intermediate metabolite, 5-oxoproline (OP), a harmful by-product of glutamic acid and its derivatives. In this study, we investigate the orthologous Pxp proteins in C. difficile to determine their roles in the regulation of sporulation and metabolism. Through deletion of the pxpAGBC operon, we show that, unlike in B. subtilis, the Pxp (Kip) proteins have no significant impact on sporulation. However, we found that the pxp operon encodes a functional oxoprolinase that facilitates detoxification of OP. Furthermore, our data demonstrate that PxpAGBC not only detoxifies OP but also allows OP to be used as a nutrient source that supports the growth of C. difficile, thereby facilitating the conversion of a toxic by-product of metabolism into an energy source.
{"title":"The Pxp Complex Detoxifies 5-Oxoproline and Promotes the Growth of Clostridioides difficile","authors":"Cheyenne D. Lee, Arshad Rizvi, Zavier A. Carter, Adrianne N. Edwards, Shonna M. McBride","doi":"10.1111/mmi.15373","DOIUrl":"https://doi.org/10.1111/mmi.15373","url":null,"abstract":"<i>Clostridioides difficile</i> is an anaerobic enteric pathogen that disseminates in the environment as a dormant spore. For <i>C. difficile</i> and other sporulating bacteria, the initiation of sporulation is a regulated process that prevents spore formation under favorable growth conditions. In <i>Bacillus subtilis</i>, one such mechanism for preventing sporulation is the prokaryotic 5-oxoprolinase, PxpB (KipI), which impedes the activation of the main sporulation kinase. In addition, PxpB functions as part of a complex that detoxifies the intermediate metabolite, 5-oxoproline (OP), a harmful by-product of glutamic acid and its derivatives. In this study, we investigate the orthologous Pxp proteins in <i>C. difficile</i> to determine their roles in the regulation of sporulation and metabolism. Through deletion of the <i>pxpAGBC</i> operon, we show that, unlike in <i>B. subtilis,</i> the Pxp (Kip) proteins have no significant impact on sporulation. However, we found that the <i>pxp</i> operon encodes a functional oxoprolinase that facilitates detoxification of OP. Furthermore, our data demonstrate that PxpAGBC not only detoxifies OP but also allows OP to be used as a nutrient source that supports the growth of <i>C. difficile</i>, thereby facilitating the conversion of a toxic by-product of metabolism into an energy source.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"37 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143920960","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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