Translesion DNA synthesis (TLS) is a fundamental biological process that enables DNA replication through various lesions to ensure genome stability and to prevent cell death due to replication fork collapse. Rev1, a member of Y-family DNA polymerase (Pol), functions in concert with a B-family enzyme Polζ in promoting TLS through various lesions. Interestingly, for such a function, the catalytic activity of Rev1 seems to be dispensable in Saccharomyces cerevisiae. Unlike Polζ, which possesses robust DNA polymerase activity, biochemical assays suggest that Rev1 predominantly incorporates a "C" opposite any templating residues, but the biological relevance of this activity of Rev1 remains elusive. Here we characterized Rev1 from Candida albicans, an opportunistic fungal pathogen responsible for maximum casualties due to systemic candidiasis in immunosuppressed individuals. Concerted genetic analyses of several Rev1 mutants in various DNA-damaging conditions suggested that in most lesion bypasses except 4-NQO-induced DNA lesions, the catalytic role of Rev1 is not important. However, simultaneous interactions of BRCT and the C-terminal domain of Rev1 with PCNA and Polζ, respectively, enable Rev1 to be essential during TLS. DNA damage recovery and mutagenesis assays further confirmed the lesion-specific roles of various domains of Rev1. Contrary to ex vivo data, animal studies suggested that CaRev1 is dispensable for systemic candidiasis development. We discuss the possible involvement of other TLS DNA polymerases in DNA damage response while C. albicans replicates and establishes itself in the host.
翻译DNA合成（transesion DNA synthesis， TLS）是一个基本的生物学过程，它使DNA能够通过各种病变进行复制，以确保基因组的稳定性，并防止复制叉崩溃导致细胞死亡。Rev1是y家族DNA聚合酶（Pol）的成员，与b家族酶Polζ协同作用，促进TLS通过各种病变。有趣的是，对于这样的功能，Rev1的催化活性似乎在酿酒酵母中是必不可少的。与Polζ不同，它具有强大的DNA聚合酶活性，生化分析表明Rev1主要包含与任何模板残基相反的“C”，但Rev1活性的生物学相关性仍然难以捉摸。在这里，我们鉴定了来自白色念珠菌的Rev1，这是一种机会性真菌病原体，在免疫抑制的个体中，由于全身念珠菌病造成的最大伤亡。对不同DNA损伤条件下几种Rev1突变体的一致遗传分析表明，除了4- nqo诱导的DNA损伤外，在大多数病变旁路中，Rev1的催化作用并不重要。然而，BRCT和Rev1的c端结构域分别与PCNA和Polζ同时相互作用，使得Rev1在TLS过程中至关重要。DNA损伤恢复和突变分析进一步证实了Rev1不同结构域的损伤特异性作用。与离体数据相反，动物研究表明，CaRev1对于全身念珠菌病的发展是必不可少的。我们讨论了当白色念珠菌复制并在宿主中建立自己时，其他TLS DNA聚合酶可能参与DNA损伤反应。
{"title":"Enzymatic and Structural Roles of Candida albicans Rev1 in DNA Damage Response and Disseminated Candidiasis.","authors":"Satya Ranjan Sahu,Sushree Subhashree Parida,Bhabasha Gyanadeep Utkalaja,Shreenath Nayak,Amrita Dalei,Narottam Acharya","doi":"10.1111/mmi.70013","DOIUrl":"https://doi.org/10.1111/mmi.70013","url":null,"abstract":"Translesion DNA synthesis (TLS) is a fundamental biological process that enables DNA replication through various lesions to ensure genome stability and to prevent cell death due to replication fork collapse. Rev1, a member of Y-family DNA polymerase (Pol), functions in concert with a B-family enzyme Polζ in promoting TLS through various lesions. Interestingly, for such a function, the catalytic activity of Rev1 seems to be dispensable in Saccharomyces cerevisiae. Unlike Polζ, which possesses robust DNA polymerase activity, biochemical assays suggest that Rev1 predominantly incorporates a \"C\" opposite any templating residues, but the biological relevance of this activity of Rev1 remains elusive. Here we characterized Rev1 from Candida albicans, an opportunistic fungal pathogen responsible for maximum casualties due to systemic candidiasis in immunosuppressed individuals. Concerted genetic analyses of several Rev1 mutants in various DNA-damaging conditions suggested that in most lesion bypasses except 4-NQO-induced DNA lesions, the catalytic role of Rev1 is not important. However, simultaneous interactions of BRCT and the C-terminal domain of Rev1 with PCNA and Polζ, respectively, enable Rev1 to be essential during TLS. DNA damage recovery and mutagenesis assays further confirmed the lesion-specific roles of various domains of Rev1. Contrary to ex vivo data, animal studies suggested that CaRev1 is dispensable for systemic candidiasis development. We discuss the possible involvement of other TLS DNA polymerases in DNA damage response while C. albicans replicates and establishes itself in the host.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"13 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144646020","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}
Bacterial cells activate stress‐sensing and stress‐mitigating pathways by employing a number of transcription regulators, some of which can bind directly to RNA polymerase to activate stress‐specific response pathways. However, mutations in the RNA polymerase genes can accumulate under certain selection conditions and activate stress‐mitigating pathways in a manner that is partly independent of pathway‐specific regulators. In this study, we characterized a novel mutation in the rpoB gene that transforms RNA polymerase into a “stringent” polymerase in the absence of one of the key stringent response (SR) activating factors (p)ppGpp, produced by the relA gene product. The mutant RNA polymerase allele, rpoB58, elevated thermotolerance and permitted growth without the key molecular chaperones (DnaKJ) and proteases (Lon, ClpP) at temperatures nonpermissive to cells expressing the wild type RNA polymerase genes. Remarkably, rpoB58 also reversed the cell division defect of ΔdnaJ at a nonpermissive temperature but could not overcome its lambda phage‐resistant phenotype. The rpoB58‐mediated rescue of the ΔdnaKJ growth defect was partly reversed in the absence of DksA, a protein that acts synergistically with (p)ppGpp to transform RNA polymerase into a stringent state. The data suggest that pre‐activated SR confers thermotolerance and chaperone independence in part by lowering protein synthesis.
{"title":"Characterization of a Novel RNA Polymerase Mutant of Escherichia coli That Confers Thermotolerance and Chaperone Independence","authors":"Melody Yeh, Keilen Kelly, Rajeev Misra","doi":"10.1111/mmi.70011","DOIUrl":"https://doi.org/10.1111/mmi.70011","url":null,"abstract":"Bacterial cells activate stress‐sensing and stress‐mitigating pathways by employing a number of transcription regulators, some of which can bind directly to RNA polymerase to activate stress‐specific response pathways. However, mutations in the RNA polymerase genes can accumulate under certain selection conditions and activate stress‐mitigating pathways in a manner that is partly independent of pathway‐specific regulators. In this study, we characterized a novel mutation in the <jats:italic>rpoB</jats:italic> gene that transforms RNA polymerase into a “stringent” polymerase in the absence of one of the key stringent response (SR) activating factors (p)ppGpp, produced by the <jats:italic>relA</jats:italic> gene product. The mutant RNA polymerase allele, <jats:italic>rpoB58</jats:italic>, elevated thermotolerance and permitted growth without the key molecular chaperones (DnaKJ) and proteases (Lon, ClpP) at temperatures nonpermissive to cells expressing the wild type RNA polymerase genes. Remarkably, <jats:italic>rpoB58</jats:italic> also reversed the cell division defect of Δ<jats:italic>dnaJ</jats:italic> at a nonpermissive temperature but could not overcome its lambda phage‐resistant phenotype. The <jats:italic>rpoB58</jats:italic>‐mediated rescue of the Δ<jats:italic>dnaKJ</jats:italic> growth defect was partly reversed in the absence of DksA, a protein that acts synergistically with (p)ppGpp to transform RNA polymerase into a stringent state. The data suggest that pre‐activated SR confers thermotolerance and chaperone independence in part by lowering protein synthesis.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"687 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144593957","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}
Jérôme Becam, Maxence Dessertine, Alexandra Vergnes, Laurent Aussel, Benjamin Ezraty
The periplasmic multicopper oxidase CueO plays a crucial role in copper detoxification in enterobacteria. CueO contains a catalytic site, the Cu‐T1 center, and a methionine‐rich (Met‐rich) domain capable of binding copper. This enzyme oxidizes cuprous ions (Cu+) to the less toxic cupric ions (Cu2+), coupled with oxygen reduction. This oxygen dependence has established CueO's role in alleviating copper stress under aerobic conditions, but its function in anaerobic environments remains uncertain. In this study, we demonstrated that under anaerobic conditions and copper stress in E. coli and S. Typhimurium, CueO is produced and contributes to copper homeostasis through its catalytic activity. Using CueO variants with either a mutated catalytic site or a deleted Met‐rich domain, we showed that CueO's catalytic activity, rather than its copper‐binding capacity, is essential for copper resistance. Additionally, we found that deleting other copper homeostasis systems in E. coli, the inner membrane transporter CopA and the efflux pump CusCBA, leads to the overproduction of CueO under anaerobic conditions. This overproduction enhances the copper resistance of the ∆copA ∆cusB strain. Overall, our findings provide evidence for CueO's role in copper detoxification under anaerobic conditions, highlighting its importance in such environments, that is, host–pathogen interactions or biofilm formation.
{"title":"Role of the Multicopper Oxidase CueO in Copper Homeostasis Under Anaerobic Conditions in Enterobacteria","authors":"Jérôme Becam, Maxence Dessertine, Alexandra Vergnes, Laurent Aussel, Benjamin Ezraty","doi":"10.1111/mmi.70010","DOIUrl":"https://doi.org/10.1111/mmi.70010","url":null,"abstract":"The periplasmic multicopper oxidase CueO plays a crucial role in copper detoxification in enterobacteria. CueO contains a catalytic site, the Cu‐T1 center, and a methionine‐rich (Met‐rich) domain capable of binding copper. This enzyme oxidizes cuprous ions (Cu<jats:sup>+</jats:sup>) to the less toxic cupric ions (Cu<jats:sup>2+</jats:sup>), coupled with oxygen reduction. This oxygen dependence has established CueO's role in alleviating copper stress under aerobic conditions, but its function in anaerobic environments remains uncertain. In this study, we demonstrated that under anaerobic conditions and copper stress in <jats:styled-content style=\"fixed-case\"><jats:italic>E. coli</jats:italic></jats:styled-content> and <jats:styled-content style=\"fixed-case\"><jats:italic>S.</jats:italic></jats:styled-content> Typhimurium, CueO is produced and contributes to copper homeostasis through its catalytic activity. Using CueO variants with either a mutated catalytic site or a deleted Met‐rich domain, we showed that CueO's catalytic activity, rather than its copper‐binding capacity, is essential for copper resistance. Additionally, we found that deleting other copper homeostasis systems in <jats:styled-content style=\"fixed-case\"><jats:italic>E. coli</jats:italic></jats:styled-content>, the inner membrane transporter CopA and the efflux pump CusCBA, leads to the overproduction of CueO under anaerobic conditions. This overproduction enhances the copper resistance of the ∆<jats:italic>copA</jats:italic> ∆<jats:italic>cusB</jats:italic> strain. Overall, our findings provide evidence for CueO's role in copper detoxification under anaerobic conditions, highlighting its importance in such environments, that is, host–pathogen interactions or biofilm formation.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"199 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577948","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}
Albertina Scattolini,Joaquín Costa,Tulio L Pianessi,Antonio D Uttaro,María C Mansilla
Lipoylation is a post-translational modification in which lipoic acid is attached to specific apoproteins of enzyme complexes, like E2 subunits of dehydrogenases or GcvH of the glycine cleavage system. A defining feature of organisms with a lipoyl-relay system is the presence of amidotransferase activity, which enables the transfer of lipoyl groups attached to intermediary proteins to the E2 subunits. In this study, we characterized the lipoate metabolism of Capsaspora owczarzaki and Plasmodium falciparum. Both organisms utilize amidotransferases in their lipoylation pathways, with the filasterian enzyme playing a key role in lipoate synthesis, while the apicomplexan counterpart, previously considered a lipoyltransferase, is essential in its lipoate salvage pathway. We also discovered that specific structural features and certain conserved domains in eukaryotic amidotransferases can significantly influence their mechanism of action and susceptibility to the lipoate analog bromooctanoate. Overall, this study highlights the metabolic strategies of C. owczarzaki and emphasizes the critical role of amidotransferases as ancestral enzymes in the evolution of lipoate metabolism, suggesting that the lipoyl relay may represent a universal pathway across diverse clades.
{"title":"Impairment of Lipoylation Mediated by Bromooctanoate Targets Eukaryotic Amidotransferases.","authors":"Albertina Scattolini,Joaquín Costa,Tulio L Pianessi,Antonio D Uttaro,María C Mansilla","doi":"10.1111/mmi.70007","DOIUrl":"https://doi.org/10.1111/mmi.70007","url":null,"abstract":"Lipoylation is a post-translational modification in which lipoic acid is attached to specific apoproteins of enzyme complexes, like E2 subunits of dehydrogenases or GcvH of the glycine cleavage system. A defining feature of organisms with a lipoyl-relay system is the presence of amidotransferase activity, which enables the transfer of lipoyl groups attached to intermediary proteins to the E2 subunits. In this study, we characterized the lipoate metabolism of Capsaspora owczarzaki and Plasmodium falciparum. Both organisms utilize amidotransferases in their lipoylation pathways, with the filasterian enzyme playing a key role in lipoate synthesis, while the apicomplexan counterpart, previously considered a lipoyltransferase, is essential in its lipoate salvage pathway. We also discovered that specific structural features and certain conserved domains in eukaryotic amidotransferases can significantly influence their mechanism of action and susceptibility to the lipoate analog bromooctanoate. Overall, this study highlights the metabolic strategies of C. owczarzaki and emphasizes the critical role of amidotransferases as ancestral enzymes in the evolution of lipoate metabolism, suggesting that the lipoyl relay may represent a universal pathway across diverse clades.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"104 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144547882","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}
Chenyu Zhang, Xinyi Liu, Song Zhang, Jun Liu, Zhengyan Wu, Qi Yang, Qing Liu, Bo Zhou, Qinlu Lin, Chenglun Tang
Chitin biosynthesis is intricately linked to the cellular growth and secondary metabolism of microorganisms. Our previous research has evaluated the role of the chs6 gene in modulating spore germination, mycelial morphology, and Monascus pigment biosynthesis in M. purpureus. However, the functions of other chs genes in theses contexts remain largely unexplored. The genes chsG, chsA, chs5, and chs2 were overexpressed in M. purpureus M183 to assess their diverse impacts on cell growth, Monascus pigments (MPs) and citrinin biosynthesis. The results indicated that chsG overexpression had the most significant effects, particularly enhancing MPs and citrinin synthesis while inhibiting transmembrane secretion. Morphological analysis revealed a substantial reduction in the length of mycelium of M. purpureus M183 following the overexpression of these chs genes. Furthermore, the surface of the mycelium pellets from these mutants displayed a more flocculent and roughened texture during SBF compared to M. purpureus M183. Notably, M. purpureus oe:chsG was characterized by conspicuously bolder mycelia, a denser cell wall, and darker cytoplasm. RT‐qPCR results demonstrated that the chsG mRNA level increased by 11.9‐fold in M. purpureus oe:chsG, and the individual overexpression of the genes chs5 and chsA triggered notable elevations in the chsG mRNA level. A comparative transcriptome analysis uncovered profound alterations in the expression patterns of genes associated with biosynthetic pathways of MPs, citrinin, fatty acid, and amino acid metabolism, as well as morphological regulation and growth, including the chitin and ergosterol biosynthetic pathways, MAPK signal pathway, global transcription factors, and peroxisomes.
{"title":"Multifaceted Roles of chs Genes in Regulating Cell Growth, Mycelial Morphology, Monascus Pigments and Citrinin Biosynthesis in Monascus purpureus","authors":"Chenyu Zhang, Xinyi Liu, Song Zhang, Jun Liu, Zhengyan Wu, Qi Yang, Qing Liu, Bo Zhou, Qinlu Lin, Chenglun Tang","doi":"10.1111/mmi.70008","DOIUrl":"https://doi.org/10.1111/mmi.70008","url":null,"abstract":"Chitin biosynthesis is intricately linked to the cellular growth and secondary metabolism of microorganisms. Our previous research has evaluated the role of the <jats:italic>chs6</jats:italic> gene in modulating spore germination, mycelial morphology, and <jats:italic>Monascus</jats:italic> pigment biosynthesis in <jats:styled-content style=\"fixed-case\"><jats:italic>M. purpureus</jats:italic></jats:styled-content>. However, the functions of other <jats:italic>chs</jats:italic> genes in theses contexts remain largely unexplored. The genes <jats:italic>chsG</jats:italic>, <jats:italic>chsA</jats:italic>, <jats:italic>chs5</jats:italic>, and <jats:italic>chs2</jats:italic> were overexpressed in <jats:styled-content style=\"fixed-case\"><jats:italic>M. purpureus</jats:italic></jats:styled-content> M183 to assess their diverse impacts on cell growth, <jats:italic>Monascus</jats:italic> pigments (MPs) and citrinin biosynthesis. The results indicated that <jats:italic>chsG</jats:italic> overexpression had the most significant effects, particularly enhancing MPs and citrinin synthesis while inhibiting transmembrane secretion. Morphological analysis revealed a substantial reduction in the length of mycelium of <jats:styled-content style=\"fixed-case\"><jats:italic>M. purpureus</jats:italic></jats:styled-content> M183 following the overexpression of these <jats:italic>chs</jats:italic> genes. Furthermore, the surface of the mycelium pellets from these mutants displayed a more flocculent and roughened texture during SBF compared to <jats:styled-content style=\"fixed-case\"><jats:italic>M. purpureus</jats:italic></jats:styled-content> M183. Notably, <jats:styled-content style=\"fixed-case\"><jats:italic>M. purpureus</jats:italic></jats:styled-content> oe:<jats:italic>chsG</jats:italic> was characterized by conspicuously bolder mycelia, a denser cell wall, and darker cytoplasm. RT‐qPCR results demonstrated that the <jats:italic>chsG</jats:italic> mRNA level increased by 11.9‐fold in <jats:styled-content style=\"fixed-case\"><jats:italic>M. purpureus</jats:italic></jats:styled-content> oe:<jats:italic>chsG</jats:italic>, and the individual overexpression of the genes <jats:italic>chs5</jats:italic> and <jats:italic>chsA</jats:italic> triggered notable elevations in the <jats:italic>chsG</jats:italic> mRNA level. A comparative transcriptome analysis uncovered profound alterations in the expression patterns of genes associated with biosynthetic pathways of MPs, citrinin, fatty acid, and amino acid metabolism, as well as morphological regulation and growth, including the chitin and ergosterol biosynthetic pathways, MAPK signal pathway, global transcription factors, and peroxisomes.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"36 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144547025","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}
Pub Date : 2025-07-01Epub Date: 2025-05-29DOI: 10.1111/mmi.15372
{"title":"Correction to \"Galactomannan Utilization by Cellvibrio japonicus Relies on a Single Essential α-Galactosidase Encoded by the aga27A Gene\".","authors":"","doi":"10.1111/mmi.15372","DOIUrl":"10.1111/mmi.15372","url":null,"abstract":"","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":" ","pages":"102"},"PeriodicalIF":2.6,"publicationDate":"2025-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144174187","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}
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":"https://doi.org/10.1111/mmi.70006","url":null,"abstract":"The bacterial cell envelope is essential for viability and host interaction. In the intracellular pathogen <jats:styled-content style=\"fixed-case\"><jats:italic>Brucella ovis</jats:italic></jats:styled-content>, 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 <jats:italic>phyK</jats:italic> (∆<jats:italic>phyK</jats:italic>) 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 <jats:styled-content style=\"fixed-case\"><jats:italic>B. ovis</jats:italic></jats:styled-content> exposed to an anionic detergent, indicating that loss of PhyK function primes cells to resist this stress. Despite its altered cell envelope properties, the ∆<jats:italic>phyK</jats:italic> 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 <jats:styled-content style=\"fixed-case\"><jats:italic>B. ovis</jats:italic></jats:styled-content> cell envelope properties that can modulate host immune interactions.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"643 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144503512","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}
Tiffany M. Zarrella, Jianle Gao, Nathan Forrest, Elijah Crosbourne, Kaibo Cui, Guangchun Bai
Competence is an important bioprocess for Streptococcus pneumoniae. Previously, we demonstrated that the bacterial second messenger cyclic di‐adenosine monophosphate (c‐di‐AMP) modulates pneumococcal competence. Surprisingly, cdaA*, a strain producing less c‐di‐AMP due to a point mutation in the diadenylate cyclase CdaA, is susceptible to competence‐stimulating peptide (CSP). In this study, we screened cdaA* suppressor mutants resistant to CSP to explore the underlying mechanism. Of 14 clones sequenced, nine clones possessed mutations in the c‐di‐AMP phosphodiesterase Pde1, indicating that the susceptibility to CSP of the cdaA* strain is correlated to c‐di‐AMP levels. Another two clones exhibited a mutation in FabT, a transcription factor controlling cell membrane fatty acid biosynthesis. We further showed that deletion of fabT, disruption of the FabT‐binding site within the PfabK promoter, deletion of a fabT activator BriC, or disruption of K+ uptake in the cdaA* mutant all rescued the growth defect of the cdaA* strain in media supplemented with CSP. Finally, we found that a c‐di‐AMP phosphodiesterase‐null mutant with high levels of c‐di‐AMP is highly sensitive to treatment with either ethanol or Triton X‐100, which could be corrected by reducing c‐di‐AMP levels through introducing point mutations in CdaA. Together, these findings indicate that c‐di‐AMP affects cell membrane integrity.
{"title":"Cyclic Di‐AMP Affects Cell Membrane Integrity of Streptococcus pneumoniae","authors":"Tiffany M. Zarrella, Jianle Gao, Nathan Forrest, Elijah Crosbourne, Kaibo Cui, Guangchun Bai","doi":"10.1111/mmi.70003","DOIUrl":"https://doi.org/10.1111/mmi.70003","url":null,"abstract":"Competence is an important bioprocess for <jats:styled-content style=\"fixed-case\"><jats:italic>Streptococcus pneumoniae</jats:italic></jats:styled-content>. Previously, we demonstrated that the bacterial second messenger cyclic di‐adenosine monophosphate (c‐di‐AMP) modulates pneumococcal competence. Surprisingly, <jats:italic>cdaA*</jats:italic>, a strain producing less c‐di‐AMP due to a point mutation in the diadenylate cyclase CdaA, is susceptible to competence‐stimulating peptide (CSP). In this study, we screened <jats:italic>cdaA</jats:italic>* suppressor mutants resistant to CSP to explore the underlying mechanism. Of 14 clones sequenced, nine clones possessed mutations in the c‐di‐AMP phosphodiesterase Pde1, indicating that the susceptibility to CSP of the <jats:italic>cdaA</jats:italic>* strain is correlated to c‐di‐AMP levels. Another two clones exhibited a mutation in FabT, a transcription factor controlling cell membrane fatty acid biosynthesis. We further showed that deletion of <jats:italic>fabT</jats:italic>, disruption of the FabT‐binding site within the P<jats:sub><jats:italic>fabK</jats:italic></jats:sub> promoter, deletion of a <jats:italic>fabT</jats:italic> activator BriC, or disruption of K<jats:sup>+</jats:sup> uptake in the <jats:italic>cdaA</jats:italic>* mutant all rescued the growth defect of the <jats:italic>cdaA</jats:italic>* strain in media supplemented with CSP. Finally, we found that a c‐di‐AMP phosphodiesterase‐null mutant with high levels of c‐di‐AMP is highly sensitive to treatment with either ethanol or Triton X‐100, which could be corrected by reducing c‐di‐AMP levels through introducing point mutations in CdaA. Together, these findings indicate that c‐di‐AMP affects cell membrane integrity.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"10 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144503509","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}
Jingjing Li, Nora E. Schmitte, Kaya Törkel, Christiane Dahl
Bacteria have evolved multiple strategies to sense and respond to the availability of inorganic reduced sulfur compounds such as thiosulfate. In Hyphomicrobium denitrificans, an obligately chemoorganoheterotrophic Alphaproteobacterium, the use of thiosulfate as a supplemental electron donor is regulated by two homologous sulfane‐sulfur‐responsive ArsR‐type transcriptional repressors, sHdrR and SoxR. Here, we provide information on the distribution and phylogeny of sHdrR, the relevance of its two conserved cysteines in vivo, and identify the genes controlled by SoxR and sHdrR not only by targeted qRT‐PCR but also by global RNA‐Seq‐based analyses of regulator‐deficient mutant strains. The absence of sHdrR and SoxR affected 165 and 170 genes, respectively, with 138 genes overlapping. SoxR regulates the sox genes for periplasmic thiosulfate oxidation and sulfane sulfur import into the cytoplasm, as well as the lip‐shdr‐lbpA genes encoding the cytoplasmic enzymes essential for sulfite formation. sHdrR affects only a subset of these genes. The transcription of sox genes remains unaltered in its absence. sHdrR and SoxR act cooperatively and their activity probably also involves interaction with other transcriptional regulators. Most importantly, sHdrR/SoxR regulation extends far beyond sulfur oxidation and deeply affects anaerobic metabolism, particularly denitrification in H. denitrificans.
{"title":"In Hyphomicrobium denitrificans Two Related Sulfane‐Sulfur Responsive Transcriptional Repressors Regulate Thiosulfate Oxidation and Have a Deep Impact on Nitrate Respiration and Anaerobic Biosyntheses","authors":"Jingjing Li, Nora E. Schmitte, Kaya Törkel, Christiane Dahl","doi":"10.1111/mmi.70002","DOIUrl":"https://doi.org/10.1111/mmi.70002","url":null,"abstract":"Bacteria have evolved multiple strategies to sense and respond to the availability of inorganic reduced sulfur compounds such as thiosulfate. In <jats:styled-content style=\"fixed-case\"><jats:italic>Hyphomicrobium denitrificans</jats:italic></jats:styled-content>, an obligately chemoorganoheterotrophic Alphaproteobacterium, the use of thiosulfate as a supplemental electron donor is regulated by two homologous sulfane‐sulfur‐responsive ArsR‐type transcriptional repressors, sHdrR and SoxR. Here, we provide information on the distribution and phylogeny of sHdrR, the relevance of its two conserved cysteines in vivo, and identify the genes controlled by SoxR and sHdrR not only by targeted qRT‐PCR but also by global RNA‐Seq‐based analyses of regulator‐deficient mutant strains. The absence of sHdrR and SoxR affected 165 and 170 genes, respectively, with 138 genes overlapping. SoxR regulates the <jats:italic>sox</jats:italic> genes for periplasmic thiosulfate oxidation and sulfane sulfur import into the cytoplasm, as well as the <jats:italic>lip‐shdr‐lbpA</jats:italic> genes encoding the cytoplasmic enzymes essential for sulfite formation. sHdrR affects only a subset of these genes. The transcription of <jats:italic>sox</jats:italic> genes remains unaltered in its absence. sHdrR and SoxR act cooperatively and their activity probably also involves interaction with other transcriptional regulators. Most importantly, sHdrR/SoxR regulation extends far beyond sulfur oxidation and deeply affects anaerobic metabolism, particularly denitrification in <jats:styled-content style=\"fixed-case\"><jats:italic>H. denitrificans</jats:italic></jats:styled-content>.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"630 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144500478","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}
{"title":"Correction to ‘TasA Fibre Interactions Are Necessary for Bacillus subtilis Biofilm Structure’","authors":"","doi":"10.1111/mmi.70004","DOIUrl":"https://doi.org/10.1111/mmi.70004","url":null,"abstract":"","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"9 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144500546","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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