During one round of DNA replication, nearly 2000 ribonucleoside monophosphates (rNMPs) are incorporated in place of their cognate deoxyribonucleoside monophosphates (dNMPs). Given their high rate of insertion, genomic DNA could contain rNMPs that are damaged or mismatched. Here, we test the activity of Bacillus subtilis and Escherichia coli RNase HII on canonical, mismatched, and damaged rNMPs. We show that E. coli RNase HII is adept at incising most rNMP variants from DNA at similar frequencies, with the exception of an oxidized rNMP, where endoribonuclease activity is sharply reduced. In contrast, B. subtilis RNase HII efficiently incises rAMP, rCMP, and rUMP but is inefficient at processing rGMP in both a canonical and mismatched base pair. We test damaged ribonucleotides and find that B. subtilis RNase HII is refractory to processing abasic and oxidized ribonucleotide lesions. Our work shows that bacterial RNase HII enzymes have different intrinsic endoribonuclease activity toward the repair of canonical, mismatched, and damaged rNMPs, demonstrating that not all rNMP errors provoke efficient resolution. Our finding that B. subtilis RNase HII is recalcitrant to repairing damaged rNMPs resembles what is observed for eukaryotic RNase H2 orthologs, suggesting that other repair processes are necessary to resolve damaged rNMPs.
{"title":" Bacillus subtilis RNase HII Is Inefficient at Processing Guanosine Monophosphate and Damaged Ribonucleotides","authors":"Julianna R. Cresti, Lyle A. Simmons","doi":"10.1111/mmi.70047","DOIUrl":"https://doi.org/10.1111/mmi.70047","url":null,"abstract":"During one round of DNA replication, nearly 2000 ribonucleoside monophosphates (rNMPs) are incorporated in place of their cognate deoxyribonucleoside monophosphates (dNMPs). Given their high rate of insertion, genomic DNA could contain rNMPs that are damaged or mismatched. Here, we test the activity of <jats:styled-content style=\"fixed-case\"> <jats:italic>Bacillus subtilis</jats:italic> </jats:styled-content> and <jats:styled-content style=\"fixed-case\"> <jats:italic>Escherichia coli</jats:italic> </jats:styled-content> RNase HII on canonical, mismatched, and damaged rNMPs. We show that <jats:styled-content style=\"fixed-case\"> <jats:italic>E. coli</jats:italic> </jats:styled-content> RNase HII is adept at incising most rNMP variants from DNA at similar frequencies, with the exception of an oxidized rNMP, where endoribonuclease activity is sharply reduced. In contrast, <jats:styled-content style=\"fixed-case\"> <jats:italic>B. subtilis</jats:italic> </jats:styled-content> RNase HII efficiently incises rAMP, rCMP, and rUMP but is inefficient at processing rGMP in both a canonical and mismatched base pair. We test damaged ribonucleotides and find that <jats:styled-content style=\"fixed-case\"> <jats:italic>B. subtilis</jats:italic> </jats:styled-content> RNase HII is refractory to processing abasic and oxidized ribonucleotide lesions. Our work shows that bacterial RNase HII enzymes have different intrinsic endoribonuclease activity toward the repair of canonical, mismatched, and damaged rNMPs, demonstrating that not all rNMP errors provoke efficient resolution. Our finding that <jats:styled-content style=\"fixed-case\"> <jats:italic>B. subtilis</jats:italic> </jats:styled-content> RNase HII is recalcitrant to repairing damaged rNMPs resembles what is observed for eukaryotic RNase H2 orthologs, suggesting that other repair processes are necessary to resolve damaged rNMPs.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"30 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145903603","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}
Luisa Pantoja, Pilar Vesga, Ziv Arbeli, Vivian Boyacá‐Vásquez, Javier Vanegas
Tecia solanivora (The Guatemalan potato tuber moth) is a major potato pest, responsible for up to 20% of crop losses and a significant economic impact. Certain Pseudomonas exhibit insecticidal activity and produce virulence factors with cytotoxic and antimicrobial properties, positioning them as promising candidates for biological control. This study evaluated seven Pseudomonas strains with insecticidal activity and identified key virulence factors involved. The strains demonstrated varying degrees of insecticidal activity, with Pseudomonas protegens strains CHA0 and 59C being the most lethal, causing over 75% mortality and triggering a systemic melanization response in the insects. Genomic analysis revealed 175 virulence‐related genes shared across all strains and 16 genes specific to the highly insecticidal ones, including genes for antimicrobial compounds and insect toxins. Mutational analysis confirmed the roles of hydrogen cyanide, 2,4‐diacetylphloroglucinol, pyoluteorin, Fit toxin, and two‐partner secretion systems in P. protegens CHA0 insecticidal activity. This strain also exhibited insecticidal effects on adult T. solanivora and delayed egg hatching and pupal emergence. In microcosm assays, P. protegens CHA0 reduced tuber damage caused by T. solanivora larvae by up to 38%. These results suggest that P. protegens CHA0 is a promising biocontrol agent, providing a sustainable alternative to chemical pesticides to control T. solanivora .
{"title":"Identification of Specific Virulence Factors of Pseudomonas Strains in the Biocontrol of the Potato Pest Tecia solanivora","authors":"Luisa Pantoja, Pilar Vesga, Ziv Arbeli, Vivian Boyacá‐Vásquez, Javier Vanegas","doi":"10.1111/mmi.70041","DOIUrl":"https://doi.org/10.1111/mmi.70041","url":null,"abstract":"<jats:italic>Tecia solanivora</jats:italic> (The Guatemalan potato tuber moth) is a major potato pest, responsible for up to 20% of crop losses and a significant economic impact. Certain <jats:italic>Pseudomonas</jats:italic> exhibit insecticidal activity and produce virulence factors with cytotoxic and antimicrobial properties, positioning them as promising candidates for biological control. This study evaluated seven <jats:italic>Pseudomonas</jats:italic> strains with insecticidal activity and identified key virulence factors involved. The strains demonstrated varying degrees of insecticidal activity, with <jats:italic>Pseudomonas protegens</jats:italic> strains CHA0 and 59C being the most lethal, causing over 75% mortality and triggering a systemic melanization response in the insects. Genomic analysis revealed 175 virulence‐related genes shared across all strains and 16 genes specific to the highly insecticidal ones, including genes for antimicrobial compounds and insect toxins. Mutational analysis confirmed the roles of hydrogen cyanide, 2,4‐diacetylphloroglucinol, pyoluteorin, Fit toxin, and two‐partner secretion systems in <jats:italic>P. protegens</jats:italic> CHA0 insecticidal activity. This strain also exhibited insecticidal effects on adult <jats:italic>T. solanivora</jats:italic> and delayed egg hatching and pupal emergence. In microcosm assays, <jats:italic>P. protegens</jats:italic> CHA0 reduced tuber damage caused by <jats:italic>T. solanivora</jats:italic> larvae by up to 38%. These results suggest that <jats:italic>P. protegens</jats:italic> CHA0 is a promising biocontrol agent, providing a sustainable alternative to chemical pesticides to control <jats:italic>T. solanivora</jats:italic> .","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"183 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145903710","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}
Gül Kilinç, Robin H. G. A. van den Biggelaar, Tom H. M. Ottenhoff, Leon H. Mei, Anno Saris
The treatment of Mycobacterium avium ( Mav ) infection, responsible for over 80% of nontuberculous mycobacterial pulmonary disease, remains challenging due to rising antibiotic resistance and unsatisfactory success rates. Hence, there is a need for a deeper understanding of host–pathogen interactions to inform the development of alternative therapeutic approaches, like host‐directed therapy (HDT), aimed at improving host antimycobacterial defenses. However, compared to Mycobacterium tuberculosis ( Mtb ) infections, knowledge of host–pathogen interactions for Mav infection is still limited. To address this knowledge gap, we performed a genome‐wide host transcriptomic analysis of Mav ‐infected primary human macrophages—the primary host cell—alongside Mtb ‐infected macrophages to leverage insights from Mtb research. Our findings show substantial overlap in the gene expression patterns between Mav ‐infected and Mtb ‐infected macrophages, including induction of cytokine responses and modulation of various G‐protein coupled receptors (GPCRs) involved in (lipid‐mediated) macrophage immune functions. Notable differences were observed in the expression of immediate early genes (IEGs), phospholipases, and genes of the GTPase of immunity‐associated protein (GIMAP) family. This study laid a foundation for identifying both shared and Mav ‐specific host response pathways, providing direction for future investigations into host–pathogen interactions during Mav infection and the identification of novel targets for HDT.
{"title":"Comparative Transcriptomic Analysis of Human Macrophages During Mycobacterium avium Versus Mycobacterium tuberculosis Infection","authors":"Gül Kilinç, Robin H. G. A. van den Biggelaar, Tom H. M. Ottenhoff, Leon H. Mei, Anno Saris","doi":"10.1111/mmi.70045","DOIUrl":"https://doi.org/10.1111/mmi.70045","url":null,"abstract":"The treatment of <jats:styled-content style=\"fixed-case\"> <jats:italic>Mycobacterium avium</jats:italic> </jats:styled-content> ( <jats:italic>Mav</jats:italic> ) infection, responsible for over 80% of nontuberculous mycobacterial pulmonary disease, remains challenging due to rising antibiotic resistance and unsatisfactory success rates. Hence, there is a need for a deeper understanding of host–pathogen interactions to inform the development of alternative therapeutic approaches, like host‐directed therapy (HDT), aimed at improving host antimycobacterial defenses. However, compared to <jats:styled-content style=\"fixed-case\"> <jats:italic>Mycobacterium tuberculosis</jats:italic> </jats:styled-content> ( <jats:italic>Mtb</jats:italic> ) infections, knowledge of host–pathogen interactions for <jats:italic>Mav</jats:italic> infection is still limited. To address this knowledge gap, we performed a genome‐wide host transcriptomic analysis of <jats:italic>Mav</jats:italic> ‐infected primary human macrophages—the primary host cell—alongside <jats:italic>Mtb</jats:italic> ‐infected macrophages to leverage insights from <jats:italic>Mtb</jats:italic> research. Our findings show substantial overlap in the gene expression patterns between <jats:italic>Mav</jats:italic> ‐infected and <jats:italic>Mtb</jats:italic> ‐infected macrophages, including induction of cytokine responses and modulation of various G‐protein coupled receptors (GPCRs) involved in (lipid‐mediated) macrophage immune functions. Notable differences were observed in the expression of immediate early genes (IEGs), phospholipases, and genes of the GTPase of immunity‐associated protein (GIMAP) family. This study laid a foundation for identifying both shared and <jats:italic>Mav</jats:italic> ‐specific host response pathways, providing direction for future investigations into host–pathogen interactions during <jats:italic>Mav</jats:italic> infection and the identification of novel targets for HDT.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"22 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2026-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145903604","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}
Enterococcus lactis , a recently established species that was previously classified as Enterococcus faecium clade B, is a common commensal in human and animal intestines. Unlike the opportunistic pathogen E. faecium , it is largely harmless and rarely associated with clinical infections. Given its recent taxonomic classification, studies on this bacterium are scarce, and the key mechanisms that facilitate its intestinal colonization remain particularly elusive. The capability to utilize different sugars is an important factor in colonizing different ecological niches, particularly in the Gastrointestinal (GI) tract, where carbohydrates are fiercely competed for by a large number of microbes. In this study, we characterized an E. lactis pts gene cluster that is highly conserved in E. lactis and E. faecium , and is widely present in numerous other bacterial species. Two gene‐deletion mutants of the putative pts gene cluster exhibited markedly reduced growth on mannose and failed to colonize the GI tract of mice. These results demonstrate the crucial role of the mannose‐utilization PTS in the intestinal colonization of E. lactis , providing new insight into the carbohydrate‐driven colonization mechanisms of gut microbes.
{"title":"The Mannose Phosphotransferase System in Enterococcus lactis Is Essential for Gastrointestinal Colonization","authors":"Linan Xu, Xiangpeng Yang, Xingshuai Li, Yufan Liu, Jia Yin, Gaoyuan Dong, Chenyan Li, Weishi Ni, Shanpeng Zhang, Bin Ye, Junfei Ma, Xinglin Zhang","doi":"10.1111/mmi.70040","DOIUrl":"https://doi.org/10.1111/mmi.70040","url":null,"abstract":"<jats:italic>Enterococcus lactis</jats:italic> , a recently established species that was previously classified as <jats:styled-content style=\"fixed-case\"> <jats:italic>Enterococcus faecium</jats:italic> </jats:styled-content> clade B, is a common commensal in human and animal intestines. Unlike the opportunistic pathogen <jats:styled-content style=\"fixed-case\"> <jats:italic>E. faecium</jats:italic> </jats:styled-content> , it is largely harmless and rarely associated with clinical infections. Given its recent taxonomic classification, studies on this bacterium are scarce, and the key mechanisms that facilitate its intestinal colonization remain particularly elusive. The capability to utilize different sugars is an important factor in colonizing different ecological niches, particularly in the Gastrointestinal (GI) tract, where carbohydrates are fiercely competed for by a large number of microbes. In this study, we characterized an <jats:italic> <jats:styled-content style=\"fixed-case\">E. lactis</jats:styled-content> pts </jats:italic> gene cluster that is highly conserved in <jats:styled-content style=\"fixed-case\"> <jats:italic>E. lactis</jats:italic> </jats:styled-content> and <jats:styled-content style=\"fixed-case\"> <jats:italic>E. faecium</jats:italic> </jats:styled-content> , and is widely present in numerous other bacterial species. Two gene‐deletion mutants of the putative <jats:italic>pts</jats:italic> gene cluster exhibited markedly reduced growth on mannose and failed to colonize the GI tract of mice. These results demonstrate the crucial role of the mannose‐utilization PTS in the intestinal colonization of <jats:styled-content style=\"fixed-case\"> <jats:italic>E. lactis</jats:italic> </jats:styled-content> , providing new insight into the carbohydrate‐driven colonization mechanisms of gut microbes.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"29 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145807840","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}
Sarah Miercke, Rabea Ghandour, Kai Papenfort, Thorsten Mascher
Under severe nutrient‐limiting conditions, Bacillus subtilis is able to form highly resilient endospores for survival. However, to avoid this irreversible process, it employs an adaptive strategy termed cannibalism, a form of programmed cell death, to outcompete siblings and delay sporulation. One of the three cannibalism toxins, the epipeptide EPE, is encoded by the epeXEPAB operon. The pre‐pro‐peptide EpeX undergoes post‐translational modification and processing to be secreted as the mature EPE toxin. While EPE production is tightly regulated at multiple levels, this study focuses on the post‐transcriptional control by the small regulatory RNA FsrA, which is transcriptionally regulated by the global iron response regulator Fur. Electrophoretic mobility shift assays and RNA structure probing revealed two binding sites of FsrA within the intergenic region between epeX and epeE flanking the annotated epeX terminator structure and potentially interfering with RNA stability and epeXEP expression. Reporter assays revealed decreased levels of EPE‐dependent stress response in the absence of FsrA, indicative of a positive FsrA effect on gene expression under iron‐limited conditions; in contrast to the normally inhibitory activity of FsrA. Together, our findings suggest that under iron starvation, FsrA promotes RNA processing and enables epeE translation, ultimately enhancing EPE production.
{"title":"The FsrA ‐Mediated Iron‐Sparing Response Regulates the Biosynthesis of the Epipeptide EPE in Bacillus subtilis","authors":"Sarah Miercke, Rabea Ghandour, Kai Papenfort, Thorsten Mascher","doi":"10.1111/mmi.70039","DOIUrl":"https://doi.org/10.1111/mmi.70039","url":null,"abstract":"Under severe nutrient‐limiting conditions, <jats:styled-content style=\"fixed-case\"> <jats:italic>Bacillus subtilis</jats:italic> </jats:styled-content> is able to form highly resilient endospores for survival. However, to avoid this irreversible process, it employs an adaptive strategy termed cannibalism, a form of programmed cell death, to outcompete siblings and delay sporulation. One of the three cannibalism toxins, the epipeptide EPE, is encoded by the <jats:italic>epeXEPAB</jats:italic> operon. The pre‐pro‐peptide EpeX undergoes post‐translational modification and processing to be secreted as the mature EPE toxin. While EPE production is tightly regulated at multiple levels, this study focuses on the post‐transcriptional control by the small regulatory RNA FsrA, which is transcriptionally regulated by the global iron response regulator Fur. Electrophoretic mobility shift assays and RNA structure probing revealed two binding sites of FsrA within the intergenic region between <jats:italic>epeX</jats:italic> and <jats:italic>epeE</jats:italic> flanking the annotated <jats:italic>epeX</jats:italic> terminator structure and potentially interfering with RNA stability and <jats:italic>epeXEP</jats:italic> expression. Reporter assays revealed decreased levels of EPE‐dependent stress response in the absence of FsrA, indicative of a positive FsrA effect on gene expression under iron‐limited conditions; in contrast to the normally inhibitory activity of FsrA. Together, our findings suggest that under iron starvation, FsrA promotes RNA processing and enables <jats:italic>epeE</jats:italic> translation, ultimately enhancing EPE production.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"23 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145770678","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 : 2025-12-01Epub Date: 2025-10-13DOI: 10.1111/mmi.70027
Lea Fuchs, Cora Lisbeth Dieterich, Elena Melgarejo Ros, Philipp Keller, Anna Sintsova, Leanid Laganenka, Thomas A Scott, Christopher Schubert, Shinichi Sunagawa, Julia A Vorholt, Jörn Piel, Wolf-Dietrich Hardt, Bidong D Nguyen
Salmonella enterica serovar Typhimurium (S. Tm) is a major cause of foodborne diarrhea. However, in healthy individuals, the microbiota typically restricts the growth of incoming pathogens, a protective mechanism termed colonization resistance (CR). To circumvent CR, Salmonella strains can utilize private nutrients that remain untapped by the resident microbiota. However, the metabolic pathways and environmental niches promoting pathogen growth are still not completely understood. Here, we investigate the significance of the gfr operon in gut colonization of S. Tm, which is essential for the utilization of fructoselysine (FL) and glucoselysine (GL). These Amadori compounds are present in heated foods with high protein and carbohydrate contents. We detected FL in both mouse chow and the intestinal tract of mice and showed that gfr mutants are attenuated during the initial phase of colonization in the murine model. Experiments in gnotobiotic mice and competition experiments with Escherichia coli suggest that gfr-dependent fitness advantage is context-dependent. We conclude that dietary Amadori products like FL can support S. Tm gut colonization, depending on the metabolic capacities of the microbiota.
{"title":"The Gfr Uptake System Provides a Context-Dependent Fitness Advantage to Salmonella Typhimurium SL1344 During the Initial Gut Colonization Phase.","authors":"Lea Fuchs, Cora Lisbeth Dieterich, Elena Melgarejo Ros, Philipp Keller, Anna Sintsova, Leanid Laganenka, Thomas A Scott, Christopher Schubert, Shinichi Sunagawa, Julia A Vorholt, Jörn Piel, Wolf-Dietrich Hardt, Bidong D Nguyen","doi":"10.1111/mmi.70027","DOIUrl":"10.1111/mmi.70027","url":null,"abstract":"<p><p>Salmonella enterica serovar Typhimurium (S. Tm) is a major cause of foodborne diarrhea. However, in healthy individuals, the microbiota typically restricts the growth of incoming pathogens, a protective mechanism termed colonization resistance (CR). To circumvent CR, Salmonella strains can utilize private nutrients that remain untapped by the resident microbiota. However, the metabolic pathways and environmental niches promoting pathogen growth are still not completely understood. Here, we investigate the significance of the gfr operon in gut colonization of S. Tm, which is essential for the utilization of fructoselysine (FL) and glucoselysine (GL). These Amadori compounds are present in heated foods with high protein and carbohydrate contents. We detected FL in both mouse chow and the intestinal tract of mice and showed that gfr mutants are attenuated during the initial phase of colonization in the murine model. Experiments in gnotobiotic mice and competition experiments with Escherichia coli suggest that gfr-dependent fitness advantage is context-dependent. We conclude that dietary Amadori products like FL can support S. Tm gut colonization, depending on the metabolic capacities of the microbiota.</p>","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":" ","pages":"507-520"},"PeriodicalIF":2.6,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12675984/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145280875","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}
Majeed Bakari-Soale, Christopher Batram, Henriette Zimmermann, Nicola G. Jones, Markus Engstler
The variant surface glycoprotein (VSG) of African trypanosomes is essential for the survival of bloodstream form parasites. These parasites undergo antigenic variation, an immune evasion strategy in which they periodically switch VSG expression from one isoform to another. The molecular processes central to the expression and regulation of the VSG are however not fully understood. In general, the regulation of gene expression in trypanosomes is largely post-transcriptional. Regulatory sequences, mostly present in the 3′ UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In T. brucei VSG genes, a 16mer motif within the 3′ UTR has been shown to be essential for the stability of VSG transcripts and abundant VSG expression. This motif is 100% conserved in the 3′ UTRs of all transcribed and non-transcribed VSG genes. As a stability-associated sequence element, the absence of nucleotide substitutions in the 16mer is however exceptional. We therefore hypothesised that the motif is involved in other essential roles/processes besides the stability of the VSG transcripts. In this study, we demonstrate that the 100% conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. We further show that the intact motif in the active VSG 3′ UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Ectopic overexpression of a second VSG, however, requires the intact 16mer motif within the ectopic VSG 3′ UTR to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The enigmatic 16mer motif therefore appears to play a dual role in transcriptional VSG switching and VSG transcript stability.
{"title":"The Dual Role of the 16mer Motif Within the 3′ Untranslated Region of the Variant Surface Glycoprotein of Trypanosoma brucei","authors":"Majeed Bakari-Soale, Christopher Batram, Henriette Zimmermann, Nicola G. Jones, Markus Engstler","doi":"10.1111/mmi.70031","DOIUrl":"https://doi.org/10.1111/mmi.70031","url":null,"abstract":"The variant surface glycoprotein (VSG) of African trypanosomes is essential for the survival of bloodstream form parasites. These parasites undergo antigenic variation, an immune evasion strategy in which they periodically switch VSG expression from one isoform to another. The molecular processes central to the expression and regulation of the VSG are however not fully understood. In general, the regulation of gene expression in trypanosomes is largely post-transcriptional. Regulatory sequences, mostly present in the 3′ UTRs, often serve as key elements in the modulation of the levels of individual mRNAs. In <i>T. brucei</i> VSG genes, a 16mer motif within the 3′ UTR has been shown to be essential for the stability of <i>VSG</i> transcripts and abundant VSG expression. This motif is 100% conserved in the 3′ UTRs of all transcribed and non-transcribed VSG genes. As a stability-associated sequence element, the absence of nucleotide substitutions in the 16mer is however exceptional. We therefore hypothesised that the motif is involved in other essential roles/processes besides the stability of the <i>VSG</i> transcripts. In this study, we demonstrate that the 100% conservation of the 16mer motif is not essential for cell viability or for the maintenance of functional VSG protein levels. We further show that the intact motif in the active VSG 3′ UTR is neither required to promote VSG silencing during switching nor is it needed during differentiation from bloodstream forms to procyclic forms. Ectopic overexpression of a second VSG, however, requires the intact 16mer motif within the ectopic VSG 3′ UTR to trigger silencing and exchange of the active VSG, suggesting a role for the motif in transcriptional VSG switching. The enigmatic 16mer motif therefore appears to play a dual role in transcriptional <i>VSG</i> switching and <i>VSG</i> transcript stability.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"14 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145531477","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}
André A. Grassmann, Melissa A. McLain, Michael R. Freeman, Melissa J. Caimano, Justin D. Radolf
BosR, the sole member of the ferric uptake regulator (FUR) family in Borrelia burgdorferi , is essential for the spirochete's transcriptional adaptation to the mammalian host environment. Although best known for activating rpoS and establishing the mammalian‐phase RpoS regulon, BosR originally was linked to regulation of genes involved in B. burgdorferi 's oxidative stress response. Here, we show that BosR governs gene expression through both RpoS‐dependent and RpoS‐independent mechanisms under in vitro and mammalian host‐adapted conditions. Using RNA‐seq and a DNA‐binding‐defective BosR‐R39A mutant, we demonstrate that DNA binding is essential for BosR's global regulatory functions. BosR activates rpoS , promotes RpoS‐dependent gene regulation, and independently modulates a distinct set of genes involved in a variety of cellular functions, including genome maintenance, chemotaxis, and virulence. Notably, canonical oxidative stress response genes previously attributed to BosR were not differentially expressed in Δ bosR strains in vitro or in mammals. Despite its broad regulatory scope, BosR does not recognize a single, conserved DNA‐binding motif, suggesting that DNA occupancy is influenced by local sequence context or DNA topology. Our findings support a bifunctional model in which BosR collaborates with RNA polymerase (RNAP)‐RpoS holoenzyme to activate and repress RpoS‐regulated genes, while functioning in a FUR‐like manner to control RpoD‐dependent genes independently of RNAP interaction.
{"title":"DNA Binding by BosR Controls RpoS ‐Dependent and ‐Independent Gene Expression in Borrelia burgdorferi","authors":"André A. Grassmann, Melissa A. McLain, Michael R. Freeman, Melissa J. Caimano, Justin D. Radolf","doi":"10.1111/mmi.70036","DOIUrl":"https://doi.org/10.1111/mmi.70036","url":null,"abstract":"BosR, the sole member of the ferric uptake regulator (FUR) family in <jats:styled-content style=\"fixed-case\"> <jats:italic>Borrelia burgdorferi</jats:italic> </jats:styled-content> , is essential for the spirochete's transcriptional adaptation to the mammalian host environment. Although best known for activating <jats:italic>rpoS</jats:italic> and establishing the mammalian‐phase RpoS regulon, BosR originally was linked to regulation of genes involved in <jats:italic>B. burgdorferi</jats:italic> 's oxidative stress response. Here, we show that BosR governs gene expression through both RpoS‐dependent and RpoS‐independent mechanisms under in vitro and mammalian host‐adapted conditions. Using RNA‐seq and a DNA‐binding‐defective BosR‐R39A mutant, we demonstrate that DNA binding is essential for BosR's global regulatory functions. BosR activates <jats:italic>rpoS</jats:italic> , promotes RpoS‐dependent gene regulation, and independently modulates a distinct set of genes involved in a variety of cellular functions, including genome maintenance, chemotaxis, and virulence. Notably, canonical oxidative stress response genes previously attributed to BosR were not differentially expressed in Δ <jats:italic>bosR</jats:italic> strains in vitro or in mammals. Despite its broad regulatory scope, BosR does not recognize a single, conserved DNA‐binding motif, suggesting that DNA occupancy is influenced by local sequence context or DNA topology. Our findings support a bifunctional model in which BosR collaborates with RNA polymerase (RNAP)‐RpoS holoenzyme to activate and repress RpoS‐regulated genes, while functioning in a FUR‐like manner to control RpoD‐dependent genes independently of RNAP interaction.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"174 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145515541","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}
Gα, Gβ, and Gγ—the heterotrimeric G protein subunits transmit signals from G-protein coupled receptors (GPCRs) to downstream pathways. The causative agent of Amoebiasis, Entamoeba, has been presumed to contain a GPCR signaling pathway playing a role in its encystation, phagocytosis, and motility. A Gα subunit, EhGα1, has been characterized earlier in Entamoeba histolytica , which is involved in its different pathogenic processes. Here, we have characterized its ortholog, EiGα1, in the reptilian model, Entamoeba invadens , which expresses both in trophozoites and during encystation. Silencing EiGα1 through trigger-mediated knockdown reduces efficiency and leads to improper cell aggregation and abnormal chitin wall formation during encystation. Downregulation of EiGα1 results in anomalous F-actin polymerization. EiGα1 silenced cells also exhibit loss of polarity and reduced motility. Furthermore, EiGα1 knockdown also results in decreased phagocytosis of bacteria. Our findings indicate that EiGα1 controls the expression of two vital proteins in Entamoeba—the atypical EiMAPK15 and the homeobox transcription factor EiHbox1—which modulates cyst-wall development and actin reorganization. In conclusion, our findings provide strong evidence for a GPCR signaling network in Entamoeba and highlight the essential function of the Gα subunit in stage conversion and actin cytoskeleton rearrangement.
{"title":"When One Is Enough: The Only Gα Subunit Governs Encystation and Other Cellular Processes in Entamoeba invadens","authors":"Shilpa Sarkar, Tiasha Chakraborty, Sudip K. Ghosh","doi":"10.1111/mmi.70035","DOIUrl":"https://doi.org/10.1111/mmi.70035","url":null,"abstract":"Gα, Gβ, and Gγ—the heterotrimeric G protein subunits transmit signals from G-protein coupled receptors (GPCRs) to downstream pathways. The causative agent of Amoebiasis, <i>Entamoeba</i>, has been presumed to contain a GPCR signaling pathway playing a role in its encystation, phagocytosis, and motility. A Gα subunit, EhGα1, has been characterized earlier in <i>Entamoeba histolytica</i> , which is involved in its different pathogenic processes. Here, we have characterized its ortholog, EiGα1, in the reptilian model, <i>Entamoeba invadens</i> , which expresses both in trophozoites and during encystation. Silencing EiGα1 through trigger-mediated knockdown reduces efficiency and leads to improper cell aggregation and abnormal chitin wall formation during encystation. Downregulation of EiGα1 results in anomalous F-actin polymerization. EiGα1 silenced cells also exhibit loss of polarity and reduced motility. Furthermore, EiGα1 knockdown also results in decreased phagocytosis of bacteria. Our findings indicate that EiGα1 controls the expression of two vital proteins in <i>Entamoeba</i>—the atypical EiMAPK15 and the homeobox transcription factor EiHbox1—which modulates cyst-wall development and actin reorganization. In conclusion, our findings provide strong evidence for a GPCR signaling network in <i>Entamoeba</i> and highlight the essential function of the Gα subunit in stage conversion and actin cytoskeleton rearrangement.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"1 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145478138","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}
The endoplasmic reticulum‐Golgi intermediate compartment (ERGIC) plays a crucial role in the secretory pathway; however, its existence and function in lower eukaryotes remain largely unexamined. In this study, we identified Emp43 (SPBC4F6.05c) of Schizosaccharomyces pombe , an orthologue of human ( Homo sapiens ) ERGIC‐53, and demonstrated its localization to an ERGIC‐like compartment. The localization of Emp43 depended on its C‐terminal KYL motif and oligomerization through the CC1 domain. Deletion of S. pombe emp43+ resulted in significant sensitivity to MgCl 2 and FK506, along with defects in septum integrity, indicating a role in cell wall maintenance. Further analysis identified Ssp120 of S. pombe , an orthologue of human MCFD2, as a functional partner of Emp43. Yeast two‐hybrid assays confirmed a strong interaction between Emp43 and Ssp120, and both proteins co‐localized within an ERGIC‐like compartment. Additionally, we identified Meu17 of S. pombe , a glucan‐α‐1,4‐glucosidase homolog, as a potential ligand for Emp43. Overexpression of Meu17 rescued MgCl 2 sensitivity in both emp43 Δ and ssp120 Δ strains, while mutations in its N‐linked glycosylation sites (N383, N409) or its predicted active site (D203) disrupted its septum localization and functional rescue capability. Our findings indicate that Emp43 forms a complex with Ssp120 to facilitate the transport of glycosylated proteins, such as Meu17, within an ERGIC‐like compartment in fission yeast S. pombe . This study provides the first evidence of an ERGIC‐like structure in S. pombe and highlights the conserved nature of ERGIC‐associated mechanisms across eukaryotes.
{"title":"Identification of an ERGIC ‐Like Compartment in Fission Yeast: Emp43 Functions as a Lectin‐Like Cargo Receptor for Glycosylated Proteins","authors":"Iori Imamura, Soma Kawaguchi, Shotaro Suzuki, Yuki Kamiya, Yuzuna Ohnishi, Juri Ueda, Kento Nashiki, Kaoru Takegawa, Mitsuaki Tabuchi, Naotaka Tanaka","doi":"10.1111/mmi.70033","DOIUrl":"https://doi.org/10.1111/mmi.70033","url":null,"abstract":"The endoplasmic reticulum‐Golgi intermediate compartment (ERGIC) plays a crucial role in the secretory pathway; however, its existence and function in lower eukaryotes remain largely unexamined. In this study, we identified Emp43 (SPBC4F6.05c) of <jats:italic>Schizosaccharomyces pombe</jats:italic> , an orthologue of human ( <jats:styled-content style=\"fixed-case\"> <jats:italic>Homo sapiens</jats:italic> </jats:styled-content> ) ERGIC‐53, and demonstrated its localization to an ERGIC‐like compartment. The localization of Emp43 depended on its C‐terminal KYL motif and oligomerization through the CC1 domain. Deletion of <jats:italic>S. pombe emp43</jats:italic> <jats:sup>+</jats:sup> resulted in significant sensitivity to MgCl <jats:sub>2</jats:sub> and FK506, along with defects in septum integrity, indicating a role in cell wall maintenance. Further analysis identified Ssp120 of <jats:italic>S. pombe</jats:italic> , an orthologue of human MCFD2, as a functional partner of Emp43. Yeast two‐hybrid assays confirmed a strong interaction between Emp43 and Ssp120, and both proteins co‐localized within an ERGIC‐like compartment. Additionally, we identified Meu17 of <jats:italic>S. pombe</jats:italic> , a glucan‐α‐1,4‐glucosidase homolog, as a potential ligand for Emp43. Overexpression of Meu17 rescued MgCl <jats:sub>2</jats:sub> sensitivity in both <jats:italic>emp43</jats:italic> Δ and <jats:italic>ssp120</jats:italic> Δ strains, while mutations in its N‐linked glycosylation sites (N383, N409) or its predicted active site (D203) disrupted its septum localization and functional rescue capability. Our findings indicate that Emp43 forms a complex with Ssp120 to facilitate the transport of glycosylated proteins, such as Meu17, within an ERGIC‐like compartment in fission yeast <jats:italic>S. pombe</jats:italic> . This study provides the first evidence of an ERGIC‐like structure in <jats:italic>S. pombe</jats:italic> and highlights the conserved nature of ERGIC‐associated mechanisms across eukaryotes.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"107 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145461942","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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