Caroline Reisacher, Estelle Saifi, Elisabeth Ageron, Robert Theodor Mallmann, Norbert Klugbauer, David Skurnik, Laurence Arbibe
MicroRNAs have recently emerged as major players in host –bacterial pathogen interactions, either as part of the host defense mechanism to neutralize infection or as a bacterial arsenal aimed at subverting host cell functions. Here, we identify the newly evolved human microRNA miR-6762-5p as a new player in the host–Shigella interplay. A microarray analysis in infected epithelial cells allowed the detection of this miRNA exclusively during the late phase of infection. Conditional expression of miR-6762-5p combined with a transcriptome analysis indicated a role in cytoskeleton remodeling. Likewise, miR-6762-5p enhanced stress fiber formation through RhoA activation, and in silico analysis identified several regulators of RhoA activity as potential direct transcriptional targets. We further showed that miR-6762-5p expression induces an increase in Shigella intercellular spreading, while miR-6762-5p inhibition reduced bacterial dissemination. We propose a model in which the expression of miR-6762-5p induces cytoskeleton modifications through RhoA activation to achieve a successful dissemination of Shigella in the host.
{"title":"The Human-Specific miR-6762-5p Is an Activator of RhoA GTPase Enhancing Shigella flexneri Intercellular Spreading","authors":"Caroline Reisacher, Estelle Saifi, Elisabeth Ageron, Robert Theodor Mallmann, Norbert Klugbauer, David Skurnik, Laurence Arbibe","doi":"10.1111/mmi.15352","DOIUrl":"https://doi.org/10.1111/mmi.15352","url":null,"abstract":"MicroRNAs have recently emerged as major players in host –bacterial pathogen interactions, either as part of the host defense mechanism to neutralize infection or as a bacterial arsenal aimed at subverting host cell functions. Here, we identify the newly evolved human microRNA miR-6762-5p as a new player in the host–<i>Shigella</i> interplay. A microarray analysis in infected epithelial cells allowed the detection of this miRNA exclusively during the late phase of infection. Conditional expression of miR-6762-5p combined with a transcriptome analysis indicated a role in cytoskeleton remodeling. Likewise, miR-6762-5p enhanced stress fiber formation through RhoA activation, and <i>in silico</i> analysis identified several regulators of RhoA activity as potential direct transcriptional targets. We further showed that miR-6762-5p expression induces an increase in <i>Shigella</i> intercellular spreading, while miR-6762-5p inhibition reduced bacterial dissemination. We propose a model in which the expression of miR-6762-5p induces cytoskeleton modifications through RhoA activation to achieve a successful dissemination of <i>Shigella</i> in the host.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"51 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143477870","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 “Bacterial Chromatin Proteins, Transcription, and DNA Topology: Inseparable Partners in the Control of Gene Expression”","authors":"","doi":"10.1111/mmi.15324","DOIUrl":"https://doi.org/10.1111/mmi.15324","url":null,"abstract":"","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"15 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143462905","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}
E. A. Abbondanzieri, A. B. Badrinarayanan, D. Barillà, S. D. Bell, F. Blombach, J. Y. Bouet, S. Bulgheresi, Q. A. D. Cao, R. T. Dame, C. Dekker, M. Demuysere, O. Espéli, P. C. M. Fogg, P. L. Freddolino, M. Ganji, T. M. Gerson, D. C. Grainger, L. W. Hamoen, J. Harju, A. Hocher, C. M. Hustmyer, J. K. Kaljevic, M. K. Karney, N. Kleckner, G. Laloux, R. Landick, V. S. Lioy, W. L. Liu, C. L. Liu, J. Mäkelä, A. S. Meyer, A. Noy, M. P. Pineau, K. Premrajka, L. R. Racki, F‐Z. M. Rashid, K. Schnetz, S. Schwab, M. Tišma, A. I. van der Sijs, T. van Heesch, R. van Raaphorst, J. Vreede, A. W. Walker, J‐C. Walter, S. C. Weber, P. A. Wiggins, H. J. Wing, J. Xiao, Z. Zhang
In September 2023, the Biology and Physics of Prokaryotic Chromosomes meeting ran at the Lorentz Center in Leiden, The Netherlands. As part of the workshop, those in attendance developed a series of discussion points centered around current challenges for the field, how these might be addressed, and how the field is likely to develop over the next 10 years. The Lorentz Center staff facilitated these discussions via tools aimed at optimizing productive interactions. This Perspective article is a summary of these discussions and reflects the state‐of‐the‐art of the field. It is expected to be of help to colleagues in advancing their own research related to prokaryotic chromosomes and inspiring novel interdisciplinary collaborations. This forward‐looking perspective highlights the open questions driving current research and builds on the impressive recent progress in these areas as represented by the accompanying reviews, perspectives, and research articles in this issue. These articles underline the multi‐disciplinary nature of the field, the multiple length scales at which chromatin is studied in vitro and in and highlight the differences and similarities of bacterial and archaeal chromatin and chromatin‐associated processes.
{"title":"Future Directions of the Prokaryotic Chromosome Field","authors":"E. A. Abbondanzieri, A. B. Badrinarayanan, D. Barillà, S. D. Bell, F. Blombach, J. Y. Bouet, S. Bulgheresi, Q. A. D. Cao, R. T. Dame, C. Dekker, M. Demuysere, O. Espéli, P. C. M. Fogg, P. L. Freddolino, M. Ganji, T. M. Gerson, D. C. Grainger, L. W. Hamoen, J. Harju, A. Hocher, C. M. Hustmyer, J. K. Kaljevic, M. K. Karney, N. Kleckner, G. Laloux, R. Landick, V. S. Lioy, W. L. Liu, C. L. Liu, J. Mäkelä, A. S. Meyer, A. Noy, M. P. Pineau, K. Premrajka, L. R. Racki, F‐Z. M. Rashid, K. Schnetz, S. Schwab, M. Tišma, A. I. van der Sijs, T. van Heesch, R. van Raaphorst, J. Vreede, A. W. Walker, J‐C. Walter, S. C. Weber, P. A. Wiggins, H. J. Wing, J. Xiao, Z. Zhang","doi":"10.1111/mmi.15347","DOIUrl":"https://doi.org/10.1111/mmi.15347","url":null,"abstract":"In September 2023, the Biology and Physics of Prokaryotic Chromosomes meeting ran at the Lorentz Center in Leiden, The Netherlands. As part of the workshop, those in attendance developed a series of discussion points centered around current challenges for the field, how these might be addressed, and how the field is likely to develop over the next 10 years. The Lorentz Center staff facilitated these discussions via tools aimed at optimizing productive interactions. This Perspective article is a summary of these discussions and reflects the state‐of‐the‐art of the field. It is expected to be of help to colleagues in advancing their own research related to prokaryotic chromosomes and inspiring novel interdisciplinary collaborations. This forward‐looking perspective highlights the open questions driving current research and builds on the impressive recent progress in these areas as represented by the accompanying reviews, perspectives, and research articles in this issue. These articles underline the multi‐disciplinary nature of the field, the multiple length scales at which chromatin is studied in vitro and in and highlight the differences and similarities of bacterial and archaeal chromatin and chromatin‐associated processes.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"20 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143462902","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}
Saher Shahid, Mateusz Balka, Daniel Lundin, Daniel O. Daley, Britt‐Marie Sjöberg, Inna Rozman Grinberg
NrdR is a universal transcriptional repressor of bacterial genes coding for ribonucleotide reductases (RNRs), essential enzymes that provide DNA building blocks in all living cells. Despite its bacterial prevalence, the NrdR mechanism has been scarcely studied. We report the biochemical, biophysical, and bioinformatical characterization of NrdR and its binding sites from two major bacterial pathogens of the phylum BacillotaListeria monocytogenes and Streptococcus pneumoniae. NrdR consists of a Zn‐ribbon domain followed by an ATP‐cone domain. We show that it forms tetramers that bind to DNA when loaded with ATP and dATP, but if loaded with only ATP, NrdR forms various oligomeric complexes unable to bind DNA. The DNA‐binding site in L. monocytogenes is a pair of NrdR boxes separated by 15–16 bp, whereas in S. pneumoniae, the NrdR boxes are separated by unusually long spacers of 25–26 bp. This observation triggered a comprehensive binding study of four NrdRs from L. monocytogenes, S. pneumoniae, Escherichia coli, and Streptomyces coelicolor to a series of dsDNA fragments where the NrdR boxes were separated by 12–27 bp. The in vitro results were confirmed in vivo in E. coli and revealed that NrdR binds most efficiently when there is an integer number of DNA turns between the center of the two NrdR boxes. The study facilitates the prediction of NrdR binding sites in bacterial genomes and suggests that the NrdR mechanism is conserved throughout the bacterial domain. It sheds light on RNR regulation in Listeria and Streptococcus, and since NrdR does not occur in eukaryotes, opens a way to the development of novel antibiotics.
{"title":"NrdR in Streptococcus and Listeria spp.: DNA Helix Phase Dependence of the Bacterial Ribonucleotide Reductase Repressor","authors":"Saher Shahid, Mateusz Balka, Daniel Lundin, Daniel O. Daley, Britt‐Marie Sjöberg, Inna Rozman Grinberg","doi":"10.1111/mmi.15349","DOIUrl":"https://doi.org/10.1111/mmi.15349","url":null,"abstract":"NrdR is a universal transcriptional repressor of bacterial genes coding for ribonucleotide reductases (RNRs), essential enzymes that provide DNA building blocks in all living cells. Despite its bacterial prevalence, the NrdR mechanism has been scarcely studied. We report the biochemical, biophysical, and bioinformatical characterization of NrdR and its binding sites from two major bacterial pathogens of the phylum <jats:italic>Bacillota</jats:italic> <jats:styled-content style=\"fixed-case\"><jats:italic>Listeria monocytogenes</jats:italic></jats:styled-content> and <jats:styled-content style=\"fixed-case\"><jats:italic>Streptococcus pneumoniae</jats:italic></jats:styled-content>. NrdR consists of a Zn‐ribbon domain followed by an ATP‐cone domain. We show that it forms tetramers that bind to DNA when loaded with ATP and dATP, but if loaded with only ATP, NrdR forms various oligomeric complexes unable to bind DNA. The DNA‐binding site in <jats:styled-content style=\"fixed-case\"><jats:italic>L. monocytogenes</jats:italic></jats:styled-content> is a pair of NrdR boxes separated by 15–16 bp, whereas in <jats:styled-content style=\"fixed-case\"><jats:italic>S. pneumoniae</jats:italic></jats:styled-content>, the NrdR boxes are separated by unusually long spacers of 25–26 bp. This observation triggered a comprehensive binding study of four NrdRs from <jats:styled-content style=\"fixed-case\"><jats:italic>L. monocytogenes</jats:italic></jats:styled-content>, <jats:styled-content style=\"fixed-case\"><jats:italic>S. pneumoniae</jats:italic></jats:styled-content>, <jats:styled-content style=\"fixed-case\"><jats:italic>Escherichia coli</jats:italic></jats:styled-content>, and <jats:styled-content style=\"fixed-case\"><jats:italic>Streptomyces coelicolor</jats:italic></jats:styled-content> to a series of dsDNA fragments where the NrdR boxes were separated by 12–27 bp. The in vitro results were confirmed in vivo in <jats:styled-content style=\"fixed-case\"><jats:italic>E. coli</jats:italic></jats:styled-content> and revealed that NrdR binds most efficiently when there is an integer number of DNA turns between the center of the two NrdR boxes. The study facilitates the prediction of NrdR binding sites in bacterial genomes and suggests that the NrdR mechanism is conserved throughout the bacterial domain. It sheds light on RNR regulation in <jats:italic>Listeria</jats:italic> and <jats:italic>Streptococcus</jats:italic>, and since NrdR does not occur in eukaryotes, opens a way to the development of novel antibiotics.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"1 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143443327","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}
Tao Huang, Xiaoling Ma, Ziqi Zhao, Danna Qin, Weiye Qin, Jinzi Wang, Baoshan Chen, Xipu He
Calnexin, a calcium-binding protein, promotes correct protein folding and prevents incompletely folded glycopolypeptides from premature oxidation and degradation. Cryphonectria parasitica, an ascomycete fungus responsible for chestnut blight, poses a significant threat to the chestnut forest or orchards worldwide. Although various aspects of calnexin have been investigated, little is known about the impact of fungal viruses. CpCne was identified and characterized in this study, encoding the calnexin in C. parasitica. Strains with deletion or interference of the CpCne gene had a significant reduction in biomass and pathogenicity, and strains with overexpression of the CpCne gene had retarded growth and reduced pathogenicity. Transcriptome analysis showed that the △CpCne mutant had significant changes in the expression of genes related to carbohydrate metabolism, cell wall polysaccharide synthesis and degradation, indicating that CpCne may reduce virulence by affecting the cell wall. Additionally, the △CpCne mutant was sensitive to endoplasmic reticulum (ER) stress, suggesting that CpCne plays an important role in maintaining ER homeostasis. Furthermore, CpCne was also involved in the interaction between C. parasitica and the CHV1-EP713. Deletion or overexpression of the CpCne gene reduced viral RNA accumulation, and deletion of the CpCne gene altered the lipid and carboxylic acid metabolic pathways, thereby interfering with virus replication and assembly. Together, we demonstrated that the homeostasis of calnexin in C. parasitica (CpCne) is essential for hyphal growth and virulence, and revealed its role in viral replication and virulence.
{"title":"Homeostasis of Calnexin Is Essential for the Growth, Virulence, and Hypovirus RNA Accumulation in the Chestnut Blight Fungus","authors":"Tao Huang, Xiaoling Ma, Ziqi Zhao, Danna Qin, Weiye Qin, Jinzi Wang, Baoshan Chen, Xipu He","doi":"10.1111/mmi.15348","DOIUrl":"https://doi.org/10.1111/mmi.15348","url":null,"abstract":"Calnexin, a calcium-binding protein, promotes correct protein folding and prevents incompletely folded glycopolypeptides from premature oxidation and degradation. <i>Cryphonectria parasitica</i>, an ascomycete fungus responsible for chestnut blight, poses a significant threat to the chestnut forest or orchards worldwide. Although various aspects of calnexin have been investigated, little is known about the impact of fungal viruses. <i>CpCne</i> was identified and characterized in this study, encoding the calnexin in <i>C. parasitica</i>. Strains with deletion or interference of the <i>CpCne</i> gene had a significant reduction in biomass and pathogenicity, and strains with overexpression of the <i>CpCne</i> gene had retarded growth and reduced pathogenicity. Transcriptome analysis showed that the △<i>CpCne</i> mutant had significant changes in the expression of genes related to carbohydrate metabolism, cell wall polysaccharide synthesis and degradation, indicating that <i>CpCne</i> may reduce virulence by affecting the cell wall. Additionally, the △<i>CpCne</i> mutant was sensitive to endoplasmic reticulum (ER) stress, suggesting that <i>CpCne</i> plays an important role in maintaining ER homeostasis. Furthermore, <i>CpCne</i> was also involved in the interaction between <i>C. parasitica</i> and the CHV1-EP713. Deletion or overexpression of the <i>CpCne</i> gene reduced viral RNA accumulation, and deletion of the <i>CpCne</i> gene altered the lipid and carboxylic acid metabolic pathways, thereby interfering with virus replication and assembly. Together, we demonstrated that the homeostasis of calnexin in <i>C. parasitica</i> (CpCne) is essential for hyphal growth and virulence, and revealed its role in viral replication and virulence.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"19 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143393300","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}
Soluble methane monooxygenase (sMMO) from methanotrophs has been extensively investigated for decades. However, major knowledge gaps persist regarding the synthesis mechanism of sMMO, particularly concerning the ambiguous roles of mmoD and mmoG in the sMMO gene cluster. Here, the functions of mmoD and mmoG were investigated in a model methanotrophic strain, Methylotuvimicrobium buryatense 5GB1C. Both genes were found to be essential for the functional expression of sMMO. Genetic and biochemical data supported the hypothesis that MmoG acts as a folding chaperone for both MmoX and MmoR, while MmoD serves as an assembly chaperone for the hydroxylase component. The functional expression of sMMO in Escherichia coli was achieved in an mmoD- and mmoG-dependent manner. In addition, deletion of mmoD dramatically reduced the transcription of the sMMO cluster in M. buryatense 5GB1C, implying that MmoD may regulate the sMMO cluster via an unknown mechanism. Knockout of neither mmoD nor mmoG abolished the essential feature of “copper switch”, indicating that they do not serve as the initial regulators of “copper switch”. These results demonstrate the crucial roles of mmoD and mmoG in sMMO synthesis and offer new insights into heterologous expression of sMMO.
{"title":"MmoD and MmoG Are Crucial for the Synthesis of Soluble Methane Monooxygenase in Methanotrophs","authors":"Minggen Cheng, Yongchuang Liu, Xin Yan","doi":"10.1111/mmi.15345","DOIUrl":"https://doi.org/10.1111/mmi.15345","url":null,"abstract":"Soluble methane monooxygenase (sMMO) from methanotrophs has been extensively investigated for decades. However, major knowledge gaps persist regarding the synthesis mechanism of sMMO, particularly concerning the ambiguous roles of <i>mmoD</i> and <i>mmoG</i> in the sMMO gene cluster. Here, the functions of <i>mmoD</i> and <i>mmoG</i> were investigated in a model methanotrophic strain, <i>Methylotuvimicrobium buryatense</i> 5GB1C. Both genes were found to be essential for the functional expression of sMMO. Genetic and biochemical data supported the hypothesis that MmoG acts as a folding chaperone for both MmoX and MmoR, while MmoD serves as an assembly chaperone for the hydroxylase component. The functional expression of sMMO in <i>Escherichia coli</i> was achieved in an <i>mmoD-</i> and <i>mmoG-</i>dependent manner. In addition, deletion of <i>mmoD</i> dramatically reduced the transcription of the sMMO cluster in <i>M. buryatense</i> 5GB1C, implying that MmoD may regulate the sMMO cluster via an unknown mechanism. Knockout of neither <i>mmoD</i> nor <i>mmoG</i> abolished the essential feature of “copper switch”, indicating that they do not serve as the initial regulators of “copper switch”. These results demonstrate the crucial roles of <i>mmoD</i> and <i>mmoG</i> in sMMO synthesis and offer new insights into heterologous expression of sMMO.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"1 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143385800","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}
Cody Cris, Monika M. A. Karney, Juniper S. Rosen, Alexander D. Karabachev, Elizabeth N. Huezo, Helen J. Wing
Classical models of bacterial transcription show regulators binding close to promoter elements to exert their effect. However, the scope for long‐range regulation exists, especially by nucleoid structuring proteins, like H‐NS. Here, long‐range regulation by VirB, a transcriptional regulator that alleviates H‐NS‐mediated silencing of key virulence genes in Shigella species, is explored in vivo to test the limits of long‐range regulation and provide further mechanistic insight. VirB‐dependent regulation of the well‐characterized icsP promoter persists if its cognate site is repositioned 1 kb, 3.3 kb, and even 4.7 kb further upstream than its native position in a plasmid reporter. VirB‐dependent regulation diminishes with binding site distance. While increasing cellular VirB pools elevated promoter activity in all constructs with wild‐type VirB binding sites, it did not generate a disproportionate increase in promoter activity from remote sites relative to the native site. Since VirB occludes a constitutively active promoter (PT5) when docked adjacent to its −35 element, we next moved the VirB binding site far outside the promoter region. We discovered that VirB still interfered with promoter activity. These findings and those generated from molecular roadblocks engineered around a distally located VirB‐binding site are reconciled with the various models of transcriptional regulation by VirB.
{"title":"Remote Regulation by VirB, the Transcriptional Anti‐Silencer of Shigella Virulence Genes, Provides Mechanistic Information","authors":"Cody Cris, Monika M. A. Karney, Juniper S. Rosen, Alexander D. Karabachev, Elizabeth N. Huezo, Helen J. Wing","doi":"10.1111/mmi.15344","DOIUrl":"https://doi.org/10.1111/mmi.15344","url":null,"abstract":"Classical models of bacterial transcription show regulators binding close to promoter elements to exert their effect. However, the scope for long‐range regulation exists, especially by nucleoid structuring proteins, like H‐NS. Here, long‐range regulation by VirB, a transcriptional regulator that alleviates H‐NS‐mediated silencing of key virulence genes in <jats:italic>Shigella</jats:italic> species, is explored in vivo to test the limits of long‐range regulation and provide further mechanistic insight. VirB‐dependent regulation of the well‐characterized <jats:italic>icsP</jats:italic> promoter persists if its cognate site is repositioned 1 kb, 3.3 kb, and even 4.7 kb further upstream than its native position in a plasmid reporter. VirB‐dependent regulation diminishes with binding site distance. While increasing cellular VirB pools elevated promoter activity in all constructs with wild‐type VirB binding sites, it did not generate a disproportionate increase in promoter activity from remote sites relative to the native site. Since VirB occludes a constitutively active promoter (PT5) when docked adjacent to its −35 element, we next moved the VirB binding site far outside the promoter region. We discovered that VirB still interfered with promoter activity. These findings and those generated from molecular roadblocks engineered around a distally located VirB‐binding site are reconciled with the various models of transcriptional regulation by VirB.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"39 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143192107","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}
Zhixing Wang, Lin Liu, Yi Pu, Yu Fang, Wenhao Lv, Weifeng Liu
Trichoderma reesei represents an important industrial workhorse for (hemi)cellulase production. However, relatively little is known about the details of its secretory pathway ensuring the extremely high-level enzyme secretion and how they might be leveraged for engineering improved protein production. Here, the functions of T. reesei ER cargo receptors p24 and Erv29 in trafficking cellulase were characterised. Whereas individual deletion of p24 or erv29 resulted in only a marginal effect on extracellular cellulase secretion, distinct intracellular trafficking pathways exist for individual hydrolytic enzyme in T. reesei. Notably, the simultaneous absence of p24 and Erv29 abolished the secreted production of cellulases but not xylanases. The secretion defect was accompanied by an apparent intracellular accumulation of cellulases. Mutations of residues on the cytosolic side of p24 and Erv29 supposed to mediate COPII coat recognition also compromised cellulase secretion although the overall ER exit sites (ERES) formation did not seem to be affected. We further revealed that a VPL motif following the signal peptide of CBH2 necessitates its efficient secretion mediated by Erv29. These results indicate that two specific ER cargo receptors complement each other to mediate the proper intracellular trafficking of cellulases and thus ensuring their extracellular secretion.
{"title":"Distinct but Redundant Roles of ER Cargo Receptors p24 and Erv29 in Facilitating Proper Secretion of Cellulases in Trichoderma reesei","authors":"Zhixing Wang, Lin Liu, Yi Pu, Yu Fang, Wenhao Lv, Weifeng Liu","doi":"10.1111/mmi.15343","DOIUrl":"https://doi.org/10.1111/mmi.15343","url":null,"abstract":"<i>Trichoderma reesei</i> represents an important industrial workhorse for (hemi)cellulase production. However, relatively little is known about the details of its secretory pathway ensuring the extremely high-level enzyme secretion and how they might be leveraged for engineering improved protein production. Here, the functions of <i>T. reesei</i> ER cargo receptors p24 and Erv29 in trafficking cellulase were characterised. Whereas individual deletion of <i>p24</i> or <i>erv29</i> resulted in only a marginal effect on extracellular cellulase secretion, distinct intracellular trafficking pathways exist for individual hydrolytic enzyme in <i>T. reesei</i>. Notably, the simultaneous absence of p24 and Erv29 abolished the secreted production of cellulases but not xylanases. The secretion defect was accompanied by an apparent intracellular accumulation of cellulases. Mutations of residues on the cytosolic side of p24 and Erv29 supposed to mediate COPII coat recognition also compromised cellulase secretion although the overall ER exit sites (ERES) formation did not seem to be affected. We further revealed that a VPL motif following the signal peptide of CBH2 necessitates its efficient secretion mediated by Erv29. These results indicate that two specific ER cargo receptors complement each other to mediate the proper intracellular trafficking of cellulases and thus ensuring their extracellular secretion.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"39 1","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143077201","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 : 2025-02-01Epub Date: 2024-03-21DOI: 10.1111/mmi.15250
Amanda M Erkelens, Bert van Erp, Wilfried J J Meijer, Remus T Dame
Bacterial genomes are folded and organized into compact yet dynamic structures, called nucleoids. Nucleoid orchestration involves many factors at multiple length scales, such as nucleoid-associated proteins and liquid-liquid phase separation, and has to be compatible with replication and transcription. Possibly, genome organization plays an intrinsic role in transcription regulation, in addition to classical transcription factors. In this review, we provide arguments supporting this view using the Gram-positive bacterium Bacillus subtilis as a model. Proteins BsSMC, HBsu and Rok all impact the structure of the B. subtilis chromosome. Particularly for Rok, there is compelling evidence that it combines its structural function with a role as global gene regulator. Many studies describe either function of Rok, but rarely both are addressed at the same time. Here, we review both sides of the coin and integrate them into one model. Rok forms unusually stable DNA-DNA bridges and this ability likely underlies its repressive effect on transcription by either preventing RNA polymerase from binding to DNA or trapping it inside DNA loops. Partner proteins are needed to change or relieve Rok-mediated gene repression. Lastly, we investigate which features characterize H-NS-like proteins, a family that, at present, lacks a clear definition.
细菌基因组折叠并组织成紧凑而动态的结构，称为核团。核糖体的组织涉及多个长度尺度上的许多因素，如核糖体相关蛋白和液-液相分离，并且必须与复制和转录兼容。除了经典的转录因子外，基因组的组织也可能在转录调控中发挥内在作用。在这篇综述中，我们以革兰氏阳性细菌枯草芽孢杆菌为模型，提供了支持这一观点的论据。蛋白质 BsSMC、HBsu 和 Rok 都会影响枯草杆菌染色体的结构。特别是 Rok，有令人信服的证据表明，它兼具结构功能和全局基因调节器的作用。许多研究都描述了 Rok 的这两种功能，但很少同时涉及这两种功能。在这里，我们回顾了硬币的两面，并将它们整合到一个模型中。Rok 可形成异常稳定的 DNA-DNA 桥，这种能力很可能是其通过阻止 RNA 聚合酶与 DNA 结合或将其困在 DNA 环路内而对转录产生抑制作用的基础。改变或缓解 Rok 介导的基因抑制作用需要伴侣蛋白。最后，我们研究了 H-NS 样蛋白的特征，目前这个家族还缺乏明确的定义。
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Pub Date : 2025-02-01Epub Date: 2024-03-21DOI: 10.1111/mmi.15251
Paul Christopher Michael Fogg
Gene transfer agents (GTAs) are genetic elements derived from ancestral bacteriophages that have become domesticated by the host. GTAs are present in diverse prokaryotic organisms, where they can facilitate horizontal gene transfer under certain conditions. Unlike typical bacteriophages, GTAs do not exhibit any preference for the replication or transfer of the genes encoding them; instead, they exhibit a remarkable capacity to package chromosomal, and sometimes extrachromosomal, DNA into virus-like capsids and disseminate it to neighboring cells. Because GTAs resemble defective prophages, identification of novel GTAs is not trivial. The detection of candidates relies on the genetic similarity to known GTAs, which has been fruitful in α-proteobacterial lineages but challenging in more distant bacteria. Here we consider several fundamental questions: What is the true prevalence of GTAs in prokaryote genomes? Given there are high costs for GTA production, what advantage do GTAs provide to the bacterial host to justify their maintenance? How is the bacterial chromosome recognized and processed for inclusion in GTA particles? This article highlights the challenges in comprehensively understanding GTAs' prevalence, function and DNA packaging method. Going forward, broad study of atypical GTAs and use of ecologically relevant conditions are required to uncover their true impact on bacterial chromosome evolution.
{"title":"Gene transfer agents: The ambiguous role of selfless viruses in genetic exchange and bacterial evolution.","authors":"Paul Christopher Michael Fogg","doi":"10.1111/mmi.15251","DOIUrl":"10.1111/mmi.15251","url":null,"abstract":"<p><p>Gene transfer agents (GTAs) are genetic elements derived from ancestral bacteriophages that have become domesticated by the host. GTAs are present in diverse prokaryotic organisms, where they can facilitate horizontal gene transfer under certain conditions. Unlike typical bacteriophages, GTAs do not exhibit any preference for the replication or transfer of the genes encoding them; instead, they exhibit a remarkable capacity to package chromosomal, and sometimes extrachromosomal, DNA into virus-like capsids and disseminate it to neighboring cells. Because GTAs resemble defective prophages, identification of novel GTAs is not trivial. The detection of candidates relies on the genetic similarity to known GTAs, which has been fruitful in α-proteobacterial lineages but challenging in more distant bacteria. Here we consider several fundamental questions: What is the true prevalence of GTAs in prokaryote genomes? Given there are high costs for GTA production, what advantage do GTAs provide to the bacterial host to justify their maintenance? How is the bacterial chromosome recognized and processed for inclusion in GTA particles? This article highlights the challenges in comprehensively understanding GTAs' prevalence, function and DNA packaging method. Going forward, broad study of atypical GTAs and use of ecologically relevant conditions are required to uncover their true impact on bacterial chromosome evolution.</p>","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":" ","pages":"124-131"},"PeriodicalIF":2.6,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11841831/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140175600","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}
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