In this issue (J Bacteriol. 206: e0014024, https://doi.org/10.1128/jb.00140-24), Ridone and Baker describe hybrids between two 5:2 heteroheptameric ion-powered motors. Chimeras were constructed between stator units of a bacterial flagellum and ExbBD of the Ton outer-membrane transport system. Only one of the 14 hybrids supported swimming in Escherichia coli. Three additional residue changes at sites distant from the hybrid region enhanced motility. This work suggests that flagellar stator units and ExbBD share an ancestor that diverged during evolution to perform different tasks.
{"title":"A very versatile molecular machine.","authors":"Michael D Manson","doi":"10.1128/jb.00351-24","DOIUrl":"https://doi.org/10.1128/jb.00351-24","url":null,"abstract":"<p><p>In this issue (J Bacteriol. 206: e0014024, https://doi.org/10.1128/jb.00140-24), Ridone and Baker describe hybrids between two 5:2 heteroheptameric ion-powered motors. Chimeras were constructed between stator units of a bacterial flagellum and ExbBD of the Ton outer-membrane transport system. Only one of the 14 hybrids supported swimming in <i>Escherichia coli</i>. Three additional residue changes at sites distant from the hybrid region enhanced motility. This work suggests that flagellar stator units and ExbBD share an ancestor that diverged during evolution to perform different tasks.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347208","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rickettsia rickettsii is an obligate intracellular, tick-borne bacterium that causes Rocky Mountain spotted fever. The demanding nature of cultivating these bacteria within host cells and the labor involved in obtaining clonal isolates have severely limited progress regarding the development of compatible genetic tools to study this pathogen. Conditional expression of genes that might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline-inducible promoter system used for Borrelia burgdorferi and one of the few characterized rickettsial promoters for the outer membrane gene, rompB (sca5). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for R. rickettsii. The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in R. rickettsii targeting the type IV secreted effector rarP2 and autotransporter peptidase rapL as examples.
Importance: This work expands upon the genetic toolbox available for R. rickettsii. We describe both an inducible promoter and CRISPRi system compatible with Rickettsia, which may provide key instruments for the development of further tools. The development of an inducible promoter system allows for the overexpression of genes, which might be toxic when expressed constitutively. The CRISPRi system enables the ability to knock down genes with specificity, and critically, genes that may be essential and could not otherwise be knocked out. These developments may provide the foundation for unlocking genetic tools for other pathogens of the order Rickettsiales, such as the Anaplasma, Orientia, and Ehrlichia for which there are currently no inducible promoters or CRISPRi platforms.
{"title":"Development of inducible promoter and CRISPRi plasmids functional in <i>Rickettsia rickettsii</i>.","authors":"Adam M Nock, Tina R Clark, Ted Hackstadt","doi":"10.1128/jb.00367-24","DOIUrl":"https://doi.org/10.1128/jb.00367-24","url":null,"abstract":"<p><p><i>Rickettsia rickettsii</i> is an obligate intracellular, tick-borne bacterium that causes Rocky Mountain spotted fever. The demanding nature of cultivating these bacteria within host cells and the labor involved in obtaining clonal isolates have severely limited progress regarding the development of compatible genetic tools to study this pathogen. Conditional expression of genes that might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline-inducible promoter system used for <i>Borrelia burgdorferi</i> and one of the few characterized rickettsial promoters for the outer membrane gene, <i>rompB</i> (<i>sca5</i>). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for <i>R. rickettsii</i>. The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in <i>R. rickettsii</i> targeting the type IV secreted effector <i>rarP2</i> and autotransporter peptidase <i>rapL</i> as examples.</p><p><strong>Importance: </strong>This work expands upon the genetic toolbox available for <i>R. rickettsii</i>. We describe both an inducible promoter and CRISPRi system compatible with <i>Rickettsia</i>, which may provide key instruments for the development of further tools. The development of an inducible promoter system allows for the overexpression of genes, which might be toxic when expressed constitutively. The CRISPRi system enables the ability to knock down genes with specificity, and critically, genes that may be essential and could not otherwise be knocked out. These developments may provide the foundation for unlocking genetic tools for other pathogens of the order Rickettsiales, such as the <i>Anaplasma</i>, <i>Orientia</i>, and <i>Ehrlichia</i> for which there are currently no inducible promoters or CRISPRi platforms.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347212","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Kexin Wang, Moontaha Mahbub, Giulia Mastroianni, Ana Valladares, Conrad W Mullineaux
Heterocyst-forming cyanobacteria such as Anabaena (Nostoc) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in Anabaena vegetative cells and developing heterocysts, using mRNA fluorescent in situ hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized "honeycomb" regions of the thylakoid membranes close to the cell poles. However, cox2 and cox3 mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest Anabaena homolog of Rbp3 in the unicellular cyanobacterium Synechocystis sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An rbpG null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent in situ hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.
{"title":"mRNA localization and thylakoid protein biogenesis in the filamentous heterocyst-forming cyanobacterium <i>Anabaena</i> sp. PCC 7120.","authors":"Kexin Wang, Moontaha Mahbub, Giulia Mastroianni, Ana Valladares, Conrad W Mullineaux","doi":"10.1128/jb.00328-24","DOIUrl":"https://doi.org/10.1128/jb.00328-24","url":null,"abstract":"<p><p>Heterocyst-forming cyanobacteria such as <i>Anabaena</i> (<i>Nostoc</i>) sp. PCC 7120 exhibit extensive remodeling of their thylakoid membranes during heterocyst differentiation. Here we investigate the sites of translation of thylakoid membrane proteins in <i>Anabaena</i> vegetative cells and developing heterocysts, using mRNA fluorescent <i>in situ</i> hybridization (FISH) to detect the location of specific mRNA species. We probed mRNAs encoding reaction center core components and the heterocyst-specific terminal oxidases Cox2 and Cox3. As in unicellular cyanobacteria, the mRNAs encoding membrane-integral thylakoid proteins are concentrated in patches at the inner face of the thylakoid membrane system, adjacent to the central cytoplasm. These patches mark the putative sites of translation and membrane insertion of these proteins. Oxidase activity in mature heterocysts is concentrated in the specialized \"honeycomb\" regions of the thylakoid membranes close to the cell poles. However, <i>cox2</i> and <i>cox3</i> mRNAs remain evenly distributed over the inner face of the thylakoids, implying that oxidase proteins migrate extensively after translation to reach their destination in the honeycomb membranes. The RNA-binding protein RbpG is the closest <i>Anabaena</i> homolog of Rbp3 in the unicellular cyanobacterium <i>Synechocystis</i> sp. PCC 6803, which we previously showed to be crucial for the correct location of photosynthetic mRNAs. An <i>rbpG</i> null mutant shows decreased cellular levels of photosynthetic mRNAs and photosynthetic complexes, coupled with perturbations to thylakoid membrane organization and lower efficiency of the Photosystem II repair cycle. This suggests that the chaperoning of photosynthetic mRNAs by RbpG is important for the correct coordination of thylakoid protein translation and assembly.IMPORTANCECyanobacteria have a complex thylakoid membrane system which is the site of the photosynthetic light reactions as well as most of the respiratory activity in the cell. Protein targeting to the thylakoids and the spatial organization of thylakoid protein biogenesis remain poorly understood. Further complexity is found in some filamentous cyanobacteria that produce heterocysts, specialized nitrogen-fixing cells in which the thylakoid membranes undergo extensive remodeling. Here we probe mRNA locations to reveal thylakoid translation sites in a heterocyst-forming cyanobacterium. We identify an RNA-binding protein important for the correct co-ordination of thylakoid protein translation and assembly, and we demonstrate the effectiveness of mRNA fluorescent <i>in situ</i> hybridization (FISH) as a way to probe cell-specific gene expression in multicellular cyanobacteria.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347225","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anubhav Basu, Amanda N D Adams, Patrick H Degnan, Carin K Vanderpool
Bacteroides species are successful colonizers of the human colon and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in polysaccharide utilization loci (PULs). While recent work has uncovered the PULs required for the use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-β-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron. Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under the control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron. Examining the genomes of other Bacteroides species, we found homologs of BT1871 in a subset and showed that representative strains of species with a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide.
Importance: The gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the microbiota in the colon since they remain undigested by the host. Here, we study how the model commensal organism, Bacteroides thetaiotaomicron utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource.
{"title":"Determinants of raffinose family oligosaccharide use in <i>Bacteroides</i> species.","authors":"Anubhav Basu, Amanda N D Adams, Patrick H Degnan, Carin K Vanderpool","doi":"10.1128/jb.00235-24","DOIUrl":"10.1128/jb.00235-24","url":null,"abstract":"<p><p><i>Bacteroides</i> species are successful colonizers of the human colon and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in polysaccharide utilization loci (PULs). While recent work has uncovered the PULs required for the use of some polysaccharides, how <i>Bacteroides</i> utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-β-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in <i>Bacteroides thetaiotaomicron</i>. Here, we identify two different types of mutations that increase <i>BT1871</i> mRNA levels and improve <i>B. thetaiotaomicron</i> growth on RFOs. First, a novel spontaneous duplication of <i>BT1872</i> and <i>BT1871</i> places these genes under the control of a ribosomal promoter, driving high <i>BT1871</i> transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase <i>BT1871</i> transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in <i>B. thetaiotaomicron</i>. Examining the genomes of other <i>Bacteroides</i> species, we found homologs of BT1871 in a subset and showed that representative strains of species with a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide.</p><p><strong>Importance: </strong>The gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the microbiota in the colon since they remain undigested by the host. Here, we study how the model commensal organism, <i>Bacteroides thetaiotaomicron</i> utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347211","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chemotaxis is the directed, flagellum-based movement of bacteria in chemoeffector gradients. Bacteria respond chemotactically to a wide range of chemoeffectors, including amino, organic, and fatty acids, sugars, polyamines, quaternary amines, purines, pyrimidines, aromatic hydrocarbons, oxygen, inorganic ions, or polysaccharides. Most frequent are chemotactic responses to amino acids (AAs), which were observed in numerous bacteria regardless of their phylogeny and lifestyle. Mostly chemoattraction responses are observed, although a number of bacteria are repelled from certain AAs. Chemoattraction is associated with the important metabolic value of AAs as growth substrates or building blocks of proteins. However, additional studies revealed that AAs are also sensed as environmental cues. Many chemoreceptors are specific for AAs, and signaling is typically initiated by direct ligand binding to their four-helix bundle or dCache ligand-binding domains. Frequently, bacteria possess multiple AA-responsive chemoreceptors that at times possess complementary AA ligand spectra. The identification of sequence motifs in the binding sites at dCache_1 domains has permitted to define an AA-specific family of dCache_1AA chemoreceptors. In addition, AAs are among the ligands recognized by broad ligand range chemoreceptors, and evidence was obtained for chemoreceptor activation by the binding of AA-loaded solute-binding proteins. The biological significance of AA chemotaxis is very ample including in biofilm formation, root and seed colonization by beneficial bacteria, plant entry of phytopathogens, colonization of the intestine, or different virulence-related features in human/animal pathogens. This review provides insights that may be helpful for the study of AA chemotaxis in other uncharacterized bacteria.
趋化作用是细菌在化学效应梯度中以鞭毛为基础的定向运动。细菌对多种化学效应物(包括氨基酸、有机酸和脂肪酸、糖、多胺、季胺、嘌呤、嘧啶、芳香烃、氧、无机离子或多糖)都会产生趋化反应。最常见的是对氨基酸(AAs)的趋化反应,在许多细菌中都观察到了这种反应,而不论其系统发育和生活方式如何。虽然一些细菌会被某些 AAs 排斥,但观察到的大多是趋化反应。趋化作用与 AAs 作为生长底物或蛋白质组成成分的重要代谢价值有关。然而,更多的研究表明,AAs 也能作为环境线索被感知。许多化学感受器对 AAs 具有特异性,信号通常是通过配体与它们的四螺旋束或 dCache 配体结合域直接结合而启动的。细菌通常具有多种 AA 响应化学感受器,这些化学感受器有时具有互补的 AA 配体谱。通过鉴定 dCache_1 结构域结合位点的序列基序,可以定义一个 AA 特异性的 dCache_1AA 化学感受器家族。此外,配体范围广泛的化学感受器也能识别 AA,而且有证据表明,化学感受器是通过与装载 AA 的溶质结合蛋白结合而激活的。AA 趋化作用的生物学意义非常广泛,包括生物膜的形成、有益细菌的根和种子定殖、植物病原体的植物进入、肠道定殖或人类/动物病原体的不同毒力相关特征。本综述提供的见解可能有助于研究其他未定性细菌的 AA 趋化性。
{"title":"Bacterial amino acid chemotaxis: a widespread strategy with multiple physiological and ecological roles.","authors":"Miguel A Matilla, Tino Krell","doi":"10.1128/jb.00300-24","DOIUrl":"https://doi.org/10.1128/jb.00300-24","url":null,"abstract":"<p><p>Chemotaxis is the directed, flagellum-based movement of bacteria in chemoeffector gradients. Bacteria respond chemotactically to a wide range of chemoeffectors, including amino, organic, and fatty acids, sugars, polyamines, quaternary amines, purines, pyrimidines, aromatic hydrocarbons, oxygen, inorganic ions, or polysaccharides. Most frequent are chemotactic responses to amino acids (AAs), which were observed in numerous bacteria regardless of their phylogeny and lifestyle. Mostly chemoattraction responses are observed, although a number of bacteria are repelled from certain AAs. Chemoattraction is associated with the important metabolic value of AAs as growth substrates or building blocks of proteins. However, additional studies revealed that AAs are also sensed as environmental cues. Many chemoreceptors are specific for AAs, and signaling is typically initiated by direct ligand binding to their four-helix bundle or dCache ligand-binding domains. Frequently, bacteria possess multiple AA-responsive chemoreceptors that at times possess complementary AA ligand spectra. The identification of sequence motifs in the binding sites at dCache_1 domains has permitted to define an AA-specific family of dCache_1AA chemoreceptors. In addition, AAs are among the ligands recognized by broad ligand range chemoreceptors, and evidence was obtained for chemoreceptor activation by the binding of AA-loaded solute-binding proteins. The biological significance of AA chemotaxis is very ample including in biofilm formation, root and seed colonization by beneficial bacteria, plant entry of phytopathogens, colonization of the intestine, or different virulence-related features in human/animal pathogens. This review provides insights that may be helpful for the study of AA chemotaxis in other uncharacterized bacteria.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Neda Habibi Arejan, Desiree R Czapski, Joseph A Buonomo, Cara C Boutte
Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis, we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 is responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation and in which the connection between Wag31, PlrA, and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate the synthesis of the different layers of the mycobacterial cell wall in changing conditions.
Importance: This study is performed in Mycobacterium smegmatis, which is used as a model to understand the basic physiology of pathogenic mycobacteria such as Mycobacterium tuberculosis. In this work, we examine the function and regulation of three proteins involved in regulating cell wall elongation in mycobacterial cells, which occurs at the cell tips or poles. We find that Wag31, a regulator of polar elongation, works partly through the regulation of MmpL3, a transporter of cell wall constituents and an important drug target. Our work suggests that, beyond its transport function, MmpL3 has another function in controlling cell wall synthesis broadly in response to stress.
{"title":"MmpL3, Wag31, and PlrA are involved in coordinating polar growth with peptidoglycan metabolism and nutrient availability.","authors":"Neda Habibi Arejan, Desiree R Czapski, Joseph A Buonomo, Cara C Boutte","doi":"10.1128/jb.00204-24","DOIUrl":"10.1128/jb.00204-24","url":null,"abstract":"<p><p>Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of <i>Mycobacterium smegmatis</i>, we identify a connection between <i>wag31</i> and trehalose monomycolate (TMM) transporter <i>mmpl3</i> in a suppressor screen and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 is responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation and in which the connection between Wag31, PlrA, and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate the synthesis of the different layers of the mycobacterial cell wall in changing conditions.</p><p><strong>Importance: </strong>This study is performed in <i>Mycobacterium smegmatis</i>, which is used as a model to understand the basic physiology of pathogenic mycobacteria such as <i>Mycobacterium tuberculosis</i>. In this work, we examine the function and regulation of three proteins involved in regulating cell wall elongation in mycobacterial cells, which occurs at the cell tips or poles. We find that Wag31, a regulator of polar elongation, works partly through the regulation of MmpL3, a transporter of cell wall constituents and an important drug target. Our work suggests that, beyond its transport function, MmpL3 has another function in controlling cell wall synthesis broadly in response to stress.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347224","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Basanti Malakar, Valdir C Barth, Julia Puffal, Nancy A Woychik, Robert N Husson
Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis, we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. In growth inhibition experiments, M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46, whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation.IMPORTANCEIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection.
{"title":"Phosphorylation of VapB antitoxins affects intermolecular interactions to regulate VapC toxin activity in <i>Mycobacterium tuberculosis</i>.","authors":"Basanti Malakar, Valdir C Barth, Julia Puffal, Nancy A Woychik, Robert N Husson","doi":"10.1128/jb.00233-24","DOIUrl":"10.1128/jb.00233-24","url":null,"abstract":"<p><p>Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the <i>Mycobacterium tuberculosis</i> complex, with 50 modules present in the <i>M. tuberculosis</i> genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the <i>vapB-vapC</i> operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in <i>M. tuberculosis</i>, we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of <i>vapB-vapC</i> operon transcription would result in increased free VapC in the <i>M. tuberculosis</i> cell. In growth inhibition experiments, <i>M. tuberculosis</i> strains expressing <i>vapB46-vapC46</i> constructs containing a phosphoablative <i>vapB</i> mutation resulted in lower toxicity compared to a strain expressing native <i>vapB46</i>, whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic <i>vapB</i> mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation.IMPORTANCEIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142307817","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In Gram-negative bacteria, LPS (lipopolysaccharide) has been thoroughly characterized and has been shown to play a major role in pathogenesis and bacterial defense. In Salmonella and Escherichia coli, LPS also influences biofilm development. However, the overall role of LPS glycoform in biofilm formation has not been conclusively settled, as there is a lack of consensus on the topic. Some studies show that LPS mutants produce less biofilm biomass than the wild-type strains, while others show that they produce more. This review summarizes current knowledge of LPS biosynthesis and explores the impact of defective steps on biofilm-related characteristics, such as motility, adhesion, auto-aggregation, and biomass production in Salmonella and E. coli. Overall, motility tends to decrease, while adhesion and auto-aggregation phenotypes tend to increase in most LPS-mutant strains. Interestingly, biofilm biomass of various LPS mutants revealed a clear pattern dependent on biofilm maturation time. Incubation times of less than 24 h resulted in a biofilm-defective phenotype compared to the wild-type, while incubation exceeding 24 h led to significantly higher levels of biofilm production. This explains conflicting results found in reports describing the same LPS mutations. It is therefore critical to consider the effect of biofilm maturation time to ascertain the effects of LPS glycoform on biofilm phenotype. Underlying reasons for such changes in biofilm kinetics may include changes in signalling systems affecting biofilm maturation and composition, and dynamic LPS modifications. A better understanding of the role of LPS in the evolution and modification of biofilms is crucial for developing strategies to disperse biofilms.
{"title":"Only time will tell: lipopolysaccharide glycoform and biofilm-formation kinetics in <i>Salmonella</i> species and <i>Escherichia coli</i>.","authors":"Magdalena Laekas-Hameder, France Daigle","doi":"10.1128/jb.00318-24","DOIUrl":"https://doi.org/10.1128/jb.00318-24","url":null,"abstract":"<p><p>In Gram-negative bacteria, LPS (lipopolysaccharide) has been thoroughly characterized and has been shown to play a major role in pathogenesis and bacterial defense. In <i>Salmonella</i> and <i>Escherichia coli</i>, LPS also influences biofilm development. However, the overall role of LPS glycoform in biofilm formation has not been conclusively settled, as there is a lack of consensus on the topic. Some studies show that LPS mutants produce less biofilm biomass than the wild-type strains, while others show that they produce more. This review summarizes current knowledge of LPS biosynthesis and explores the impact of defective steps on biofilm-related characteristics, such as motility, adhesion, auto-aggregation, and biomass production in <i>Salmonella</i> and <i>E. coli</i>. Overall, motility tends to decrease, while adhesion and auto-aggregation phenotypes tend to increase in most LPS-mutant strains. Interestingly, biofilm biomass of various LPS mutants revealed a clear pattern dependent on biofilm maturation time. Incubation times of less than 24 h resulted in a biofilm-defective phenotype compared to the wild-type, while incubation exceeding 24 h led to significantly higher levels of biofilm production. This explains conflicting results found in reports describing the same LPS mutations. It is therefore critical to consider the effect of biofilm maturation time to ascertain the effects of LPS glycoform on biofilm phenotype. Underlying reasons for such changes in biofilm kinetics may include changes in signalling systems affecting biofilm maturation and composition, and dynamic LPS modifications. A better understanding of the role of LPS in the evolution and modification of biofilms is crucial for developing strategies to disperse biofilms.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142307816","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hayden A Bender, Roger Huynh, Charles Puerner, Jennifer Pelaez, Craig Sadowski, Elijah N Kissman, Julia Barbano, Karla B Schallies, Katherine E Gibson
The cell cycle is a fundamental process involved in bacterial reproduction and cellular differentiation. For Sinorhizobium meliloti, cell cycle outcomes depend on its growth environment. This bacterium shows a tight coupling of DNA replication initiation with cell division during free-living growth. In contrast, it undergoes a novel program of endoreduplication and terminal differentiation during symbiosis within its host. While several DivK regulators at the top of its CtrA pathway have been shown to play an important role in this differentiation process, there is a lack of resolution regarding the downstream molecular activities required and whether they could be unique to the symbiosis cell cycle. The DivK kinase CbrA is a negative regulator of CtrA activity and is required for successful symbiosis. In this work, spontaneous symbiosis suppressors of ΔcbrA were identified as alleles of divL and cckA. In addition to rescuing symbiotic development, they restore wild-type cell cycle progression to free-living ΔcbrA cells. Biochemical characterization of the S. meliloti hybrid histidine kinase CckA in vitro demonstrates that it has both kinase and phosphatase activities. Specifically, CckA on its own has autophosphorylation activity, and phosphatase activity is induced by the second messenger c-di-GMP. Importantly, the CckAA373S suppressor protein of ΔcbrA has a significant loss in kinase activity, and this is predicted to cause decreased CtrA activity in vivo. These findings deepen our understanding of the CbrA regulatory pathway and open new avenues for further molecular characterization of a network pivotal to the free-living cell cycle and symbiotic differentiation of S. meliloti.IMPORTANCESinorhizobium meliloti is a soil bacterium able to form a nitrogen-fixing symbiosis with certain legumes, including the agriculturally important Medicago sativa. It provides ammonia to plants growing in nitrogen-poor soils and is therefore of agricultural and environmental significance as this symbiosis negates the need for industrial fertilizers. Understanding mechanisms governing symbiotic development is essential to either engineer a more effective symbiosis or extend its potential to non-leguminous crops. Here, we identify mutations within cell cycle regulators and find that they control cell cycle outcomes during both symbiosis and free-living growth. As regulators within the CtrA two-component signal transduction pathway, this study deepens our understanding of a regulatory network shaping host colonization, cell cycle differentiation, and symbiosis in an important model organism.
{"title":"The <i>Sinorhizobium meliloti</i> nitrogen-fixing symbiosis requires CbrA-dependent regulation of a DivL and CckA phosphorelay.","authors":"Hayden A Bender, Roger Huynh, Charles Puerner, Jennifer Pelaez, Craig Sadowski, Elijah N Kissman, Julia Barbano, Karla B Schallies, Katherine E Gibson","doi":"10.1128/jb.00399-23","DOIUrl":"https://doi.org/10.1128/jb.00399-23","url":null,"abstract":"<p><p>The cell cycle is a fundamental process involved in bacterial reproduction and cellular differentiation. For <i>Sinorhizobium meliloti</i>, cell cycle outcomes depend on its growth environment. This bacterium shows a tight coupling of DNA replication initiation with cell division during free-living growth. In contrast, it undergoes a novel program of endoreduplication and terminal differentiation during symbiosis within its host. While several DivK regulators at the top of its CtrA pathway have been shown to play an important role in this differentiation process, there is a lack of resolution regarding the downstream molecular activities required and whether they could be unique to the symbiosis cell cycle. The DivK kinase CbrA is a negative regulator of CtrA activity and is required for successful symbiosis. In this work, spontaneous symbiosis suppressors of Δ<i>cbrA</i> were identified as alleles of <i>divL</i> and <i>cckA</i>. In addition to rescuing symbiotic development, they restore wild-type cell cycle progression to free-living Δ<i>cbrA</i> cells. Biochemical characterization of the <i>S. meliloti</i> hybrid histidine kinase CckA <i>in vitro</i> demonstrates that it has both kinase and phosphatase activities. Specifically, CckA on its own has autophosphorylation activity, and phosphatase activity is induced by the second messenger c-di-GMP. Importantly, the CckA<sup>A373S</sup> suppressor protein of Δ<i>cbrA</i> has a significant loss in kinase activity, and this is predicted to cause decreased CtrA activity <i>in vivo</i>. These findings deepen our understanding of the CbrA regulatory pathway and open new avenues for further molecular characterization of a network pivotal to the free-living cell cycle and symbiotic differentiation of <i>S. meliloti</i>.IMPORTANCE<i>Sinorhizobium meliloti</i> is a soil bacterium able to form a nitrogen-fixing symbiosis with certain legumes, including the agriculturally important <i>Medicago sativa</i>. It provides ammonia to plants growing in nitrogen-poor soils and is therefore of agricultural and environmental significance as this symbiosis negates the need for industrial fertilizers. Understanding mechanisms governing symbiotic development is essential to either engineer a more effective symbiosis or extend its potential to non-leguminous crops. Here, we identify mutations within cell cycle regulators and find that they control cell cycle outcomes during both symbiosis and free-living growth. As regulators within the CtrA two-component signal transduction pathway, this study deepens our understanding of a regulatory network shaping host colonization, cell cycle differentiation, and symbiosis in an important model organism.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142307818","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Stephanie L Cooper, Ryan M Requijo, Aaron L Lucius, David A Schneider
Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb). While eukaryotic species employ several specialized RNA polymerases (Pols) to fulfill the RNA synthesis requirements of the cell, bacterial species use a single RNA polymerase (RNAP). To contribute to the foundational understanding of how Mtb and the related non-pathogenic mycobacterial species, Mycobacterium smegmatis (Msm), perform the essential function of RNA synthesis, we performed a series of in vitro transcription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs. In this study, we characterize the mechanism of nucleotide addition used by these bacterial RNAPs with comparisons to previously characterized eukaryotic Pols I, II, and III. We show that Mtb RNAP and Msm RNAP demonstrate similar enzymatic properties and nucleotide addition kinetics to each other but diverge significantly from eukaryotic Pols. We also show that Mtb RNAP and Msm RNAP uniquely bind a nucleotide analog with significantly higher affinity than canonical nucleotides, in contrast to eukaryotic RNA polymerase II. This affinity for analogs may reveal a vulnerability for selective inhibition of the pathogenic bacterial enzyme.IMPORTANCETuberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a severe global health threat. The World Health Organization (WHO) has reported that tuberculosis is second only to COVID-19 as the most lethal infection worldwide, with more annual deaths than HIV and AIDS (WHO.int). The first-line treatment for tuberculosis, Rifampin (or Rifampicin), specifically targets the Mtb RNA polymerase. This drug has been used for decades, leading to increased numbers of multi-drug-resistant infections (Stephanie, et al). To effectively treat tuberculosis, there is an urgent need for new therapeutics that selectively target vulnerabilities of the bacteria and not the host. Characterization of the differences between Mtb enzymes and host enzymes is critical to inform these ongoing drug design efforts.
{"title":"Biochemical characterization of <i>Mycobacterial</i> RNA polymerases.","authors":"Stephanie L Cooper, Ryan M Requijo, Aaron L Lucius, David A Schneider","doi":"10.1128/jb.00256-24","DOIUrl":"10.1128/jb.00256-24","url":null,"abstract":"<p><p>Tuberculosis is caused by the bacterium <i>Mycobacterium tuberculosis</i> (Mtb). While eukaryotic species employ several specialized RNA polymerases (Pols) to fulfill the RNA synthesis requirements of the cell, bacterial species use a single RNA polymerase (RNAP). To contribute to the foundational understanding of how Mtb and the related non-pathogenic mycobacterial species, <i>Mycobacterium smegmatis</i> (Msm), perform the essential function of RNA synthesis, we performed a series of <i>in vitro</i> transcription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs. In this study, we characterize the mechanism of nucleotide addition used by these bacterial RNAPs with comparisons to previously characterized eukaryotic Pols I, II, and III. We show that Mtb RNAP and Msm RNAP demonstrate similar enzymatic properties and nucleotide addition kinetics to each other but diverge significantly from eukaryotic Pols. We also show that Mtb RNAP and Msm RNAP uniquely bind a nucleotide analog with significantly higher affinity than canonical nucleotides, in contrast to eukaryotic RNA polymerase II. This affinity for analogs may reveal a vulnerability for selective inhibition of the pathogenic bacterial enzyme.IMPORTANCETuberculosis, caused by the bacterium <i>Mycobacterium tuberculosis</i> (Mtb), remains a severe global health threat. The World Health Organization (WHO) has reported that tuberculosis is second only to COVID-19 as the most lethal infection worldwide, with more annual deaths than HIV and AIDS (WHO.int). The first-line treatment for tuberculosis, Rifampin (or Rifampicin), specifically targets the Mtb RNA polymerase. This drug has been used for decades, leading to increased numbers of multi-drug-resistant infections (Stephanie, <i>et al</i>). To effectively treat tuberculosis, there is an urgent need for new therapeutics that selectively target vulnerabilities of the bacteria and not the host. Characterization of the differences between Mtb enzymes and host enzymes is critical to inform these ongoing drug design efforts.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142307814","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}