Natalie C. Bamford, Ryan J. Morris, Alan Prescott, Paul Murphy, Elliot Erskine, Cait E. MacPhee, Nicola R. Stanley-Wall
The extracellular matrix of biofilms provides crucial structural support to the community and protection from environmental perturbations. TasA, a key Bacillus subtilis biofilm matrix protein, forms both amyloid and non-amyloid fibrils. Non-amyloid TasA fibrils are formed via a strand-exchange mechanism, whereas the amyloid-like form involves non-specific self-assembly. We performed mutagenesis of the N-terminus to assess the role of non-amyloid fibrils in biofilm development. We find that the N-terminal tail is essential for the formation of structured biofilms, providing evidence that the strand-exchange fibrils are the active form in the biofilm matrix. Furthermore, we demonstrate that fibre formation alone is not sufficient to give structure to the biofilm. We build an interactome of TasA with other extracellular protein components, and identify important interaction sites. Our results provide insight into how protein–matrix interactions modulate biofilm development.
生物膜的细胞外基质为生物群落提供重要的结构支持，并保护其免受环境干扰。TasA 是枯草芽孢杆菌生物膜基质的一种关键蛋白质，可形成淀粉样和非淀粉样纤维。非淀粉样 TasA 纤维是通过链交换机制形成的，而淀粉样形式则涉及非特异性自组装。我们对 N 端进行了诱变，以评估非淀粉样纤维在生物膜发育中的作用。我们发现，N 端尾部对于结构化生物膜的形成至关重要，这为链交换纤维是生物膜基质中的活性形式提供了证据。此外，我们还证明，仅形成纤维不足以使生物膜具有结构。我们建立了 TasA 与其他细胞外蛋白质成分的相互作用组，并确定了重要的相互作用位点。我们的研究结果为了解蛋白质与基质之间的相互作用如何调节生物膜的发展提供了启示。
{"title":"TasA Fibre Interactions Are Necessary for Bacillus subtilis Biofilm Structure","authors":"Natalie C. Bamford, Ryan J. Morris, Alan Prescott, Paul Murphy, Elliot Erskine, Cait E. MacPhee, Nicola R. Stanley-Wall","doi":"10.1111/mmi.15315","DOIUrl":"https://doi.org/10.1111/mmi.15315","url":null,"abstract":"The extracellular matrix of biofilms provides crucial structural support to the community and protection from environmental perturbations. TasA, a key <i>Bacillus subtilis</i> biofilm matrix protein, forms both amyloid and non-amyloid fibrils. Non-amyloid TasA fibrils are formed via a strand-exchange mechanism, whereas the amyloid-like form involves non-specific self-assembly. We performed mutagenesis of the N-terminus to assess the role of non-amyloid fibrils in biofilm development. We find that the N-terminal tail is essential for the formation of structured biofilms, providing evidence that the strand-exchange fibrils are the active form in the biofilm matrix. Furthermore, we demonstrate that fibre formation alone is not sufficient to give structure to the biofilm. We build an interactome of TasA with other extracellular protein components, and identify important interaction sites. Our results provide insight into how protein–matrix interactions modulate biofilm development.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142330270","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}
Xinwei Liu, Gabriela Boelter, Waldemar Vollmer, Manuel Banzhaf, Tanneke den Blaauwen
Escherichia coli has many periplasmic hydrolases to degrade and modify peptidoglycan (PG). However, the redundancy of eight PG endopeptidases makes it challenging to define specific roles to individual enzymes. Therefore, the cellular role of PBP7 (encoded by pbpG) is not clearly defined. In this work, we show that PBP7 localizes in the lateral cell envelope and at midcell. The C-terminal α-helix of PBP7 is crucial for midcell localization but not for its activity, which is dispensable for this localization. Additionally, midcell localization of PBP7 relies on the assembly of FtsZ up to FtsN in the divisome, and on the activity of PBP3. PBP7 was found to affect the assembly timing of FtsZ and FtsN in the divisome. The absence of PBP7 slows down the assembly of FtsN at midcell. The ΔpbpG mutant exhibited a weaker incorporation of the fluorescent D-amino acid HADA, reporting on transpeptidase activity, compared to wild-type cells. This could indicate reduced PG synthesis at the septum of the ΔpbpG strain, explaining the slower accumulation of FtsN and suggesting that endopeptidase-mediated PG cleavage may be a rate-limiting step for septal PG synthesis.
{"title":"Peptidoglycan Endopeptidase PBP7 Facilitates the Recruitment of FtsN to the Divisome and Promotes Peptidoglycan Synthesis in Escherichia coli","authors":"Xinwei Liu, Gabriela Boelter, Waldemar Vollmer, Manuel Banzhaf, Tanneke den Blaauwen","doi":"10.1111/mmi.15321","DOIUrl":"https://doi.org/10.1111/mmi.15321","url":null,"abstract":"<i>Escherichia coli</i> has many periplasmic hydrolases to degrade and modify peptidoglycan (PG). However, the redundancy of eight PG endopeptidases makes it challenging to define specific roles to individual enzymes. Therefore, the cellular role of PBP7 (encoded by <i>pbpG</i>) is not clearly defined. In this work, we show that PBP7 localizes in the lateral cell envelope and at midcell. The C-terminal α-helix of PBP7 is crucial for midcell localization but not for its activity, which is dispensable for this localization. Additionally, midcell localization of PBP7 relies on the assembly of FtsZ up to FtsN in the divisome, and on the activity of PBP3. PBP7 was found to affect the assembly timing of FtsZ and FtsN in the divisome. The absence of PBP7 slows down the assembly of FtsN at midcell. The Δ<i>pbpG</i> mutant exhibited a weaker incorporation of the fluorescent D-amino acid HADA, reporting on transpeptidase activity, compared to wild-type cells. This could indicate reduced PG synthesis at the septum of the Δ<i>pbpG</i> strain, explaining the slower accumulation of FtsN and suggesting that endopeptidase-mediated PG cleavage may be a rate-limiting step for septal PG synthesis.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142330267","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}
Two-component systems (TCSs) are vital signal transduction pathways ubiquitous among bacteria, facilitating their responses to diverse environmental stimuli. In Bacillus subtilis, the DesK histidine kinase thermosensor, together with the response regulator DesR, constitute a TCS dedicated to membrane lipid homeostasis maintenance. This TCS orchestrates the transcriptional regulation of the des gene, encoding the sole desaturase in these bacteria, Δ5-Des. Additionally, B. subtilis possesses a paralog TCS, YvfT/YvfU, with unknown target gene(s). In this work, we show that YvfT/YvfU controls the expression of the yvfRS operon that codes for an ABC transporter. Interestingly, we found that this regulation also involves the action of DesK/DesR. Notably, opposite to des, yvfRS transcription is induced at 37°C and not at 25°C. Our in vivo and in vitro experiments demonstrate that both YvfU and DesR directly bind to the operon promoter region, with DesR exerting its control over yvfRS expression in its unphosphorylated state. Our study uncovers an intriguing case of cross-regulation where two homologous TCSs interact closely to finely tune gene expression in response to environmental cues. These findings shed light on the complexity of bacterial signal transduction systems and their critical role in bacterial adaptability.
{"title":"Unveiling the Coordinated Action of DesK/DesR and YvfT/YvfU to Control the Expression of an ABC Transporter in Bacillus subtilis","authors":"Pilar Fernández, Lucía Porrini, Julián Ignacio Pereyra, Daniela Albanesi, María Cecilia Mansilla","doi":"10.1111/mmi.15320","DOIUrl":"https://doi.org/10.1111/mmi.15320","url":null,"abstract":"Two-component systems (TCSs) are vital signal transduction pathways ubiquitous among bacteria, facilitating their responses to diverse environmental stimuli. In <i>Bacillus subtilis</i>, the DesK histidine kinase thermosensor, together with the response regulator DesR, constitute a TCS dedicated to membrane lipid homeostasis maintenance. This TCS orchestrates the transcriptional regulation of the <i>des</i> gene, encoding the sole desaturase in these bacteria, Δ5-Des. Additionally, <i>B. subtilis</i> possesses a paralog TCS, YvfT/YvfU, with unknown target gene(s). In this work, we show that YvfT/YvfU controls the expression of the <i>yvfRS</i> operon that codes for an ABC transporter. Interestingly, we found that this regulation also involves the action of DesK/DesR. Notably, opposite to <i>des</i>, <i>yvfRS</i> transcription is induced at 37°C and not at 25°C. Our in vivo and in vitro experiments demonstrate that both YvfU and DesR directly bind to the operon promoter region, with DesR exerting its control over <i>yvfRS</i> expression in its unphosphorylated state. Our study uncovers an intriguing case of cross-regulation where two homologous TCSs interact closely to finely tune gene expression in response to environmental cues. These findings shed light on the complexity of bacterial signal transduction systems and their critical role in bacterial adaptability.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142330268","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}
Sierra George, Connor Waldron, Christina Thompson, Zhiming Ouyang
In Borrelia burgdorferi, BB0556 was annotated as a conserved hypothetical protein. We herein investigated gene expression and the importance of this protein during infection. Our data support that bb0556 forms an operon with five other genes. A transcriptional start site and the associated σ70-type promoter were identified in the sequences upstream of bb0554, and luciferase reporter assays indicated that this promoter is functional in B. burgdorferi. Furthermore, the sequences upstream of bb0556 contain an internal promoter to drive gene expression. bb0556 expression was affected by various environmental factors such as changes in temperature, pH, and cell density when B. burgdorferi was grown in vitro. Surprisingly, significant differences were observed for bb0556 expression between B. burgdorferi strains B31-A3 and CE162, likely due to the different cis- and trans-acting factors in these strains. Moreover, bb0556 was found to be highly expressed by B. burgdorferi in infected mice tissues, suggesting that this gene plays an important role during animal infection. To test this hypothesis, we generated a bb0556 deletion mutant in a virulent bioluminescent B. burgdorferi strain. The mutant grew normally in the medium and displayed no defect in the resistance to environmental stresses such as reactive oxygen species, reactive nitrogen species, and osmotic stress. However, when the infectivity was compared between the mutant and its parental strain using in vivo bioluminescence imaging as well as analyses of spirochete recovery and bacterial burdens in animal tissues, our data showed that, contrary to the parental strain, the mutant was unable to infect mice. Complementation of bb0556 in cis fully restored the infectious phenotype to wild-type levels. Taken together, our study demonstrates that the hypothetical protein BB0556 is a novel virulence factor essential for B. burgdorferi mammalian infection.
在鲍曼不动杆菌中，BB0556 被注释为一种保守的假定性蛋白。我们在此研究了该蛋白在感染过程中的基因表达及其重要性。我们的数据支持 bb0556 与其他五个基因形成一个操作子。在 bb0554 的上游序列中发现了一个转录起始位点和相关的 σ70 型启动子，荧光素酶报告实验表明该启动子在 B. burgdorferi 中具有功能。此外，bb0556 的上游序列包含一个内部启动子，可驱动基因表达。在 B. burgdorferi 体外生长时，bb0556 的表达受各种环境因素的影响，如温度、pH 值和细胞密度的变化。令人惊讶的是，在 B. burgdorferi 菌株 B31-A3 和 CE162 之间观察到了 bb0556 表达的显著差异，这可能是由于这些菌株中的顺式和反式作用因子不同所致。此外，我们还发现 bb0556 在受感染的小鼠组织中高度表达，这表明该基因在动物感染过程中发挥着重要作用。为了验证这一假设，我们在一株毒性生物发光杆菌中产生了一个 bb0556 缺失突变体。该突变体在培养基中生长正常，对活性氧、活性氮和渗透压等环境胁迫的抵抗力也没有缺陷。然而，当我们使用体内生物发光成像以及螺旋体恢复和动物组织中细菌负荷分析来比较突变体及其亲本菌株的感染性时，我们的数据显示，与亲本菌株相反，突变体无法感染小鼠。bb0556 的顺式互补可将感染表型完全恢复到野生型水平。综上所述，我们的研究表明，假说蛋白 BB0556 是一种新型毒力因子，对 B. burgdorferi 感染哺乳动物至关重要。
{"title":"Analysis of bb0556 Expression and Its Role During Borrelia burgdorferi Mammalian Infection","authors":"Sierra George, Connor Waldron, Christina Thompson, Zhiming Ouyang","doi":"10.1111/mmi.15319","DOIUrl":"https://doi.org/10.1111/mmi.15319","url":null,"abstract":"In <i>Borrelia burgdorferi</i>, BB0556 was annotated as a conserved hypothetical protein. We herein investigated gene expression and the importance of this protein during infection. Our data support that <i>bb0556</i> forms an operon with five other genes. A transcriptional start site and the associated σ<sup>70</sup>-type promoter were identified in the sequences upstream of <i>bb0554</i>, and luciferase reporter assays indicated that this promoter is functional in <i>B. burgdorferi</i>. Furthermore, the sequences upstream of <i>bb0556</i> contain an internal promoter to drive gene expression. <i>bb0556</i> expression was affected by various environmental factors such as changes in temperature, pH, and cell density when <i>B. burgdorferi</i> was grown in vitro. Surprisingly, significant differences were observed for <i>bb0556</i> expression between <i>B. burgdorferi</i> strains B31-A3 and CE162, likely due to the different <i>cis-</i> and <i>trans</i>-acting factors in these strains. Moreover, <i>bb0556</i> was found to be highly expressed by <i>B. burgdorferi</i> in infected mice tissues, suggesting that this gene plays an important role during animal infection. To test this hypothesis, we generated a <i>bb0556</i> deletion mutant in a virulent bioluminescent <i>B. burgdorferi</i> strain. The mutant grew normally in the medium and displayed no defect in the resistance to environmental stresses such as reactive oxygen species, reactive nitrogen species, and osmotic stress. However, when the infectivity was compared between the mutant and its parental strain using in vivo bioluminescence imaging as well as analyses of spirochete recovery and bacterial burdens in animal tissues, our data showed that, contrary to the parental strain, the mutant was unable to infect mice. Complementation of <i>bb0556</i> in <i>cis</i> fully restored the infectious phenotype to wild-type levels. Taken together, our study demonstrates that the hypothetical protein BB0556 is a novel virulence factor essential for <i>B. burgdorferi</i> mammalian infection.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142275659","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}
Hsp70:J-domain protein (JDP) machineries play pivotal roles in maintaining cellular proteostasis and governing various aspects of fungal physiology. While Hsp70 is known for its involvement in conferring tolerance to diverse antifungal drugs, the specific contribution of JDPs remains unclear. In this study, we examined the sensitivity of cytosolic JDP deletion strains of budding yeast to amphotericin B (AmB), a polyene antifungal agent widely utilized in fungal disease treatment due to its ability to disrupt the fungal plasma membrane (PM). Deleting Caj1, a PM-associated class II JDP, heightened susceptibility to AmB, and the protection conferred by Caj1 against AmB necessitated both its N-terminal J-domain and C-terminal lipid binding domain. Moreover, Caj1 deficiency compromised PM integrity as evidenced by increased phosphate efflux and exacerbated AmB sensitivity, particularly at elevated temperatures. Notably, phytosphingosine (PHS) addition as well as overexpression of PMP3, a positive PM integrity regulator, significantly rescued AmB sensitivity of caj1Δ cells. Our results align with the notion that Caj1 associates with the PM and cooperates with Hsp70 to regulate PM proteostasis, thereby influencing PM integrity in budding yeast. Loss of Caj1 function at the PM compromises PM protein quality control, thereby rendering yeast cells more susceptible to AmB.
Hsp70：J-结构域蛋白（JDP）机制在维持细胞蛋白稳态和管理真菌生理的各个方面发挥着关键作用。众所周知，Hsp70 参与赋予真菌对多种抗真菌药物的耐受性，但 JDPs 的具体贡献仍不清楚。在这项研究中，我们检测了芽殖酵母细胞膜 JDP 缺失菌株对两性霉素 B（AmB）的敏感性，两性霉素 B 是一种多烯类抗真菌药，因其具有破坏真菌质膜（PM）的能力而被广泛用于真菌疾病的治疗。删除与 PM 相关的 II 类 JDP Caj1 会增加对 AmB 的敏感性，而 Caj1 对 AmB 的保护作用需要其 N 端 J 域和 C 端脂质结合域。此外，Caj1 的缺乏会损害 PM 的完整性，表现为磷酸盐外流增加和对 AmB 的敏感性加剧，尤其是在温度升高时。值得注意的是，植物鞘磷脂（PHS）的添加以及PMP3（一种积极的PM完整性调节因子）的过表达能显著缓解caj1Δ细胞对AmB的敏感性。我们的研究结果与以下观点一致：Caj1与PM结合，并与Hsp70合作调节PM的蛋白稳态，从而影响芽殖酵母中PM的完整性。失去 Caj1 在 PM 上的功能会影响 PM 蛋白质的质量控制，从而使酵母细胞更容易受到 AmB 的影响。
{"title":"Specificity of Membrane-Associated J-Domain Protein, Caj1, in Amphotericin B Tolerance in Budding Yeast","authors":"Preeti Sagarika, Neha Dobriyal, Pakirisamy Deepsika, Avanti Vairagkar, Ankita Das, Chandan Sahi","doi":"10.1111/mmi.15318","DOIUrl":"https://doi.org/10.1111/mmi.15318","url":null,"abstract":"Hsp70:J-domain protein (JDP) machineries play pivotal roles in maintaining cellular proteostasis and governing various aspects of fungal physiology. While Hsp70 is known for its involvement in conferring tolerance to diverse antifungal drugs, the specific contribution of JDPs remains unclear. In this study, we examined the sensitivity of cytosolic JDP deletion strains of budding yeast to amphotericin B (AmB), a polyene antifungal agent widely utilized in fungal disease treatment due to its ability to disrupt the fungal plasma membrane (PM). Deleting Caj1, a PM-associated class II JDP, heightened susceptibility to AmB, and the protection conferred by Caj1 against AmB necessitated both its N-terminal J-domain and C-terminal lipid binding domain. Moreover, Caj1 deficiency compromised PM integrity as evidenced by increased phosphate efflux and exacerbated AmB sensitivity, particularly at elevated temperatures. Notably, phytosphingosine (PHS) addition as well as overexpression of <i>PMP3</i>, a positive PM integrity regulator, significantly rescued AmB sensitivity of <i>caj1Δ</i> cells. Our results align with the notion that Caj1 associates with the PM and cooperates with Hsp70 to regulate PM proteostasis, thereby influencing PM integrity in budding yeast. Loss of Caj1 function at the PM compromises PM protein quality control, thereby rendering yeast cells more susceptible to AmB.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142237016","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}
María Trinidad Gallegos, Matías Garavaglia, Claudio Valverde
Bacteria of the genus Pseudomonas are ubiquitous on Earth due to their great metabolic versatility and adaptation to fluctuating environments and different hosts. Some groups are important animal/human and plant pathogens, whereas others are studied for their biotechnological applications, including bioremediation, biological control of phytopathogens and plant growth promotion. Notably, their adaptability is mediated by various signal transduction systems, with the post-transcriptional Gac-Rsm cascade playing a key role. This pervasive Pseudomonas pathway controls major transitions at the population level, such as motile/sessile lifestyle, primary/secondary metabolism or replicative/infective behaviour. A hallmark of the Gac-Rsm cascade is the participation of small, regulatory, non-coding RNAs of the Rsm clan. These RNAs are synthetised in response to cell-density-dependent autoinducer signals channelled through the GacS/GacA two-component system, and they counteract, by molecular mimicry, the translational control that RNA-binding proteins of the RsmA family exert over hundreds of mRNAs. Rsm RNAs have been investigated in a few Pseudomonas model species, evidencing the presence of a variable number and families of genes depending on the taxonomic clade. However, the global picture of the distribution of these riboregulators at the genus level was unknown until now. We have undertaken a comprehensive survey and annotation of the vast array of gene sequences encoding members of the Rsm RNA clan in 245 complete genomes that cover 28 phylogenomic clades across the entire genus. The properties of the different families of rsm genes, their phylogenetic radiation, as well as the features of their promoters and adjacent regions, are discussed. The novel insights presented in our manuscript will significantly boost research on the biology of these prevalent RNAs in understudied species of the genus Pseudomonas and closely related genera.
{"title":"Small Regulatory RNAs of the Rsm Clan in Pseudomonas","authors":"María Trinidad Gallegos, Matías Garavaglia, Claudio Valverde","doi":"10.1111/mmi.15313","DOIUrl":"https://doi.org/10.1111/mmi.15313","url":null,"abstract":"Bacteria of the genus <i>Pseudomonas</i> are ubiquitous on Earth due to their great metabolic versatility and adaptation to fluctuating environments and different hosts. Some groups are important animal/human and plant pathogens, whereas others are studied for their biotechnological applications, including bioremediation, biological control of phytopathogens and plant growth promotion. Notably, their adaptability is mediated by various signal transduction systems, with the post-transcriptional Gac-Rsm cascade playing a key role. This pervasive <i>Pseudomonas</i> pathway controls major transitions at the population level, such as motile/sessile lifestyle, primary/secondary metabolism or replicative/infective behaviour. A hallmark of the Gac-Rsm cascade is the participation of small, regulatory, non-coding RNAs of the Rsm clan. These RNAs are synthetised in response to cell-density-dependent autoinducer signals channelled through the GacS/GacA two-component system, and they counteract, by molecular mimicry, the translational control that RNA-binding proteins of the RsmA family exert over hundreds of mRNAs. Rsm RNAs have been investigated in a few <i>Pseudomonas</i> model species, evidencing the presence of a variable number and families of genes depending on the taxonomic clade. However, the global picture of the distribution of these riboregulators at the genus level was unknown until now. We have undertaken a comprehensive survey and annotation of the vast array of gene sequences encoding members of the Rsm RNA clan in 245 complete genomes that cover 28 phylogenomic clades across the entire genus. The properties of the different families of <i>rsm</i> genes, their phylogenetic radiation, as well as the features of their promoters and adjacent regions, are discussed. The novel insights presented in our manuscript will significantly boost research on the biology of these prevalent RNAs in understudied species of the genus <i>Pseudomonas</i> and closely related genera.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142234053","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}
In E. coli K-12, the absence of unphosphorylated PtsN (unphospho-PtsN) has been proposed to cause an L-leucine-sensitive growth phenotype (LeuS) by hyperactivated K+ uptake mediated impairment of the expression of the ilvBN operon, encoding subunits of the L-valine (Val)-sensitive acetohydroxyacid synthase I (AHAS I) that renders residual AHAS activity susceptible to inhibition by Leu and K+. This leads to AHAS insufficiency and a requirement for L-isoleucine (Ile). Herein, we provide an alternate mechanism for the LeuS of the ∆ptsN mutant. Genetic and physiological studies with suppressors of the LeuS indicate that impaired expression of the ilvBN operon jointly caused by the absence of unphospho-PtsN and the presence of Leu coupled to Leu-mediated repression of expression of AHAS III leads to AHAS insufficiency rendering residual AHAS activity susceptible to chronic Val stress that may be generated by exogenous Leu. Hyperactivated K+ uptake and an elevated α-ketobutyrate level mediate elevation of ilvBN expression and alleviate the LeuS. The requirement of unphospho-PtsN as a positive regulator of ilvBN expression may buffer Ile biosynthesis against Leu-mediated AHAS insufficiency and protect AHAS I function from chronic endogenous Val generated by Leu and could be realized in certain environments that impair AHAS function.
在大肠杆菌 K-12 中，未磷酸化 PtsN（unphospho-PtsN）的缺失被认为会导致对 L-亮氨酸敏感的生长表型（LeuS），其原因是 K+摄取过度活化介导的 ilvBN 操作子表达受损，该操作子编码对 L-缬氨酸（Val）敏感的乙酰羟基酸合成酶 I（AHAS I）的亚基，使残余的 AHAS 活性易受 Leu 和 K+的抑制。这导致了 AHAS 的不足和对 L-异亮氨酸（Ile）的需求。在此，我们为 ∆ptsN 突变体的 LeuS 提供了另一种机制。利用 LeuS 抑制剂进行的遗传学和生理学研究表明，由于缺乏非磷酸-PtsN 和存在 Leu，再加上 Leu 介导的 AHAS III 表达抑制，共同导致 ilvBN 操作子表达受损，从而导致 AHAS 不足，使残余的 AHAS 活性易受外源 Leu 可能产生的慢性 Val 压力的影响。超活化的 K+ 摄取和升高的 α-酮丁酸水平介导了 ilvBN 表达的升高并缓解了 LeuS。作为 ilvBN 表达的正向调节因子，unphospho-PtsN 的需要可能会缓冲 Ile 生物合成，防止 Leu 介导的 AHAS 不足，并保护 AHAS I 的功能免受 Leu 产生的慢性内源性 Val 的影响，在某些损害 AHAS 功能的环境中可能会实现这一点。
{"title":"Multiple Effects of L-Leucine in Escherichia coli Lead to L-Leucine-Sensitive Growth in the Absence of Unphosphorylated PtsN","authors":"Neeraj Kumar, Abhijit A. Sardesai","doi":"10.1111/mmi.15317","DOIUrl":"https://doi.org/10.1111/mmi.15317","url":null,"abstract":"In <i>E. coli</i> K-12, the absence of unphosphorylated PtsN (unphospho-PtsN) has been proposed to cause an L-leucine-sensitive growth phenotype (Leu<sup>S</sup>) by hyperactivated K<sup>+</sup> uptake mediated impairment of the expression of the <i>ilvBN</i> operon, encoding subunits of the L-valine (Val)-sensitive acetohydroxyacid synthase I (AHAS I) that renders residual AHAS activity susceptible to inhibition by Leu and K<sup>+</sup>. This leads to AHAS insufficiency and a requirement for L-isoleucine (Ile). Herein, we provide an alternate mechanism for the Leu<sup>S</sup> of the ∆<i>ptsN</i> mutant. Genetic and physiological studies with suppressors of the Leu<sup>S</sup> indicate that impaired expression of the <i>ilvBN</i> operon jointly caused by the absence of unphospho-PtsN and the presence of Leu coupled to Leu-mediated repression of expression of AHAS III leads to AHAS insufficiency rendering residual AHAS activity susceptible to chronic Val stress that may be generated by exogenous Leu. Hyperactivated K<sup>+</sup> uptake and an elevated α-ketobutyrate level mediate elevation of <i>ilvBN</i> expression and alleviate the Leu<sup>S</sup>. The requirement of unphospho-PtsN as a positive regulator of <i>ilvBN</i> expression may buffer Ile biosynthesis against Leu-mediated AHAS insufficiency and protect AHAS I function from chronic endogenous Val generated by Leu and could be realized in certain environments that impair AHAS function.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142231820","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}
Morgan S. Osborne, Joshua N. Brehm, Carmen Olivença, Alicia M. Cochran, Mónica Serrano, Adriano O. Henriques, Joseph A. Sorg
YabG is a sporulation‐specific protease that is conserved among sporulating bacteria. Clostridioides difficile YabG processes the cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co‐germinant receptor, mutations in yabG were found which altered the co‐germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabGC207A (a catalytically inactive allele), C. difficile yabGA46D, C. difficile yabGG37E, and C. difficile yabGP153L strains germinated in response to taurocholic acid alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the presequence from preproSleC. Interestingly, only YabGA46D showed any activity toward purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP‐tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.
{"title":"The Impact of YabG Mutations on Clostridioides difficile Spore Germination and Processing of Spore Substrates","authors":"Morgan S. Osborne, Joshua N. Brehm, Carmen Olivença, Alicia M. Cochran, Mónica Serrano, Adriano O. Henriques, Joseph A. Sorg","doi":"10.1111/mmi.15316","DOIUrl":"https://doi.org/10.1111/mmi.15316","url":null,"abstract":"YabG is a sporulation‐specific protease that is conserved among sporulating bacteria. <jats:italic>Clostridioides difficile</jats:italic> YabG processes the cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co‐germinant receptor, mutations in <jats:italic>yabG</jats:italic> were found which altered the co‐germinants required to initiate spore germination. To understand how these mutations in the <jats:italic>yabG</jats:italic> locus contribute to <jats:italic>C</jats:italic>. <jats:italic>difficile</jats:italic> spore germination, we introduced these mutations into an isogenic background. Spores derived from <jats:italic>C</jats:italic>. <jats:italic>difficile yabG</jats:italic><jats:sub>C207A</jats:sub> (a catalytically inactive allele), <jats:italic>C</jats:italic>. <jats:italic>difficile yabG</jats:italic><jats:sub>A46D</jats:sub>, <jats:italic>C</jats:italic>. <jats:italic>difficile yabG</jats:italic><jats:sub>G37E</jats:sub>, and <jats:italic>C</jats:italic>. <jats:italic>difficile yabG</jats:italic><jats:sub>P153L</jats:sub> strains germinated in response to taurocholic acid alone. Recombinantly expressed and purified preproSleC incubated with <jats:italic>E</jats:italic>. <jats:italic>coli</jats:italic> lysate expressing wild type YabG resulted in the removal of the presequence from preproSleC. Interestingly, only YabG<jats:sub>A46D</jats:sub> showed any activity toward purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in <jats:italic>yabG</jats:italic> expression under the mutant promoters were analyzed using a SNAP‐tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142170937","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}
Sónia Castanheira, David López-Escarpa, Alberto Paradela, Francisco García-Del Portillo
Bacterial cell division is orchestrated by proteins that assemble in dynamic complexes collectively known as the divisome. Essential monofunctional enzymes with glycosyltransferase or transpeptidase (TPase) activities, FtsW and FtsI respectively, engage in the synthesis of septal peptidoglycan (sPG). Enigmatically, Salmonella has two TPases that can promote cell division independently: FtsI (PBP3) and the pathogen-specific paralogue PBP3SAL. How Salmonella regulates the assembly of the sPG synthase complex with these two TPases, is unknown. Here, we characterized Salmonella division complexes in wild-type cells and isogenic mutants lacking PBP3 or PBP3SAL. The complexes were cross-linked in vivo and pulled down with antibodies recognizing each enzyme. Proteomics of the immunoprecipitates showed that PBP3 and PBP3SAL do not extensively cross-link in wild type cells, supporting the presence of independent complexes. More than 40 proteins cross-link in complexes in which these two TPases are present. Those identified with high scores include FtsA, FtsK, FtsQLB, FtsW, PBP1B, SPOR domain-containing proteins (FtsN, DedD, RlpA, DamX), amidase activators (FtsX, EnvC, NlpD) and Tol-Pal proteins. Other cross-linked proteins are the protease Prc, the elongasome TPase PBP2 and, D,D-endo- and D,D-carboxypeptidases. PBP3 and PBP3SAL localize at midcell and compete for occupying the division complex in response to environmental cues. Thus, a catalytic-dead PBP3SAL-S300A variant impairs cell division in a high osmolarity and acidic condition in which it is produced at levels exceeding those of PBP3. Salmonella may therefore exploit an 'adjustable' divisome to exchange TPases for ensuring cell division in distinct environments and, in this manner, expand its colonization capacities.
{"title":"In Vivo Cross-Linking Sheds Light on the Salmonella Divisome in Which PBP3 and PBP3<sub>SAL</sub> Compete for Occupancy.","authors":"Sónia Castanheira, David López-Escarpa, Alberto Paradela, Francisco García-Del Portillo","doi":"10.1111/mmi.15309","DOIUrl":"https://doi.org/10.1111/mmi.15309","url":null,"abstract":"<p><p>Bacterial cell division is orchestrated by proteins that assemble in dynamic complexes collectively known as the divisome. Essential monofunctional enzymes with glycosyltransferase or transpeptidase (TPase) activities, FtsW and FtsI respectively, engage in the synthesis of septal peptidoglycan (sPG). Enigmatically, Salmonella has two TPases that can promote cell division independently: FtsI (PBP3) and the pathogen-specific paralogue PBP3<sub>SAL</sub>. How Salmonella regulates the assembly of the sPG synthase complex with these two TPases, is unknown. Here, we characterized Salmonella division complexes in wild-type cells and isogenic mutants lacking PBP3 or PBP3<sub>SAL</sub>. The complexes were cross-linked in vivo and pulled down with antibodies recognizing each enzyme. Proteomics of the immunoprecipitates showed that PBP3 and PBP3<sub>SAL</sub> do not extensively cross-link in wild type cells, supporting the presence of independent complexes. More than 40 proteins cross-link in complexes in which these two TPases are present. Those identified with high scores include FtsA, FtsK, FtsQLB, FtsW, PBP1B, SPOR domain-containing proteins (FtsN, DedD, RlpA, DamX), amidase activators (FtsX, EnvC, NlpD) and Tol-Pal proteins. Other cross-linked proteins are the protease Prc, the elongasome TPase PBP2 and, D,D-endo- and D,D-carboxypeptidases. PBP3 and PBP3<sub>SAL</sub> localize at midcell and compete for occupying the division complex in response to environmental cues. Thus, a catalytic-dead PBP3<sub>SAL</sub>-S300A variant impairs cell division in a high osmolarity and acidic condition in which it is produced at levels exceeding those of PBP3. Salmonella may therefore exploit an 'adjustable' divisome to exchange TPases for ensuring cell division in distinct environments and, in this manner, expand its colonization capacities.</p>","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133258","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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