Journal of Bacteriology最新文献

{"title":"Identification of a novel NADPH generation reaction in the pentose phosphate pathway in <i>Escherichia coli</i> using mBFP.","authors":"Koichiro Ueno, Shogo Sawada, Mai Ishibashi, Yoshiki Kanda, Hiroshi Shimizu, Yoshihiro Toya","doi":"10.1128/jb.00276-24","DOIUrl":"10.1128/jb.00276-24","url":null,"abstract":"<p><p>NADPH is a redox cofactor that drives the anabolic reactions. Although major NADPH generation reactions have been identified in <i>Escherichia coli</i>, some minor reactions have not been identified. In the present study, we explored novel NADPH generation reactions by monitoring the fluorescence dynamics after the addition of carbon sources to starved cells, using a metagenome-derived blue fluorescent protein (mBFP) as an intracellular NADPH reporter. Perturbation analyses were performed on a glucose-6-phosphate isomerase (PGI) deletion strain and its parental strain. Interestingly, mBFP fluorescence increased not only in the parental strain but also in the ΔPGI strain after the addition of xylose. Because the ΔPGI strain cannot metabolize xylose through the oxidative pentose phosphate pathway, this suggests that an unexpected NADPH generation reaction contributes to an increase in fluorescence. To unravel this mystery, we deleted the NADPH generation enzymes including transhydrogenase, isocitrate dehydrogenase, NADP⁺-dependent malic enzyme, glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH) in the ΔPGI strain, and revealed that G6PDH and 6PGDH contribute to an increase in fluorescence under xylose conditions. <i>In vitro</i> assays using purified enzymes showed that G6PDH can produce NADPH using erythrose-4-phosphate (E4P) as a substitute for glucose-6-phosphate. Because the <i>Km</i> (0.65 mM) for E4P was much higher than the reported intracellular E4P concentrations in <i>E. coli</i>, little E4P must be metabolized through this bypass in the parental strain. However, the flux would increase when E4P accumulates in the cells owing to genetic modifications. This finding provides a metabolic engineering strategy for generating NADPH to produce useful compounds using xylose as a carbon source.IMPORTANCEBecause NADPH is consumed during the synthesis of various useful compounds, enhancing NADPH regeneration is highly desirable in metabolic engineering. In this study, we explored novel NADPH generation reactions in <i>Escherichia coli</i> using a fluorescent NADPH reporter and found that glucose-6-phosphate dehydrogenase can produce NADPH using erythrose-4-phosphate as a substrate under xylose conditions. Xylose is an abundant sugar in nature and is an attractive carbon source for bioproduction. Therefore, this finding contributes to novel pathway engineering strategies using a xylose carbon source in <i>E. coli</i> to produce useful compounds that consume NADPH for their synthesis.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0027624"},"PeriodicalIF":2.7,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11580446/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142466182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A flagellar accessory protein links chemotaxis to surface sensing.","authors":"Rachel I Salemi, Ana K Cruz, David M Hershey","doi":"10.1128/jb.00404-24","DOIUrl":"10.1128/jb.00404-24","url":null,"abstract":"<p><p>Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. <i>Caulobacter crescentus</i> adheres to surfaces using a polysaccharide adhesin called the holdfast. In <i>C. crescentus</i>, disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several <i>C. crescentus</i> genes involved in flagellar surface sensing. One of these, <i>fssF</i>, codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting <i>fssF</i> results in a severe motility defect, which we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that <i>fssF</i> promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of <i>fssF</i> or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network (<i>pleD</i>, <i>motB</i>, and <i>dgcB</i>) contribute to both ∆<i>flgH-</i>dependent and ∆<i>fssF-</i>dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion.IMPORTANCEBacterial biofilms pose a threat in clinical and industrial settings. Surface sensing is one of the first steps in biofilm formation. Studying surface sensing can improve our understanding of biofilm formation and develop preventative strategies. In this study, we use the freshwater bacterium <i>Caulobacter crescentus</i> to study surface sensing and the regulation of surface attachment. We characterize a previously unstudied gene, <i>fssF</i>, and find that it localizes to the cell pole in the presence of three proteins that make up a component of the flagellum called the C-ring. Additionally, we find that <i>fssF</i> is required for chemotaxis behavior but dispensable for swimming motility. Lastly, our results indicate that deletion of <i>fssF</i> and other genes required for chemotaxis results in a hyperadhesive phenotype. These results support that surface sensing requires chemotaxis for a robust response to a surface.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0040424"},"PeriodicalIF":2.7,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11580411/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142466178","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovery and synthesis of leaderless bacteriocins from the Actinomycetota.","authors":"David Hourigan, Felipe Miceli de Farias, Paula M O'Connor, Colin Hill, R Paul Ross","doi":"10.1128/jb.00298-24","DOIUrl":"10.1128/jb.00298-24","url":null,"abstract":"<p><p>Leaderless bacteriocins are a unique class of bacteriocins that possess antimicrobial activity after translation and have few cases of documented resistance. Aureocin A53 and lacticin Q are considered two of the most well-studied leaderless bacteriocins. Here, we used <i>in silico</i> genome mining to search for novel aureocin A53-like leaderless bacteriocins in GenBank and MGnify. We identified 757 core peptides across 430 genomes with 75 species found currently without characterized leaderless bacteriocin production. These include putative novel species containing bacteriocin gene clusters (BGCs) from the genera <i>Streptomyces</i> (sp. NBC_00237) and <i>Agrococcus</i> (sp. SL85). To date, all characterized leaderless bacteriocins have been found within the phylum Bacillota, but this study identified 97 core peptides within the phylum Actinomycetota. Members of this phylum are traditionally associated with the production of antibiotics, such is the case with the genus <i>Streptomyces</i>. Actinomycetota is an underexplored phylum in terms of bacteriocin production with no characterized leaderless bacteriocin production to date. The two novel leaderless bacteriocins arcanocin and arachnicin from Actinomycetota members <i>Arcanobacterium</i> sp. and <i>Arachnia</i> sp., respectively, were chemically synthesized and antimicrobial activity was verified. These peptides were encoded in human gut (PRJNA485056) and oral (PRJEB43277) microbiomes, respectively. This research highlights the biosynthetic potential of Actinomycetota in terms of leaderless bacteriocin production and describes the first antimicrobial peptides encoded in the genera <i>Arcanobacterium</i> and <i>Arachnia</i>.IMPORTANCEBacteriocins are gathering attention as alternatives to current antibiotics given the increasing incidence of antimicrobial resistance. Leaderless bacteriocins are considered a commercially attractive subclass of bacteriocins due to the ability to synthesize active peptide and low levels of documented resistance. Therefore, in this work, we mined publicly available data to determine how widespread and diverse leaderless bacteriocins are within the domain of bacteria. Actinomycetota, known for its antibiotic producers but lacking described and characterized bacteriocins, proved to be a rich source of leaderless bacteriocins-97 in total. Two such peptides, arcanocin and arachnicin, were chemically synthesized and have antimicrobial activity. These bacteriocins may provide a novel source of novel antimicrobials that could aid in the development of future alternative antimicrobials and highlight that the Actinomycetota are an underexplored resource of bacteriocin peptides.</p>","PeriodicalId":15107,"journal":{"name":"Journal of Bacteriology","volume":" ","pages":"e0029824"},"PeriodicalIF":2.7,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11580447/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142466181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"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":"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":" ","pages":"e0031824"},"PeriodicalIF":2.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11500611/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"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":" ","pages":"e0023324"},"PeriodicalIF":2.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11500542/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"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":" ","pages":"e0020424"},"PeriodicalIF":2.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11500546/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"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":"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^A373S 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":" ","pages":"e0039923"},"PeriodicalIF":2.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11500502/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"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":"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":" ","pages":"e0032824"},"PeriodicalIF":2.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11500504/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"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":" ","pages":"e0025624"},"PeriodicalIF":2.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11505635/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"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":" ","pages":"e0023524"},"PeriodicalIF":2.7,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11501099/pdf/","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}