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1.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360615

RESUMO

In contrast to Bacillus subtilis, Streptomyces coelicolor A3(2) contains nine homologues of stress response sigma factor SigB with a major role in differentiation and osmotic stress response. The aim of this study was to further characterize these SigB homologues. We previously established a two-plasmid system to identify promoters recognized by sigma factors and used it to identify promoters recognized by the three SigB homologues, SigF, SigG, and SigH from S. coelicolor A3(2). Here, we used this system to identify 14 promoters recognized by SigB. The promoters were verified in vivo in S. coelicolor A3(2) under osmotic stress conditions in sigB and sigH operon mutants, indicating some cross-recognition of these promoters by these two SigB homologues. This two-plasmid system was used to examine the recognition of all identified SigB-, SigF-, SigG-, and SigH-dependent promoters with all nine SigB homologues. The results confirmed this cross-recognition. Almost all 24 investigated promoters were recognized by two or more SigB homologues and data suggested some distinguishing groups of promoters recognized by these sigma factors. However, analysis of the promoters did not reveal any specific sequence characteristics for these recognition groups. All promoters showed high similarity in the -35 and -10 regions. Immunoblot analysis revealed the presence of SigB under osmotic stress conditions and SigH during morphological differentiation. Together with the phenotypic analysis of sigB and sigH operon mutants in S. coelicolor A3(2), the results suggest a dominant role for SigB in the osmotic stress response and a dual role for SigH in the osmotic stress response and morphological differentiation. These data suggest a complex regulation of the osmotic stress response in relation to morphological differentiation in S. coelicolor A3(2).


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Regiões Promotoras Genéticas , Fator sigma/genética , Streptomyces coelicolor/genética , Transcrição Genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Fator sigma/metabolismo , Streptomyces coelicolor/crescimento & desenvolvimento , Streptomyces coelicolor/metabolismo
2.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299177

RESUMO

Adaptation of bacteria to a changing environment is often accompanied by remodeling of the transcriptome. In the facultative phototroph Rhodobacter sphaeroides the alternative sigma factors RpoE, RpoHI and RpoHII play an important role in a variety of stress responses, including heat, oxidative stress and nutrient limitation. Photooxidative stress caused by the simultaneous presence of chlorophylls, light and oxygen is a special challenge for phototrophic organisms. Like alternative sigma factors, several non-coding sRNAs have important roles in the defense against photooxidative stress. RNAseq-based transcriptome data pointed to an influence of the stationary phase-induced StsR sRNA on levels of mRNAs and sRNAs with a role in the photooxidative stress response. Furthermore, StsR also affects expression of photosynthesis genes and of genes for regulators of photosynthesis genes. In vivo and in vitro interaction studies revealed that StsR, that is under control of the RpoHI and RpoHII sigma factors, targets rpoE mRNA and affects its abundance by altering its stability. RpoE regulates expression of the rpoHII gene and, consequently, expression of stsR. These data provide new insights into a complex regulatory network of protein regulators and sRNAs involved in defense against photooxidative stress and the regulation of photosynthesis genes.


Assuntos
Proteínas de Bactérias/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , RNA Bacteriano/genética , Rhodobacter sphaeroides/crescimento & desenvolvimento , Fator sigma/metabolismo , Transcriptoma , Proteínas de Bactérias/genética , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Fator sigma/genética
3.
Int J Food Microbiol ; 351: 109269, 2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34102570

RESUMO

Microbial population heterogeneity contributes to differences in stress response between individual cells in a population, and can lead to the selection of genetically stable variants with increased stress resistance. We previously provided evidence that the multiple-stress resistant Listeria monocytogenes LO28 variant 15, carries a point mutation in the rpsU gene, resulting in an arginine-proline substitution in ribosomal protein RpsU (RpsU17Arg-Pro). Here, we investigated the trade-off between general stress sigma factor SigB-mediated stress resistance and fitness in variant 15 using experimental evolution. By selecting for higher fitness in two parallel evolving cultures, we identified two evolved variants: 15EV1 and 15EV2. Whole genome sequencing and SNP analysis showed that both parallel lines mutated in the same codon in rpsU as the original mutation resulting in RpsU17Pro-His (15EV1) and RpsU17Pro-Thr (15EV2). Using a combined phenotyping and proteomics approach, we assessed the resistance of the evolved variants to both heat and acid stress, and found that in both lines reversion to WT-like fitness also resulted in WT-like stress sensitivity. Proteome analysis of L. monocytogenes LO28 WT, variant 15, 15EV1, and 15EV2 revealed high level expression of SigB regulon members only in variant 15, whereas protein profiles of both evolved variants were highly similar to that of the LO28 WT. Experiments with constructed RpsU17Arg-Pro mutants in L. monocytogenes LO28 and EGDe, and RpsU17Arg-His and RpsU17Arg-Thr in LO28, confirmed that single amino acid substitutions in RpsU enable switching between multiple-stress resistant and high fitness states in L. monocytogenes.


Assuntos
Adaptação Fisiológica/genética , Proteínas de Bactérias/genética , Listeria monocytogenes/fisiologia , Proteínas Ribossômicas/genética , Ácidos/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/metabolismo , Evolução Molecular Direcionada , Genoma Bacteriano/genética , Temperatura Alta , Listeria monocytogenes/genética , Mutação , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo , Fator sigma/genética , Fator sigma/metabolismo
4.
PLoS Biol ; 19(6): e3001306, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34170902

RESUMO

Free-living bacteria adapt to environmental change by reprogramming gene expression through precise interactions of hundreds of DNA-binding proteins. A predictive understanding of bacterial physiology requires us to globally monitor all such protein-DNA interactions across a range of environmental and genetic perturbations. Here, we show that such global observations are possible using an optimized version of in vivo protein occupancy display technology (in vivo protein occupancy display-high resolution, IPOD-HR) and present a pilot application to Escherichia coli. We observe that the E. coli protein-DNA interactome organizes into 2 distinct prototypic features: (1) highly dynamic condition-dependent transcription factor (TF) occupancy; and (2) robust kilobase scale occupancy by nucleoid factors, forming silencing domains analogous to eukaryotic heterochromatin. We show that occupancy dynamics across a range of conditions can rapidly reveal the global transcriptional regulatory organization of a bacterium. Beyond discovery of previously hidden regulatory logic, we show that these observations can be utilized to computationally determine sequence specificity models for the majority of active TFs. Our study demonstrates that global observations of protein occupancy combined with statistical inference can rapidly and systematically reveal the transcriptional regulatory and structural features of a bacterial genome. This capacity is particularly crucial for non-model bacteria that are not amenable to routine genetic manipulation.


Assuntos
Cromossomos Bacterianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Ligação a DNA/metabolismo , Meio Ambiente , Escherichia coli/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fator sigma/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
5.
Mol Cell ; 81(14): 2887-2900.e5, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34171298

RESUMO

WhiB7 represents a distinct subclass of transcription factors in the WhiB-Like (Wbl) family, a unique group of iron-sulfur (4Fe-4S] cluster-containing proteins exclusive to the phylum of Actinobacteria. In Mycobacterium tuberculosis (Mtb), WhiB7 interacts with domain 4 of the primary sigma factor (σA4) in the RNA polymerase holoenzyme and activates genes involved in multiple drug resistance and redox homeostasis. Here, we report crystal structures of the WhiB7:σA4 complex alone and bound to its target promoter DNA at 1.55-Å and 2.6-Å resolution, respectively. These structures show how WhiB7 regulates gene expression by interacting with both σA4 and the AT-rich sequence upstream of the -35 promoter DNA via its C-terminal DNA-binding motif, the AT-hook. By combining comparative structural analysis of the two high-resolution σA4-bound Wbl structures with molecular and biochemical approaches, we identify the structural basis of the functional divergence between the two distinct subclasses of Wbl proteins in Mtb.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Proteínas Ferro-Enxofre/genética , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas/genética , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética
6.
Microb Pathog ; 156: 104917, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33940135

RESUMO

The obligate intracellular bacterium Chlamydia trachomatis is an important human pathogen with a biphasic developmental cycle comprised of an infectious elementary body (EB) and a replicative reticulate body (RB). Whereas σ66, the primary sigma factor, is necessary for transcription of most chlamydial genes throughout the developmental cycle, σ28 is required for expression of some late genes. We previously showed that the Chlamydia-specific transcription factor GrgA physically interacts with both of these sigma factors and activates transcription from σ66- and σ28-dependent promoters in vitro. Here, we investigated the organismal functions of GrgA. We show that overexpression of GrgA slows EB-to-RB conversion, decreases RB proliferation, and reduces progeny EB production. In contrast, overexpression of a GrgA variant without the σ28-binding domain shows significantly less severe inhibitory effects, while overexpression of a variant without the σ66-binding domain demonstrates no adverse effects. These findings indicate that GrgA plays important roles in the expression regulation of both σ66-dependent genes and σ28-dependent genes during the chlamydial developmental cycle.


Assuntos
Chlamydia trachomatis , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Humanos , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Appl Environ Microbiol ; 87(12): e0039721, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-33811030

RESUMO

Listeria monocytogenes is a ubiquitous environmental bacterium and intracellular pathogen that responds to stress using predominantly the alternative sigma factor SigB. Stress is sensed by a multiprotein complex, the stressosome, extensively studied in bacteria grown in nutrient media. Following signal perception, the stressosome triggers a phosphorylation cascade that releases SigB from its anti-sigma factor. Whether the stressosome is activated during the intracellular infection is unknown. Here, we analyzed the subcellular distribution of stressosome proteins in L. monocytogenes located inside epithelial cells following their immunodetection in membrane and cytosolic fractions prepared from intracellular bacteria. Unlike bacteria in laboratory media, intracellular bacteria have a large proportion of the core stressosome protein RsbR1 associated with the membrane. However, another core protein, RsbS, is undetectable. Despite the absence of RsbS, a SigB-dependent reporter revealed that SigB activity increases gradually from early (1 h) to late (6 h) postinfection times. We also found that RsbR1 paralogues attenuate the intensity of the SigB response and that the miniprotein Prli42, reported to tether the stressosome to the membrane in response to oxidative stress, plays no role in associating RsbR1 to the membrane of intracellular bacteria. Altogether, these data indicate that, once inside host cells, the L. monocytogenes stressosome may adopt a unique configuration to sense stress and to activate SigB in the intracellular eukaryotic niche. IMPORTANCE The response to stress mediated by the alternative sigma factor SigB has been extensively characterized in Bacillus subtilis and Listeria monocytogenes. These bacteria sense stress using a supramacromolecular complex, the stressosome, which triggers a cascade that releases SigB from its anti-sigma factor. Despite the fact that many structural data on the complex are available and analyses have been performed in mutants lacking components of the stressosome or the signaling cascade, the integration of the stress signal and the dynamics of stressosome proteins following environmental changes remain poorly understood. Our study provides data at the protein level on essential stressosome components and SigB activity when L. monocytogenes, normally a saprophytic bacterium, adapts to an intracellular lifestyle. Our results support activation of the stressosome complex in intracellular bacteria. The apparent loss of the stressosome core protein RsbS in intracellular L. monocytogenes also challenges current models, favoring the idea of a unique stressosome architecture responding to intracellular host cues.


Assuntos
Proteínas de Bactérias/metabolismo , Células Epiteliais/microbiologia , Listeria monocytogenes/metabolismo , Fator sigma/metabolismo , Estresse Fisiológico , Linhagem Celular , Proliferação de Células , Células Eucarióticas , Humanos
8.
J Biol Chem ; 296: 100673, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33865858

RESUMO

Escherichia coli RseP, a member of the site-2 protease family of intramembrane proteases, is involved in the activation of the σE extracytoplasmic stress response and elimination of signal peptides from the cytoplasmic membrane. However, whether RseP has additional cellular functions is unclear. In this study, we used mass spectrometry-based quantitative proteomic analysis to search for new substrates that might reveal unknown physiological roles for RseP. Our data showed that the levels of several Fec system proteins encoded by the fecABCDE operon (fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with a fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate-dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. A shortened FecR construct was not dependent on RseP for activation, confirming this cleavage step is the essential and sufficient role of RseP. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.


Assuntos
Membrana Celular/metabolismo , Endopeptidases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Compostos Férricos/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fator sigma/metabolismo , Transporte Biológico , Endopeptidases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Fator sigma/genética
9.
World J Microbiol Biotechnol ; 37(5): 82, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33855623

RESUMO

Many volatile compounds secreted by bacteria play an important role in the interactions of microorganisms, can inhibit the growth of phytopathogenic bacteria and fungi, can suppress or stimulate plant growth and serve as infochemicals presenting a new type of interspecies communication. In this work, we investigated the effect of total pools of volatile substances and individual volatile organic compounds (VOCs) synthesized by the rhizosphere bacteria Pseudomonas chlororaphis 449 and Serratia plymuthica IC1270, the soil-borne strain P. fluorescens B-4117 and the spoiled meat isolate S. proteamaculans 94 on Arabidopsis thaliana plants. We showed that total gas mixtures secreted by these strains during their growth on Luria-Bertani agar inhibited A. thaliana growth. Hydrogen cyanide synthesis was unnecessary for the growth suppression. A decrease in the inhibition level was observed for the strain P. chlororaphis 449 with a mutation in the gacS gene, while inactivation of the rpoS gene had no effect. Individual VOCs synthesized by these bacteria (1-indecene, ketones 2-nonanone, 2-heptanone, 2-undecanone, and dimethyl disulfide) inhibited the growth of plants or killed them. Older A. thaliana seedlings were more resistant to VOCs than younger seedlings. The results indicated that the ability of some volatiles emitted by the rhizosphere and soil bacteria to inhibit plant growth should be considered when assessing the potential of such bacteria for the biocontrol of plant diseases.


Assuntos
Arabidopsis/efeitos dos fármacos , Pseudomonas chlororaphis/química , Pseudomonas chlororaphis/genética , Pseudomonas fluorescens/química , Serratia/química , Compostos Orgânicos Voláteis/toxicidade , Arabidopsis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianeto de Hidrogênio/metabolismo , Carne/microbiologia , Mutação , Pseudomonas chlororaphis/metabolismo , Pseudomonas fluorescens/metabolismo , Rizosfera , Plântula/efeitos dos fármacos , Serratia/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Microbiologia do Solo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Compostos Orgânicos Voláteis/química
10.
PLoS One ; 16(4): e0249379, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33857164

RESUMO

Tuberculosis caused by the pathogen Mycobacterium tuberculosis (MTB), remains a significant threat to global health. Elucidating the mechanisms of essential MTB genes provides an important theoretical basis for drug exploitation. Gene mtsp17 is essential and is conserved in the Mycobacterium genus. Although Mtsp17 has a structure closely resembling typical steroidogenic acute regulatory protein-related lipid transfer (START) family proteins, its biological function is different. This study characterizes the transcriptomes of Mycobacterium smegmatis to explore the consequences of mtsp17 downregulation on gene expression. Suppression of the mtsp17 gene resulted in significant down-regulation of 3% and upregulation of 1% of all protein-coding genes. Expression of desA1, an essential gene involved in mycolic acid synthesis, and the anti-SigF antagonist MSMEG_0586 were down-regulated in the conditional Mtsp17 knockout mutant and up-regulated in the Mtsp17 over-expression strain. Trends in the changes of 70 of the 79 differentially expressed genes (Log2 fold change > 1.5) in the conditional Mtsp17 knockout strain were the same as in the SigF knockout strain. Our data suggest that Mtsp17 is likely an activator of desA1 and Mtsp17 regulates the SigF regulon by SigF regulatory pathways through the anti-SigF antagonist MSMEG_0586. Our findings indicate the role of Mtsp17 may be in transcriptional regulation, provide new insights into the molecular mechanisms of START family proteins, and uncover a new node in the regulatory network of mycobacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Ativação Transcricional , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Mycobacterium smegmatis/genética , Domínios Proteicos , Fator sigma/genética , Fator sigma/metabolismo
11.
J Bacteriol ; 203(12): e0012721, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33820796

RESUMO

The intracellular bacterial pathogen Brucella causes persistent infections in various mammalian species. To survive and replicate within macrophages, these bacteria must be able to withstand oxidative stresses and express the type IV secretion system (T4SS) to evade host immune responses. The extracytoplasmic function (ECF) sigma factor system is a major signal transduction mechanism in bacteria that senses environmental cues and responds by regulating gene expression. In this study, we defined an ECF σ bcrS and its cognate anti-σ factor abcS in Brucella melitensis M28 by conserved domain analysis and a protein interaction assay. BcrS directly activates an adjacent operon, bcrXQP, that encodes a methionine-rich peptide and a putative methionine sulfoxide reductase system, whereas AbcS is a negative regulator of bcrS and bcrXQP. The bcrS-abcS and bcrXQP operons can be induced by hypochlorous acid and contribute to hypochlorous acid resistance in vitro. Next, RNA sequencing analysis and genome-wide recognition sequence search identified the regulons of BcrS and AbcS. Interestingly, we found that BcrS positively influences T4SS expression in an AbcS-dependent manner and that AbcS also affects T4SS expression independently of BcrS. Last, we demonstrate that abcS is required for the maintenance of persistent infection, while bcrS is dispensable in a mouse infection model. Collectively, we conclude that BcrS and AbcS influence expression of multiple genes responsible for Brucella virulence traits. IMPORTANCE Brucella is a notorious intracellular pathogen that induces chronic infections in animals and humans. To survive and replicate within macrophages, these bacteria require a capacity to withstand oxidative stresses and to express the type IV secretion system (T4SS) to combat host immune responses. In this study, we characterized an extracytoplasmic function sigma/anti-sigma factor system that regulates resistance to reactive chlorine species and T4SS expression, thereby establishing a potential link between two crucial virulence traits of Brucella. Furthermore, the anti-sigma factor AbcS contributes to Brucella persistent infection of mice. Thus, this work provides novel insights into Brucella virulence regulation as well as a potential drug target for fighting Brucella infections.


Assuntos
Brucella melitensis/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ácido Hipocloroso/farmacologia , Fator sigma/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Sequência de Bases , Modelos Moleculares , Conformação Proteica , Fator sigma/genética , Sistemas de Secreção Tipo IV/genética
12.
Int J Mol Sci ; 22(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918849

RESUMO

The ability of bacterial core RNA polymerase (RNAP) to interact with different σ factors, thereby forming a variety of holoenzymes with different specificities, represents a powerful tool to coordinately reprogram gene expression. Extracytoplasmic function σ factors (ECFs), which are the largest and most diverse family of alternative σ factors, frequently participate in stress responses. The classification of ECFs in 157 different groups according to their phylogenetic relationships and genomic context has revealed their diversity. Here, we have clustered 55 ECF groups with experimentally studied representatives into two broad classes of stress responses. The remaining 102 groups still lack any mechanistic or functional insight, representing a myriad of systems yet to explore. In this work, we review the main features of ECFs and discuss the different mechanisms controlling their production and activity, and how they lead to a functional stress response. Finally, we focus in more detail on two well-characterized ECFs, for which the mechanisms to detect and respond to stress are complex and completely different: Escherichia coli RpoE, which is the best characterized ECF and whose structural and functional studies have provided key insights into the transcription initiation by ECF-RNAP holoenzymes, and the ECF15-type EcfG, the master regulator of the general stress response in Alphaproteobacteria.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Fator sigma/genética , Fator sigma/metabolismo , Estresse Fisiológico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Espaço Extracelular , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Ligação Proteica , Fator sigma/classificação , Transdução de Sinais , Iniciação da Transcrição Genética
13.
mBio ; 12(2)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758089

RESUMO

ß-Lactams are a class of antibiotics that target the synthesis of peptidoglycan, an essential component of the cell wall. ß-Lactams inhibit the function of penicillin-binding proteins (PBPs), which form the cross-links between strands of peptidoglycan. Resistance to ß-lactams complicates the treatment of bacterial infections. In recent years, the spread of ß-lactam resistance has increased with growing intensity. Resistance is often conferred by ß-lactamases, which inactivate ß-lactams, or the expression of alternative ß-lactam-resistant PBPs. σP is an extracytoplasmic function (ECF) σ factor that controls ß-lactam resistance in the species Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis σP is normally held inactive by the anti-σ factor RsiP. σP is activated by ß-lactams that trigger the proteolytic destruction of RsiP. Here, we identify the penicillin-binding protein PbpP and demonstrate its essential role in the activation of σP Our data show that PbpP is required for σP activation and RsiP degradation. Our data suggest that PbpP acts as a ß-lactam sensor since the binding of a subset of ß-lactams to PbpP is required for σP activation. We find that PbpP likely directly or indirectly controls site 1 cleavage of RsiP, which results in the degradation of RsiP and, thus, σP activation. σP activation results in increased expression of ß-lactamases and, thus, increased ß-lactam resistance. This work is the first report of a PBP acting as a sensor for ß-lactams and controlling the activation of an ECF σ factor.IMPORTANCE The bacterial cell envelope is the target for numerous antibiotics. Many antibiotics target the synthesis of peptidoglycan, which is a central metabolic pathway essential for bacterial survival. One of the most important classes of antibiotics has been ß-lactams, which inhibit the transpeptidase activity of penicillin-binding proteins to decrease the cross-linking of peptidoglycan and the strength of the cell wall. While ß-lactam antibiotics have historically proven to be effective, resistance to ß-lactams is a growing problem. The ECF σ factor σP is required for ß-lactam resistance in B. thuringiensis and close relatives, including B. anthracis Here, we provide insight into the mechanism of activation of σP by ß-lactams.


Assuntos
Antibacterianos/farmacologia , Bacillus thuringiensis/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , beta-Lactamas/farmacologia , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas de Ligação às Penicilinas/classificação , Resistência beta-Lactâmica , beta-Lactamases/metabolismo
14.
Int J Mol Sci ; 22(4)2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33672733

RESUMO

Sigma factor C (SigC) contributes to Mycobacterium tuberculosis virulence in various animal models, but the stress response coordinated by this transcription factor was undefined. The results presented here indicate that SigC prevents copper starvation. Whole genome expression studies demonstrate short-term (4-h) induction of sigC, controlled from a tetracycline-inducible promoter, upregulates ctpB and genes in the nonribosomal peptide synthase (nrp) operon. These genes are expressed at higher levels after 48-h sigC induction, but also elevated are genes encoding copper-responsive regulator RicR and RicR-regulated copper toxicity response operon genes rv0846-rv0850, suggesting prolonged sigC induction results in excessive copper uptake. No growth and global transcriptional differences are observed between a sigC null mutant relative to its parent strain in 7H9 medium. In a copper-deficient medium, however, growth of the sigC deletion strain lags the parent, and 40 genes (including those in the nrp operon) are differentially expressed. Copper supplementation reverses the growth defect and silences most transcriptional differences. Together, these data support SigC as a transcriptional regulator of copper acquisition when the metal is scarce. Attenuation of sigC mutants in severe combined immunodeficient mice is consistent with an inability to overcome innate host defenses that sequester copper ions to deprive invading microbes of this essential micronutrient.


Assuntos
Cobre/farmacologia , Imunidade/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Fator sigma/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Transporte Biológico/efeitos dos fármacos , Sulfato de Cobre/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Camundongos SCID , Viabilidade Microbiana/efeitos dos fármacos , Mutação/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fenótipo , Transcrição Genética/efeitos dos fármacos , Virulência/efeitos dos fármacos , Virulência/genética
15.
Science ; 371(6535): 1225-1232, 2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33737482

RESUMO

Early life is thought to have required the self-replication of RNA by RNA replicases. However, how such replicases evolved and subsequently enabled gene expression remains largely unexplored. We engineered and selected a holopolymerase ribozyme that uses a sigma factor-like specificity primer to first recognize an RNA promoter sequence and then, in a second step, rearrange to a processive elongation form. Using its own sequence, the polymerase can also program itself to polymerize from certain RNA promoters and not others. This selective promoter-based polymerization could allow an RNA replicase ribozyme to define "self" from "nonself," an important development for the avoidance of replicative parasites. Moreover, the clamp-like mechanism of this polymerase could eventually enable strand invasion, a critical requirement for replication in the early evolution of life.


Assuntos
Regiões Promotoras Genéticas , RNA Catalítico , RNA Polimerase Dependente de RNA , RNA/química , RNA/metabolismo , Evolução Molecular Direcionada , Evolução Molecular , Mutação , Conformação de Ácido Nucleico , Polimerização , Domínios Proteicos , RNA/genética , RNA Catalítico/química , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Fator sigma/metabolismo
16.
Food Microbiol ; 97: 103755, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33653528

RESUMO

Shewanella baltica is a typical specific spoilage organism causing the deterioration of seafood, but the exact regulation of its adaptive and competitive dominance in diverse environments remains undefined. In this study, the regulatory function of two sigma factors, RpoS and RpoN, in environmental adaptation and spoilage potential were evaluated in S. baltica SB02. Two in-frame deletion mutants, ΔrpoS and ΔrpoN, were constructed to explore the roles in their motility, biofilm formation, stress response and spoilage potential, as well as antibiotics by comparing the phenotypes and transcription with those of wild type (WT) strain. Compared with WT strain, the ΔrpoN showed the slower growth and weaker motility due to loss of flagella, while swimming of the ΔrpoS was increased. Deletion of rpoN significantly decreased biofilm biomass, and production of exopolysaccharide and pellicle, resulting in a thinner biofilm structure, while ΔrpoS formed the looser aggregation in biofilm. Resistance of S. baltica to NaCl, heat, ethanol and three oxidizing disinfectants apparently declined in the two mutants compared to WT strain. The ΔrpoN mutant decreased sensory score, accumulation of trimethylamine, putrescine and TVB-N and protease activity, while a weaker effect was observed in ΔrpoS. The two mutants had significantly higher susceptibility to antibiotics than WT strain, especially ΔrpoN. Deficiency of rpoN and rpoS significantly repressed the activities of two diketopiperazines related to quorum sensing (QS). Furthermore, transcriptome analyses revealed that RpoN was involved in the regulation of the expression of 143 genes, mostly including flagellar assembly, nitrogen and amino acid metabolism, ABC transporters. Transcript changes of seven differentially expressed coding sequences were in agreement with the phenotypes observed in the two mutants. Our findings reveal that RpoN, as a central regulator, controls the fitness and bacterial spoilage in S. baltica, while RpoS is a key regulatory factor of stress response. Characterization of these two sigma regulons in Shewanella has expanded current understanding of a possible co-regulatory mechanism with QS for adaptation and spoilage potential.


Assuntos
Proteínas de Bactérias/metabolismo , Perciformes/microbiologia , Shewanella/fisiologia , Fator sigma/metabolismo , Adaptação Fisiológica , Animais , Proteínas de Bactérias/genética , Biofilmes , Contaminação de Alimentos/análise , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Regulon , Shewanella/genética , Fator sigma/genética
17.
J Bacteriol ; 203(10)2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33649146

RESUMO

Lytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan must be unfailingly stable to preserve cell integrity, but must also be dynamically remodeled for the cell to grow, divide, and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG, and flagellar insertion is aided by localized activity of a dedicated PG lyase in Gram-negative bacteria. To date, there is no known dedicated lyase in Gram-positive bacteria for the insertion of flagella. Here, we take a reverse-genetic candidate-gene approach and find that cells mutated for the lytic transglycosylase CwlQ exhibit a severe defect in flagellum-dependent swarming motility. We further show that CwlQ is expressed by the motility sigma factor SigD and is secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells with mutations of CwlQ remain proficient for flagellar biosynthesis even when mutated in combination with four other lyases related to motility (LytC, LytD, LytF, and CwlO). The PG lyase (or lyases) essential for flagellar synthesis in B. subtilis, if any, remains unknown.IMPORTANCE Bacteria are surrounded by a wall of peptidoglycan and early work in Bacillus subtilis was the first to suggest that bacteria needed to enzymatically remodel the wall to permit insertion of the flagellum. No PG remodeling enzyme alone or in combination, however, has been found to be essential for flagellar assembly in B. subtilis Here, we take a reverse-genetic candidate-gene approach and find that the PG lytic transglycosylase CwlQ is required for swarming motility. Subsequent characterization determined that while CwlQ was coexpressed with motility genes and is secreted by the flagellar secretion apparatus, it was not required for flagellar synthesis. The PG lyase needed for flagellar assembly in B. subtilis remains unknown.


Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/fisiologia , Flagelos/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Movimento , Mutação , Peptidoglicano Glicosiltransferase/genética , Fator sigma/metabolismo , Sistemas de Secreção Tipo III/metabolismo
18.
J Bacteriol ; 203(7)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33526615

RESUMO

To enable accurate and efficient translation, sulfur modifications are introduced posttranscriptionally into nucleosides in tRNAs. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems for the incorporation of sulfur atoms in different nucleosides of tRNA. One of the proteins that is involved in inserting the sulfur for 5-methylaminomethyl-2-thiouridine (mnm5s2U34) modifications in tRNAs is the TusA protein. TusA, however, is a versatile protein that is also involved in numerous other cellular pathways. Despite its role as a sulfur transfer protein for the 2-thiouridine formation in tRNA, a fundamental role of TusA in the general physiology of Escherichia coli has also been discovered. Poor viability, a defect in cell division, and a filamentous cell morphology have been described previously for tusA-deficient cells. In this report, we aimed to dissect the role of TusA for cell viability. We were able to show that the lack of the thiolation status of wobble uridine (U34) nucleotides present on Lys, Gln, or Glu in tRNAs has a major consequence on the translation efficiency of proteins; among the affected targets are the proteins RpoS and Fis. Both proteins are major regulatory factors, and the deregulation of their abundance consequently has a major effect on the cellular regulatory network, with one consequence being a defect in cell division by regulating the FtsZ ring formation.IMPORTANCE More than 100 different modifications are found in RNAs. One of these modifications is the mnm5s2U modification at the wobble position 34 of tRNAs for Lys, Gln, and Glu. The functional significance of U34 modifications is substantial since it restricts the conformational flexibility of the anticodon, thus providing translational fidelity. We show that in an Escherichia coli TusA mutant strain, involved in sulfur transfer for the mnm5s2U34 thio modifications, the translation efficiency of RpoS and Fis, two major cellular regulatory proteins, is altered. Therefore, in addition to the transcriptional regulation and the factors that influence protein stability, tRNA modifications that ensure the translational efficiency provide an additional crucial regulatory factor for protein synthesis.


Assuntos
Divisão Celular , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação , RNA de Transferência/genética , RNA de Transferência/metabolismo , Fator sigma/genética , Fator sigma/metabolismo
19.
World J Microbiol Biotechnol ; 37(3): 38, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33544236

RESUMO

Oxidative stress can have lethal consequences if organisms do not respond and remediate the damage to DNA, proteins and lipids. Bacterial species respond to oxidative stress by activating transcriptional profiles that include biochemical functions to reduce oxidized cellular components, regenerate pools of reducing molecules, and detoxify harmful metabolites. Interestingly, the general stress response in Gram positive bacteria controlled by SigB is induced by oxidative stress from reactive oxygen and electrophilic species. The upregulation of SigB regulated genes during exposure to electrophilic and oxidative compounds suggests SigB contributes directly to the adaptations required for oxidative stress survival. A subset of the functions of SigB regulated genes can be categorized with antioxidant biochemical activities, such as redoxins, reductases and dehydrogenases, including regulation of low molecular weight thiols, yet their exact cellular role is not fully understood. Here, we present an overview of the predicted antioxidant biochemical functions regulated by SigB, with potential for biomedical research given the prevalence of oxidative stress during bacterial infection, as well as during industrial applications of large-scale production of compounds by microbes.


Assuntos
Proteínas de Bactérias/metabolismo , Bactérias Gram-Positivas/fisiologia , Fator sigma/metabolismo , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Positivas/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio , Estresse Fisiológico
20.
Protein Sci ; 30(4): 899-907, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33599047

RESUMO

In the model organism Escherichia coli and related species, the general stress response relies on tight regulation of the intracellular levels of the promoter specificity subunit RpoS. RpoS turnover is exclusively dependent on RssB, a two-domain response regulator that functions as an adaptor that delivers RpoS to ClpXP for proteolysis. Here, we report crystal structures of the receiver domain of RssB both in its unphosphorylated form and bound to the phosphomimic BeF3 - . Surprisingly, we find only modest differences between these two structures, suggesting that truncating RssB may partially activate the receiver domain to a "meta-active" state. Our structural and sequence analysis points to RssB proteins not conforming to either the Y-T coupling scheme for signaling seen in prototypical response regulators, such as CheY, or to the signaling model of the less understood FATGUY proteins.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Transdução de Sinais , Fatores de Transcrição/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endopeptidase Clp/química , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator sigma/química , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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