RESUMO
The murine model is invaluable for studying intricate interactions among gut microbes; hosts; and diseases. However; the impact of genetic variations in the murine microbiome; especially in disease contexts such as Klebsiella pneumoniae (Kp) infection; still needs to be explored. Kp; an opportunistic global pathogen; is becoming increasingly prevalent in regions like Asia; especially China. This study explored the role of the gut microbiota during Kp infection using mouse model; including wild-type and rpoS mutants of Kp138; KpC4; and KpE4 from human; maize; and ditch water; respectively. Under stress conditions; RpoS reconfigures global gene expression in bacteria; shifting the cells from active growth to survival mode. Our study examined notable differences in microbiome composition; finding that Lactobacillus and Klebsiella (particularly in WKp138) were the most abundant genera in mice guts at the genus level in all wild-type treated mice. In contrast; Firmicutes were predominant in the healthy control mice. Furthermore; Clostridium was the dominant genus in all mutants; mainly in ∆KpC4; and was absent in wild-type treated mice. Differential abundance analysis identified that these candidate taxa potentially influence disease progression and pathogen virulence. Functional prediction analysis showed that most bacterial groups were functionally involved in biosynthesis; precursor metabolites; degradation; energy generation; and metabolic cluster formation. These findings challenge the conventional understanding and highlight the need for nuanced interpretations in murine studies. Additionally; this study sheds light on microbiome-immune interactions in K. pneumoniae infection and proposes new potential therapeutic strategies.
Assuntos
Proteínas de Bactérias , Microbioma Gastrointestinal , Infecções por Klebsiella , Klebsiella pneumoniae , Fator sigma , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Animais , Microbioma Gastrointestinal/genética , Camundongos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/genética , Humanos , Regulação Bacteriana da Expressão Gênica , Modelos Animais de Doenças , Feminino , Virulência/genéticaRESUMO
Auranofin (AF), a former rheumatoid polyarthritis treatment, gained renewed interest for its use as an antimicrobial. AF is an inhibitor of thioredoxin reductase (TrxB), a thiol and protein repair enzyme, with an antibacterial activity against several bacteria including C. difficile, an enteropathogen causing post-antibiotic diarrhea. Several studies demonstrated the effect of AF on C. difficile physiology, but the crucial questions of resistance mechanisms and impact on microbiota remain unaddressed. We explored potential resistance mechanisms by studying the impact of TrxB multiplicity and by generating and characterizing adaptive mutations. We showed that if mutants inactivated for trxB genes have a lower MIC of AF, the number of TrxBs naturally present in clinical strains does not impact the MIC. All stable mutations isolated after AF long-term exposure were in the anti-sigma factor of σB and strongly affect physiology. Finally, we showed that AF has less impact on human gut microbiota than vancomycin.
Assuntos
Antibacterianos , Auranofina , Clostridioides difficile , Microbioma Gastrointestinal , Testes de Sensibilidade Microbiana , Humanos , Auranofina/farmacologia , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Antibacterianos/farmacologia , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Mutação , Farmacorresistência Bacteriana , Adaptação Fisiológica , Fator sigma/genética , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vancomicina/farmacologiaRESUMO
The functional amyloid of Pseudomonas (Fap) is essential for the formation of macrocolony biofilms, pellicles, and solid surface-associated (SSA) biofilms of Pseudomonas fluorescens PF07, an isolate from refrigerated marine fish. However, limited information on the expression regulation of fap genes is available. Herein, we found that a novel bacterial enhancer-binding protein (bEBP), BrfA, regulated Fap-dependent biofilm formation by directly sensing cyclic diguanosine monophosphate (c-di-GMP). Our in vivo data showed that the REC domain deletion of BrfA promoted fap gene expression and biofilm formation, and c-di-GMP positively regulated the transcription of fapA in a BrfA-dependent manner. In in vitro experiments, we found that the ATPase activity of BrfA was inhibited by the REC domain and was activated by c-di-GMP. BrfA and the sigma factor RpoN bound to the upstream region of fapA, and the binding ability of BrfA was not affected by either deletion of the REC domain or c-di-GMP. BrfA specifically bound to the three enhancer sites upstream of the fapA promoter, which contain the consensus sequence CA-(N4)-TGA(A/T)ACACC. In vivo experiments using a lacZ fusion reporter indicated that all three BrfA enhancer sites were essential for the activation of fapA transcription. Overall, these findings reveal that BrfA is a new type of c-di-GMP-responsive transcription factor that directly controls the transcription of Fap biosynthesis genes in P. fluorescens. Fap functional amyloids and BrfA-type transcription factors are widespread in Pseudomonas species. The novel insights into the c-di-GMP- and BrfA-dependent expression regulation of fap provided by this work will contribute to the development of antibiofilm strategies.
Assuntos
Proteínas de Bactérias , Biofilmes , GMP Cíclico , Regulação Bacteriana da Expressão Gênica , Pseudomonas fluorescens , Biofilmes/crescimento & desenvolvimento , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Pseudomonas fluorescens/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Amiloide/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fator sigma/genética , Fator sigma/metabolismoRESUMO
After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli (E. coli) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 (σ70) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α-C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ-independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation.
Assuntos
RNA Polimerases Dirigidas por DNA , Proteínas de Escherichia coli , Escherichia coli , Regiões Promotoras Genéticas , Fator sigma , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Fator sigma/metabolismo , Fator sigma/genética , Fator sigma/química , Transcrição Gênica , Fatores de Transcrição/metabolismo , Domínios Proteicos , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Alongamento de Peptídeos/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/química , Repressores Lac/metabolismo , Repressores Lac/genéticaRESUMO
Membrane vesicles (MVs) are produced by species across all domains of life and have diverse physiological functions as well as promising applications. While the mechanisms for vesiculation in Gram-negative bacteria are well-established, the genetic determinants and regulatory factors responsible for MV biogenesis in Gram-positive bacteria remain largely unknown. Here, we demonstrate that a Q225P substitution in the alternative sigma factor B (SigB) triggers MV production in Staphylococcus aureus strain Newman by hindering the specific binding of SigB to the asp23 promoter, thereby repressing expression of alkaline shock protein 23 (Asp23). Isogenic deletion of asp23 also promotes MV formation in Newman, confirming the critical roles played by sigB and asp23 in modulating S. aureus vesiculation. While bacterial growth and cytoplasmic membrane fluidity are not impaired, mutation of asp23 weakens the cell wall and enhances autolysis, consistent with decreased expression of alpha-type psm and lrgAB that modulate murein hydrolase activity. TEM and proteomic analysis show that Newman and asp23 deletion mutant generate MVs with nearly identical morphology and composition, but virulence-associated factors are significantly enriched in MVs from the asp23 mutant. Overall, this study reveals novel genetic determinants underlying S. aureus vesiculation and advances the understanding of the physiology of MV biogenesis in S. aureus.
Assuntos
Proteínas de Bactérias , Parede Celular , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Fator sigma/metabolismo , Fator sigma/genética , Regulação Bacteriana da Expressão Gênica , Vesículas Extracelulares/metabolismoRESUMO
Although RNA structures play important roles in regulating gene expression, the mechanism and function of mRNA folding in plant bacterial pathogens remain elusive. Therefore, we perform dimethyl sulfate sequencing (DMS-seq) on the Pseudomonas syringae under nutrition-rich and -deficient conditions, revealing that the mRNA structure changes substantially in the minimal medium (MM) that tunes global translation efficiency (TE), thereby inducing virulence. This process is led by the increased expression of hfq, which is directly activated by transcription regulators RpoS and CysB. The co-occurrence of Hfq and RpoS in diverse bacteria and the deep conservation of Hfq Y25 is critical for RNA-mediated regulation and implicates the wider biological importance of mRNA structure and feedback loops in the control of global gene expression.
Assuntos
Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro , Pseudomonas syringae , Transcriptoma , Fator Proteico 1 do Hospedeiro/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Virulência/genética , Transcriptoma/genética , Pseudomonas syringae/patogenicidade , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Fator sigma/metabolismo , Fator sigma/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Doenças das Plantas/microbiologiaRESUMO
The metabolic state of bacteria significantly contributes to their resistance to antibiotics; however, the specific metabolic mechanisms conferring antimicrobial resistance in Helicobacter pylori remain largely understudied. Employing transcriptomic and non-targeted metabolomics, we characterized the metabolic reprogramming of H. pylori when challenged with antibiotic agents. We observed a notable increase in both genetic and key proteomic components involved in fatty acid biosynthesis. Inhibition of this pathway significantly enhanced the antibiotic susceptibility of the sensitive and multidrug-resistant H. pylori strains while also disrupting their biofilm-forming capacities. Further analysis revealed that antibiotic treatment induced a stringent response, triggering the expression of the hp0560-hp0557 operon regulated by Sigma28 (σ28). This activation in turn stimulated the fatty acid biosynthetic pathway, thereby enhancing the antibiotic tolerance of H. pylori. Our findings reveal a novel adaptive strategy employed by H. pylori to withstand antibiotic stress.
Assuntos
Antibacterianos , Proteínas de Bactérias , Biofilmes , Farmacorresistência Bacteriana Múltipla , Ácidos Graxos , Helicobacter pylori , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana Múltipla/genética , Ácidos Graxos/biossíntese , Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Testes de Sensibilidade Microbiana , Óperon , Fator sigma/genética , Fator sigma/metabolismoRESUMO
Effector proteins secreted by bacteria that infect mammalian and plant cells often subdue eukaryotic host cell defenses by simultaneously affecting multiple targets. However, instances when a bacterial effector injected in the competing bacteria sabotage more than a single target have not been reported. Here, we demonstrate that the effector protein, LtaE, translocated by the type IV secretion system from the soil bacterium Lysobacter enzymogenes into the competing bacterium, Pseudomonas protegens, affects several targets, thus disabling the antibacterial defenses of the competitor. One LtaE target is the transcription factor, LuxR1, that regulates biosynthesis of the antimicrobial compound, orfamide A. Another target is the sigma factor, PvdS, required for biosynthesis of another antimicrobial compound, pyoverdine. Deletion of the genes involved in orfamide A and pyoverdine biosynthesis disabled the antibacterial activity of P. protegens, whereas expression of LtaE in P. protegens resulted in the near-complete loss of the antibacterial activity against L. enzymogenes. Mechanistically, LtaE inhibits the assembly of the RNA polymerase complexes with each of these proteins. The ability of LtaE to bind to LuxR1 and PvdS homologs from several Pseudomonas species suggests that it can sabotage defenses of various competitors present in the soil or on plant matter. Our study thus reveals that the multi-target effectors have evolved to subdue cell defenses not only in eukaryotic hosts but also in bacterial competitors.
Assuntos
Proteínas de Bactérias , Lysobacter , Pseudomonas , Sistemas de Secreção Tipo IV , Pseudomonas/genética , Pseudomonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lysobacter/genética , Lysobacter/metabolismo , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo , Regulação Bacteriana da Expressão Gênica , Oligopeptídeos/metabolismo , Oligopeptídeos/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator sigma/genética , Fator sigma/metabolismoRESUMO
As an efficient and safe industrial bacterium, Corynebacterium glutamicum has extensive application in amino acid production. However, it often faces oxidative stress induced by reactive oxygen species (ROS), leading to diminished production efficiency. To enhance the robustness of C. glutamicum, numerous studies have focused on elucidating its regulatory mechanisms under various stress conditions such as heat, acid, and sulfur stress. However, a comprehensive review of its defense mechanisms against oxidative stress is needed. This review offers an in-depth overview of the mechanisms C. glutamicum employs to manage oxidative stress. It covers both enzymatic and non-enzymatic systems, including antioxidant enzymes, regulatory protein families, sigma factors involved in transcription, and physiological redox reduction pathways. This review provides insights for advancing research on the antioxidant mechanisms of C. glutamicum and sheds light on its potential applications in industrial production.
Assuntos
Antioxidantes , Proteínas de Bactérias , Corynebacterium glutamicum , Regulação Bacteriana da Expressão Gênica , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio , Fator sigma , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/genética , Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Espécies Reativas de Oxigênio/metabolismo , Fator sigma/metabolismo , Fator sigma/genéticaRESUMO
BACKGROUND: Ethanol shock significantly affects expression of over 1200 genes in Streptomyces venezuelae NRRL B-65,442, including those involved in secondary metabolite biosynthesis and a cryptic gene pepX, which encodes a 19-amino acid peptide with an unknown function. RESULTS: To establish a possible correlation between the PepX peptide and secondary metabolism in S. venezuelae, its gene was deleted, followed by analyses of the transcriptome and secondary metabolome of the mutant. Although the secondary metabolome of the pepX mutant was not strongly affected, pepX deletion, similar to ethanol shock, mostly resulted in downregulated expression of secondary metabolite biosynthesis gene clusters (BGCs). At the same time, there was a reverse correlation between the expression of certain extracytoplasmic function sigma factors (ECFs) and several BGCs. Individual deletions of three selected ECF-coding genes conserved in Streptomyces that were upregulated upon both pepX deletion and ethanol shock, had a profound positive effect on the expression of BGCs, which also correlated with the overproduction of specific secondary metabolites. Deletion of one such ECF-coding gene in a marine sponge-derived Streptomyces sp. also significantly altered the secondary metabolite profile, suggesting an important role of this ECF in the regulation of secondary metabolism. CONCLUSIONS: These findings pave the way for the activation or upregulation of BGCs in Streptomyces bacteria harboring genes for ECFs homologous to those identified in this study, hereby assisting in the discovery of novel bioactive secondary metabolites.
Assuntos
Metabolismo Secundário , Fator sigma , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Metabolismo Secundário/genética , Fator sigma/genética , Fator sigma/metabolismo , Regulação Bacteriana da Expressão Gênica , Deleção de Genes , Família Multigênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Etanol/metabolismo , TranscriptomaRESUMO
In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.
Assuntos
Proteínas de Bactérias , Clostridioides difficile , N-Acetil-Muramil-L-Alanina Amidase , Peptidoglicano , Esporos Bacterianos , Esporos Bacterianos/metabolismo , Esporos Bacterianos/genética , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Clostridioides difficile/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/genética , Peptidoglicano/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma/metabolismo , Fator sigma/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/enzimologia , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genéticaRESUMO
The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer layer of mycolic acids called the mycomembrane (MM) and a cell wall composed of peptidoglycan and arabinogalactan. This envelope architecture is unique among bacteria and contributes significantly to the virulence of pathogenic Mycobacteriales like Mycobacterium tuberculosis. Characterization of pathways that govern envelope biogenesis in these organisms is therefore critical in understanding their biology and for identifying new antibiotic targets. To better understand MM biogenesis, we developed a cell sorting-based screen for mutants defective in the surface exposure of a porin normally embedded in the MM of the model organism Corynebacterium glutamicum. The results revealed a requirement for the conserved σD envelope stress response in porin export and identified MarP as the site-1 protease, respectively, that activate the response by cleaving the membrane-embedded anti-sigma factor. A reporter system revealed that the σD pathway responds to defects in mycolic acid and arabinogalactan biosynthesis, suggesting that the stress response has the unusual property of being induced by activating signals that arise from defects in the assembly of two distinct envelope layers. Our results thus provide new insights into how C. glutamicum and related bacteria monitor envelope integrity and suggest a potential role for members of the σD regulon in protein export to the MM.
Assuntos
Membrana Celular , Parede Celular , Corynebacterium glutamicum , Ácidos Micólicos , Fator sigma , Parede Celular/metabolismo , Parede Celular/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácidos Micólicos/metabolismo , Fator sigma/metabolismo , Fator sigma/genética , Membrana Celular/metabolismo , Estresse Fisiológico , Porinas/metabolismo , Porinas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Galactanos/metabolismo , Regulação Bacteriana da Expressão Gênica , Peptidoglicano/metabolismoRESUMO
Although enteric bacteria normally reside within the animal intestine, the ability to persist extraintestinally is an essential part of their overall lifestyle, and it might contribute to transmission between hosts. Despite this potential importance, few genetic determinants of extraintestinal growth and survival have been identified, even for the best-studied model, Escherichia coli. In this work, we thus used a genome-wide library of barcoded transposon insertions to systematically identify functional clusters of genes that are crucial for E. coli fitness in lake water. Our results revealed that inactivation of pathways involved in maintaining outer membrane integrity, nucleotide biosynthesis, and chemotaxis negatively affected E. coli growth or survival in this extraintestinal environment. In contrast, inactivation of another group of genes apparently benefited E. coli growth or persistence in filtered lake water, resulting in higher abundance of these mutants. This group included rpoS, which encodes the general stress response sigma factor, as well as genes encoding several other global transcriptional regulators and RNA chaperones, along with several poorly annotated genes. Based on this co-enrichment, we identified these gene products as novel positive regulators of RpoS activity. We further observed that, despite their enhanced growth, E. coli mutants with inactive RpoS had reduced viability in lake water, and they were not enriched in the presence of the autochthonous microbiota. This highlights the duality of the general stress response pathway for E. coli growth outside the host.
Assuntos
Escherichia coli , Lagos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Lagos/microbiologia , Fator sigma/genética , Fator sigma/metabolismo , Genoma Bacteriano , Elementos de DNA Transponíveis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Microbiologia da ÁguaRESUMO
Bacterial RNAP needs to form holoenzyme with σ factors to initiate transcription. While Staphylococcus aureus σA controls housekeeping functions, S. aureus σB regulates virulence, biofilm formation, persistence, cell internalization, membrane transport, and antimicrobial resistance. Besides the sequence difference, the spacers between the -35 element and -10 element of σB regulated promoters are shorter than those of σA regulated promoters. Therefore, how σB recognizes and initiates transcription from target promoters can not be inferred from that of the well studied σ. Here, we report the cryo-EM structures of S. aureus RNAP-promoter open complexes comprising σA and σB, respectively. Structural analyses, in combination with biochemical experiments, reveal the structural basis for the promoter specificity of S. aureus transcription. Although the -10 element of σA regulated promoters is recognized by domain σA2 as single-stranded DNA, the -10 element of σB regulated promoters is co-recognized by domains σB2 and σB3 as double-stranded DNA, accounting for the short spacers of σB regulated promoters. S. aureus RNAP is a validated target of antibiotics, and our structures pave the way for rational drug design targeting S. aureus RNAP.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA , Regiões Promotoras Genéticas , Fator sigma , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/química , Fator sigma/metabolismo , Fator sigma/genética , Fator sigma/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Transcrição Gênica , Ligação ProteicaRESUMO
Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in E. coli has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, E. coli must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the protein's central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations' impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.
Assuntos
RNA Polimerases Dirigidas por DNA , Escherichia coli , Mutação , Estresse Fisiológico , Transcriptoma , Escherichia coli/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Estresse Fisiológico/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Adaptação Fisiológica/genéticaRESUMO
BACKGROUND: Bacterial RNA polymerase holoenzyme requires sigma70 factors to start transcription by identifying promoter elements. Cyanobacteria possess multiple sigma70 factors to adapt to a wide variety of ecological niches. These factors are grouped into two categories: primary sigma factor initiates transcription of housekeeping genes during normal growth conditions, while alternative sigma factors initiate transcription of specific genes under particular conditions. However, the present classification does not consider the modular organization of their structural domains, introducing therefore multiple functional and structural biases. A comprehensive analysis of this protein family in cyanobacteria is needed to address these limitations. RESULTS: We investigated the structure and evolution of sigma70 factors in cyanobacteria, analyzing their modular architecture and variation among unicellular, filamentous, and heterocyst-forming morphotypes. 4,193 sigma70 homologs were found with 59 distinct modular patterns, including six essential and 29 accessory domains, such as DUF6596. 90% of cyanobacteria typically have 5 to 17 sigma70 homologs and this number likely depends on the strain morphotype, the taxonomic order and the genome size. We classified sigma70 factors into 12 clans and 36 families. According to taxonomic orders and phenotypic traits, the number of homologs within the 14 main families was variable, with the A.1 family including the primary sigma factor since this family was found in all cyanobacterial species. The A.1, A.5, C.1, E.1, J.1, and K.1 families were found to be key sigma families that distinguish heterocyst-forming strains. To explain the diversification and evolution of sigma70, we propose an evolutionary scenario rooted in the diversification of a common ancestor of the A1 family. This scenario is characterized by evolutionary events including domain losses, gains, insertions, and modifications. The high occurrence of the DUF6596 domain in bacterial sigma70 proteins, and its association with the highest prevalence observed in Actinobacteria, suggests that this domain might be important for sigma70 function. It also implies that the domain could have emerged in Actinobacteria and been transferred through horizontal gene transfer. CONCLUSION: Our analysis provides detailed insights into the modular domain architecture of sigma70, introducing a novel robust classification. It also proposes an evolutionary scenario explaining their diversity across different taxonomical orders.
Assuntos
Cianobactérias , Evolução Molecular , Filogenia , Fator sigma , Fator sigma/genética , Fator sigma/metabolismo , Cianobactérias/genética , Cianobactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Variação GenéticaRESUMO
Small non-coding 6S RNA mimics DNA promoters and binds to the σ70 holoenzyme of bacterial RNA polymerase (RNAP) to suppress transcription of various genes mainly during the stationary phase of cell growth or starvation. This inhibition can be relieved upon synthesis of short product RNA (pRNA) performed by RNAP from the 6S RNA template. Here, we have shown that pRNA synthesis depends on specific contacts of 6S RNA with RNAP and interactions of the σ finger with the RNA template in the active site of RNAP, and is also modulated by the secondary channel factors. We have adapted a molecular beacon assay with fluorescently labeled σ70 to analyze 6S RNA release during pRNA synthesis. We found the kinetics of 6S RNA release to be oppositely affected by mutations in the σ finger and in the CRE pocket of core RNAP, similarly to the reported role of these regions in promoter-dependent transcription. Secondary channel factors, DksA and GreB, inhibit pRNA synthesis and 6S RNA release from RNAP, suggesting that they may contribute to the 6S RNA-mediated switch in transcription during stringent response. Our results demonstrate that pRNA synthesis depends on a similar set of contacts between RNAP and 6S RNA as in the case of promoter-dependent transcription initiation and reveal that both processes can be regulated by universal transcription factors acting on RNAP.
Assuntos
RNA Polimerases Dirigidas por DNA , Proteínas de Escherichia coli , RNA Bacteriano , Fator sigma , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/metabolismo , Fator sigma/metabolismo , Fator sigma/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Regiões Promotoras Genéticas , RNA não Traduzido/metabolismo , RNA não Traduzido/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , Fatores de Elongação da TranscriçãoRESUMO
Leptospirosis is a widespread zoonotic infectious disease of human and veterinary concern caused by pathogenic spirochetes of the genus Leptospira. To date, little progress towards understanding leptospiral pathogenesis and identification of virulence factors has been made, which is the main bottleneck for developing effective measures against the disease. Some leptospiral proteins, including LipL32, Lig proteins, LipL45, and LipL21, are being considered as potential virulence factors or vaccine candidates. However, their function remains to be established. LipL45 is the most expressed membrane lipoprotein in leptospires, upregulated when the bacteria are transferred to temperatures resembling the host, expressed during infection, suppressed after culture attenuation, and known to suffer processing in vivo and in vitro, generating fragments. Based on body of evidence, we hypothesized that the LipL45 processing might occur by an auto-cleavage event, deriving two fragments. The results presented here, based on bioinformatics, structure modeling analysis, and experimental data, corroborate that LipL45 processing probably includes a self-catalyzed non-proteolytic event and suggest the participation of LipL45 in cell-surface signaling pathways, as the protein shares structural similarities with bacterial sigma regulators. Our data indicate that LipL45 might play an important role in response to environmental conditions, with possible function in the adaptation to the host.
Assuntos
Leptospira , Lipoproteínas , Lipoproteínas/metabolismo , Lipoproteínas/química , Lipoproteínas/genética , Leptospira/metabolismo , Leptospira/química , Fator sigma/metabolismo , Fator sigma/química , Fator sigma/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Modelos Moleculares , Leptospirose/metabolismo , Leptospirose/microbiologiaRESUMO
RNA polymerase sigma factors are indispensable in the process of bacterial transcription. They are responsible for a given gene's promoter region recognition on template DNA and hence determine specificity of RNA polymerase and play a significant role in gene expression regulation. Here, we present a simple and unified protocol for purification of all seven Escherichia coli RNA polymerase sigma factors. In our approach, we took advantage of the His8-SUMO tag, known to increase protein solubilization. Sigma factors were first purified in N-terminal fusions with this tag, which was followed by tag removal with Ulp1 protease. This allowed to obtain proteins in their native form. In addition, the procedure is simple and requires only one resin type. With the general protocol we employed, we were able to successfully purify σD, σE, σS, and σN. Final step modification was required for σF, while for σH and σFecI, denaturing conditions had to be applied. All seven sigma factors were fully functional in forming an active holoenzyme with core RNA polymerase which we demonstrated with EMSA studies.
Assuntos
RNA Polimerases Dirigidas por DNA , Proteínas de Escherichia coli , Escherichia coli , Fator sigma , Fator sigma/genética , Escherichia coli/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The WRKY transcription factors play essential roles in a variety of plant signaling pathways associated with biotic and abiotic stress response. The transcriptional activity of many WRKY members are regulated by a class of intrinsically disordered VQ proteins. While it is known that VQ proteins interact with the WRKY DNA-binding domains (DBDs), also termed as the WRKY domains, structural information regarding VQ-WRKY interaction is lacking and the regulation mechanism remains unknown. Herein we report a solution NMR study of the interaction between Arabidopsis WRKY33 and its regulatory VQ protein partner SIB1. We uncover a SIB1 minimal sequence neccessary for forming a stable complex with WRKY33 DBD, which comprises not only the consensus "FxxhVQxhTG" VQ motif but also its preceding region. We demonstrate that the ßN-strand and the extended ßN-ß1 loop of WRKY33 DBD form the SIB1 docking site, and build a structural model of the complex based on the NMR paramagnetic relaxation enhancement and mutagenesis data. Based on this model, we further identify a cluster of positively-charged residues in the N-terminal region of SIB1 to be essential for the formation of a SIB1-WRKY33-DNA ternary complex. These results provide a framework for the mechanism of SIB1-enhanced WRKY33 transcriptional activity.