RESUMO
Divergent transcription from within bacterial intergenic regions frequently involves promoters dependent on alternative σ-factors. This is the case for the non-overlapping σ70- and σ54-dependent promoters that control production of the substrate-responsive regulator and enzymes for (methyl)phenol catabolism. Here, using an array of in vivo and in vitro assays, we identify transcription-driven supercoiling arising from the σ54-promoter as the mechanism underlying inter-promoter communication that results in stimulation of the activity of the σ70-promoter. The non-overlapping 'back-to-back' configuration of a powerful σ54-promoter and weak σ70-promoter within this system offers a previously unknown means of inter-sigmulon communication that renders the σ70-promoter subservient to signals that elicit σ54-dependent transcription without it possessing a cognate binding site for the σ54-RNA polymerase holoenzyme. This mode of control has the potential to be a prevalent, but hitherto unappreciated, mechanism by which bacteria adjust promoter activity to gain appropriate transcriptional control.
Assuntos
Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Expressão Gênica , Genes Reporter , Óperon , Ligação ProteicaRESUMO
The nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, the Escherichia coli (p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purified E. coli components, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to ß-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.
Assuntos
Antibacterianos/farmacologia , Tolerância a Medicamentos , Guanosina Tetrafosfato/análogos & derivados , Ligases/genética , Tioestreptona/farmacologia , beta-Lactamas/farmacologia , Cloranfenicol/farmacologia , Interações Medicamentosas , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Isoleucina-tRNA Ligase/genética , Ligases/antagonistas & inibidores , Ligases/metabolismo , Mupirocina/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Frações Subcelulares/química , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Tetraciclina/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/farmacologiaRESUMO
Alternative bacterial sigma factors bind the catalytic core RNA polymerase to confer promoter selectivity on the holoenzyme. The different holoenzymes are thus programmed to recognize the distinct promoter classes in the genome to allow coordinated activation of discrete sets of genes needed for adaptive responses. To form the holoenzymes, the different sigma factors must be available to compete for their common substrate (core RNA polymerase). This review highlights (a) the roles of antisigma factors in controlling the availability of alternative sigma factors and (b) the involvement of diverse regulatory molecules that promote the use of alternative sigma factors through subversion of the domineering housekeeping σ(70). The latter include the nucleotide alarmone ppGpp and small proteins (DksA, Rsd, and Crl), which directly target the transcriptional machinery to mediate their effects.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Ligação Proteica , Fator sigma/antagonistas & inibidores , Fator sigma/genéticaRESUMO
Expression of pathways for dissimilation of toxic aromatic compounds such as (methyl)phenols interfaces both stress-response and carbon catabolite repression control cascades. In Pseudomonas putida, carbon catabolite repression is mediated by the protein Crc - a translational repressor that counteracts utilization of less-preferred carbon sources as growth substrates until they are needed. In this work we dissect the regulatory role of the 5'-leader region (5'-LR) of the dmpR gene that encodes the master regulator of (methyl)phenol catabolism. Using deletion and substitution mutants combined with artificial manipulations of Crc availability in P. putida, we present evidence that a DNA motif within the 5'-leader region is critical for inhibition of the output from the Pr promoter that drives transcription of dmpR, while the RNA chaperone Hfq facilitates Crc-mediated translation repression through the 5'-leader region of the dmpR mRNA. The results are discussed in the light of a model in which Hfq assists Crc to target a sequence within a loop formed by secondary structure of the 5'-LR mRNA. Our results support the idea that Crc functions as a global translational inhibitor to co-ordinate hierarchical carbon utilization in Pseudomonads.
Assuntos
Regiões 5' não Traduzidas , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Pseudomonas putida/genética , Sequências Reguladoras de Ácido Ribonucleico , Transativadores/genética , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Genes Reguladores , Fator Proteico 1 do Hospedeiro/metabolismo , Regiões Promotoras Genéticas , Pseudomonas putida/metabolismo , RNA Bacteriano/metabolismo , Proteínas Repressoras/metabolismoRESUMO
The Pr promoter is the first verified member of a class of bacterial σ(70)-promoters that only possess a single match to consensus within its -10 element. In its native context, the activity of this promoter determines the ability of Pseudomonas putida CF600 to degrade phenolic compounds, which provides proof-of-principle for the significance of such promoters. Lack of identity within the -10 element leads to non-detection of Pr-like promoters by current search engines, because of their bias for detection of the -10 motif. Here, we report a mutagenesis analysis of Pr that reveals strict sequence requirements for its activity that includes an essential -15 element and preservation of non-consensus bases within its -35 and -10 elements. We found that highly similar promoters control plasmid- and chromosomally- encoded phenol degradative systems in various Pseudomonads. However, using a purpose-designed promoter-search algorithm and activity analysis of potential candidate promoters, no bona fide Pr-like promoter could be found in the entire genome of P. putida KT2440. Hence, Pr-like σ(70)-promoters, which have the potential to be a widely distributed class of previously unrecognized promoters, are in fact highly restricted and remain in a class of their own.
Assuntos
DNA Bacteriano/química , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Mutagênese , Motivos de Nucleotídeos , Fenóis/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismoRESUMO
The activities of promoters can be temporally and conditionally regulated by mechanisms other than classical DNA-binding repressors and activators. One example is the inherently weak σ(70)-dependent Pr promoter that ultimately controls catabolism of phenolic compounds. The activity of Pr is up-regulated through the joint action of ppGpp and DksA that enhance the performance of RNA polymerase at this promoter. Here, we report a mutagenesis analysis that revealed substantial differences between Pr and other ppGpp/DksA co-stimulated promoters. In vitro transcription and RNA polymerase binding assays show that it is the T at the -11 position of the extremely suboptimal -10 element of Pr that underlies both poor binding of σ(70)-RNAP and a slow rate of open complex formation--the process that is accelerated by ppGpp and DksA. Our findings support the idea that collaborative action of ppGpp and DksA lowers the rate-limiting transition energy required for conversion between intermediates on the road to open complex formation.
Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/metabolismo , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Bactérias/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutagênese , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Adaptation to shifting temperatures is crucial for the survival of the bacterial pathogen Vibrio cholerae. Here, we show that colony rugosity, a biofilm-associated phenotype, is regulated by temperature in V. cholerae strains that naturally lack the master biofilm transcriptional regulator HapR. Using transposon-insertion mutagenesis, we found the V. cholerae ortholog of BipA, a conserved ribosome-associated GTPase, is critical for this temperature-dependent phenomenon. Proteomic analyses revealed that loss of BipA alters the synthesis of >300 proteins in V. cholerae at 22°C, increasing the production of biofilm-related proteins including the key transcriptional activators VpsR and VpsT, as well as proteins important for diverse cellular processes. At low temperatures, BipA protein levels increase and are required for optimal ribosome assembly in V. cholerae, suggesting that control of BipA abundance is a mechanism by which bacteria can remodel their proteomes. Our study reveals a remarkable new facet of V. cholerae's complex biofilm regulatory network.
Assuntos
Proteínas de Bactérias/genética , Biofilmes , GTP Fosfo-Hidrolases/genética , Vibrio cholerae/fisiologia , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , GTP Fosfo-Hidrolases/metabolismo , Fenótipo , Temperatura , Vibrio cholerae/genéticaRESUMO
Pseudomonas sp. strain Y2 degrades styrene through oxidation to phenylacetic acid via the styABCD operon-encoded enzymes, whose expression is induced in response to styrene by the StyS/StyR two-component regulatory system. Further transformation of phenylacetic acid to tricarboxylic acid cycle intermediates is mediated by the enzymes of paa catabolic genes, whose expression is regulated by the PaaX repressor. The first step of this paa degradation pathway is catalysed by paaF-encoded phenylacetyl-coenzyme A ligases that produce phenylacetyl-coenzyme A. This metabolic intermediate, upon being bound by PaaX, inactivates PaaX-mediated repression of both the paa genes and the styABCD operon. Strain Y2 is unique in having three paaF genes located within two complete copies of the paa gene clusters. Expression of both paaF and paaF3 is controlled by the PaaX repressor. Here we use specific mutants in combination with in vivo and in vitro assays to demonstrate that paaF2, adjacent to the StyS/StyR regulatory genes, belongs to the StyR regulon and is not subject to repression by PaaX. We propose that this unexpected styrene-responsive regulatory strategy for the otherwise metabolically redundant PaaF2 auxiliary enzyme provides a system for rapid co-ordinate de-repression of the two sets of catabolic genes required for styrene degradation.
Assuntos
Proteínas de Bactérias/metabolismo , Coenzima A Ligases/biossíntese , Regulação Bacteriana da Expressão Gênica , Pseudomonas/enzimologia , Pseudomonas/fisiologia , Estireno/metabolismo , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Sequência de Bases , Coenzima A Ligases/genética , Deleção de Genes , Ordem dos Genes , Genes Reporter , Redes e Vias Metabólicas/genética , Dados de Sequência Molecular , Família Multigênica , Regiões Promotoras Genéticas , Pseudomonas/crescimento & desenvolvimento , Regulon , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sítio de Iniciação de Transcrição , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
The P(styA) promoter of Pseudomonas sp. strain Y2 controls expression of the styABCD genes, which are required for the conversion of styrene to phenylacetate, which is further catabolized by the products of two paa gene clusters. Two PaaX repressor proteins (PaaX1 and PaaX2) regulate transcription of the paa gene clusters of this strain. In silico analysis of the P(styA) promoter region revealed a sequence located just within styA that is similar to the reported PaaX binding sites of Escherichia coli and the proposed PaaX binding sites of the paa genes of Pseudomonas species. Here we show that protein extracts from some Pseudomonas strains that have paaX genes, but not from a paaX mutant strain, can bind and retard the migration of a P(styA) specific probe. Purified maltose-binding protein (MBP)-PaaX1 fusion protein specifically binds the P(styA) promoter proximal PaaX site, and this binding is eliminated by the addition of phenylacetyl-coenzyme A. The sequence protected by MBP-PaaX1 binding was defined by DNase I footprinting. Moreover, MBP-PaaX1 represses transcription from the P(styA) promoter in a phenylacetyl-coenzyme A-dependent manner in vitro. Finally, the inactivation of both paaX gene copies of Pseudomonas sp. strain Y2 leads to a higher level of transcription from the P(styA) promoter, while heterologous expression of the PaaX1 in E. coli greatly decreases transcription from the P(styA) promoter. These findings reveal a control mechanism that integrates regulation of styrene catabolism by coordinating the expression of the styrene upper catabolic operon to that of the paa-encoded central pathway and support a role for PaaX as a major regulatory protein in the phenylacetyl-coenzyme A catabolon through its response to the levels of this central metabolite.