RESUMO
In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP), we deep sequenced an E. coli genomic library enriched for RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD, greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis-acting RNA signals that globally control bacterial transcription.
Assuntos
Aptâmeros de Nucleotídeos/genética , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Técnica de Seleção de Aptâmeros , Terminação da Transcrição Genética , Aptâmeros de Nucleotídeos/metabolismo , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Fases de Leitura Aberta , Ribossomos/metabolismo , Fatores de TempoRESUMO
RNA polymerase-binding RNA aptamers (RAPs) are natural RNA elements that control transcription in cis by directly contacting RNA polymerase. Many RAPs inhibit transcription by inducing Rho-dependent termination in Escherichia coli. Here, we studied the role of inhibitory RAPs (iRAPs) in modulation of antisense transcription (AT) using in silico and in vivo approaches. We revisited the antisense transcriptome in cells with impaired AT regulators (Rho, H-NS and RNaseIII) and searched for the presence of RAPs within antisense RNAs. Many of these RAPs were found at key genomic positions where they terminate AT. By exploring the activity of several RAPs both in a reporter system and in their natural genomic context, we confirmed their significant role in AT regulation. RAPs coordinate Rho activity at the antisense strand and terminate antisense transcripts. In some cases, they stimulated sense expression by alleviating ongoing transcriptional interference. Essentially, our data postulate RAPs as key determinants of Rho-mediated AT regulation in E. coli.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , RNA Antissenso/metabolismo , Transcrição Gênica , Regulação Bacteriana da Expressão GênicaRESUMO
The role of Acinetobacter baumannii ATCC 17978 UmuDC homologs A1S_0636-A1S_0637, A1S_1174-A1S_1173, and A1S_1389 (UmuDAb) in antibiotic resistance acquired through UV-induced mutagenesis was evaluated. Neither the growth rate nor the UV-related survival of any of the three mutants was significantly different from that of the wild-type parental strain. However, all mutants, and especially the umuDAb mutant, were less able to acquire resistance to rifampin and streptomycin through the activities of their error-prone DNA polymerases. Furthermore, in the A. baumannii mutant defective in the umuDAb gene, the spectrum of mutations included a dramatic reduction in the frequency of transition mutations, the mutagenic signature of the DNA polymerase V encoded by umuDC.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Dano ao DNA/genética , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Dano ao DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/genética , Farmacorresistência Bacteriana/genética , Mutagênese/genética , Rifampina/farmacologia , Estreptomicina/farmacologia , Raios UltravioletaRESUMO
The bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes.IMPORTANCE Cholera is an infectious disease that is caused by the bacterium Vibrio cholerae Best known for causing disease in humans, the bacterium is most commonly found in aquatic ecosystems. Hence, humans acquire cholera following ingestion of food or water contaminated with V. cholerae Transition between an aquatic environment and a human host triggers a lifestyle switch that involves reprogramming of V. cholerae gene expression patterns. This process is controlled by a network of transcription factors. In this paper, we show that the cAMP receptor protein (CRP) is a key regulator of V. cholerae gene expression in response to lifestyle changes.