RESUMO
Small non-coding RNAs (sRNAs) are well-established post-transcriptional regulators of gene expression in bacteria that respond to a variety of environmental stimuli. They usually act by base-pairing with their target mRNAs, which is commonly facilitated by the RNA chaperone Hfq. In this study we initiated the analysis of the sRNA FnrS of Neisseria gonorrhoeae, which is induced under anaerobic conditions. We identified four putative FnrS target genes using bioinformatics approaches and validated these target genes using translational reporter gene fusions in both Escherichia coli and N. gonorrhoeae, thereby demonstrating their downregulation by direct base-pairing between the respective mRNA and FnrS. We demonstrate deregulation of target mRNAs upon deletion of fnrS and provide evidence that the isc gene cluster required for iron-sulfur cluster biosynthesis, which harbours iscS, which is a direct target of FnrS, is coordinately downregulated by the sRNA. By mutational analysis we show that, surprisingly, three distinct regions of FnrS are employed for interaction with different target genes.
Assuntos
Regulação Bacteriana da Expressão Gênica , Neisseria gonorrhoeae/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Família Multigênica , Neisseria gonorrhoeae/metabolismo , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genéticaRESUMO
Neisseria gonorrhoeae is the causative agent of gonorrhoea, the second most common bacterial sexually transmitted disease. Riboregulation mediated by small regulatory RNAs (sRNAs) is increasingly recognized as an important means of gene expression control in this human-restricted pathogen. sRNAs act at the post-transcriptional level by base-pairing with their target mRNAs which affects translation initiation and/or mRNA stability. In this study we initiated the characterization of a pair of highly conserved sRNAs of N. gonorrhoeae which exhibit redundant functions in the control of a common set of target genes. The identified targets of the sibling sRNAs NgncR_162 and NgncR_163 participate in basic metabolic processes including the methylcitrate and citrate cycle, aa uptake and degradation, and also in transcription regulation. Our data indicate that the sibling sRNAs control their targets via direct base-pairing between the same single-stranded domain(s) of the sRNA and the ribosome binding site in the 5'-untranslated region of the mRNA.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Neisseria gonorrhoeae/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Regiões 5' não Traduzidas/genética , Sítios de Ligação , Redes Reguladoras de Genes , Genes Bacterianos/genética , Redes e Vias Metabólicas/genética , Mutação , Neisseria gonorrhoeae/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/classificação , Sequências Reguladoras de Ácido Ribonucleico/genéticaRESUMO
The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes.