RESUMEN
The ability of Neisseria gonorrhoeae to reduce nitric oxide (NO) may have important immunomodulatory effects on the host during infection. Therefore, a comprehensive understanding of the regulatory mechanism of the nitric oxide reductase gene (norB) needs to be elucidated. To accomplish this, we analysed the functional regions of the norB upstream region. The promoter contains an extended -10 motif (TGNTACAAT) that is required for high-level expression. Deletion and substitution analysis of the norB upstream region revealed that no sequence upstream of the -10 motif is involved in norB regulation under anaerobic conditions or in the presence of NO. However, replacement of a 29 bp inverted repeat sequence immediately downstream of the extended -10 motif gave high levels of aerobic expression of a norB : : lacZ fusion. Insertional inactivation of gonococcal nsrR, predicted to bind to this inverted repeat sequence, resulted in the loss of norB repression and eliminated NO induction capacity. Single-copy complementation of nsrR in trans restored regulation of both norB transcription and NorB activity by NO. In Escherichia coli, expression of a gonococcal nsrR gene repressed gonococcal norB; induction of norB occurred in the presence of exogenously added NO. NsrR also regulates aniA and dnrN, as well as its own expression. We also determined that Fur regulates norB by a novel indirect activation method, by preventing the binding of a gonococcal ArsR homologue, a second repressor whose putative binding site overlaps the Fur binding site.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Neisseria gonorrhoeae/genética , Oxidorreductasas/genética , Regiones Promotoras Genéticas , Proteínas Represoras/fisiología , Antígenos Bacterianos/biosíntesis , Fusión Artificial Génica , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Genes Reporteros , Prueba de Complementación Genética , Modelos Biológicos , Mutagénesis Insercional , Neisseria gonorrhoeae/enzimología , Mutación Puntual , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Eliminación de Secuencia , Transactivadores/metabolismo , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Iron is both essential for bacterial growth and toxic at higher concentrations; thus, iron homeostasis is tightly regulated. In Neisseria meningitidis the majority of iron-responsive gene regulation is mediated by the ferric uptake regulator protein (Fur), a protein classically defined as a transcriptional repressor. Recently, however, microarray studies have identified a number of genes in N. meningitidis that are iron and Fur activated, demonstrating a new role for Fur as a transcriptional activator. Since Fur has been shown to indirectly activate gene transcription through the repression of small regulatory RNA molecules in other organisms, we hypothesized that a similar mechanism could account for Fur-dependent, iron-activated gene transcription in N. meningitidis. In this study, we used a bioinformatics approach to screen for the presence of Fur-regulated small RNA molecules in N. meningitidis MC58. This screen identified one small RNA, herein named NrrF (for neisserial regulatory RNA responsive to iron [Fe]), which was demonstrated to be both iron responsive and Fur regulated and which has a well-conserved orthologue in N. gonorrhoeae. In addition, this screen identified a number of other likely, novel small RNA transcripts. Lastly, we utilized a new bioinformatics approach to predict regulatory targets of the NrrF small RNA. This analysis led to the identification of the sdhA and sdhC genes, which were subsequently demonstrated to be under NrrF regulation in an nrrF mutant. This study is the first report of small RNAs in N. meningitidis and the first to use a bioinformatics approach to identify, a priori, regulatory targets of a small RNA.
Asunto(s)
Proteínas Bacterianas/genética , Hierro/metabolismo , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , ARN Bacteriano/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Flavoproteínas/genética , Regulación Bacteriana de la Expresión Génica , Homeostasis/fisiología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/fisiología , ARN Bacteriano/química , Proteínas Represoras/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Our whole-genome microarray studies of Neisseria meningitidis MC58 previously identified a set of 153 genes whose transcription was activated during growth in iron. In this study, Fur-mediated regulation of the iron-activated nspA gene was confirmed, whereas iron-activated regulation of the secY gene was demonstrated to be Fur independent. Analysis of the Fur binding sequences in the nspA gene and an additional iron-activated and Fur-regulated gene identified a hexameric (G/T)ATAAT unit in the operator regions of these genes similar to that observed in Fur- and iron-repressed genes. These studies indicate that the expression of the iron-activated nspA and secY genes in N. meningitidis occur by Fur-dependent and -independent mechanisms, respectively.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Neisseria meningitidis Serogrupo B/genética , Proteínas Represoras/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Huella de ADN , Ensayo de Cambio de Movilidad Electroforética , Perfilación de la Expresión Génica , Genes Bacterianos/genética , Hierro/metabolismo , Neisseria meningitidis Serogrupo B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Microbial flavohaemoglobins are proteins with homology to haemoglobins from higher organisms, but clearly linked to nitric oxide (NO) metabolism by bacteria and yeast. hmp mutant strains of several bacteria are hypersensitive to NO and related compounds and hmp genes are up-regulated by the presence of NO. The regulatory mechanisms involved in hmp induction by NO and the superoxide-generating agent, methyl viologen (paraquat; PQ), are complex, but progressively being resolved. Here we show for the first time that, in Salmonella enterica serovar Typhimurium, hmp transcription is increased on exposure to PQ and demonstrate that RamA, a homologue of MarA is responsible for most of the hmp paraquat regulation. In addition we demonstrate NO-dependent elevation of Salmonella hmp transcription and Hmp accumulation. In both Escherichia coli and Salmonella modest transcriptional repression of hmp is exerted by the iron responsive transcriptional repressor Fur. Finally, in contrast to previous reports, we show that in E. coli and Salmonella, hmp induction by both paraquat and sodium nitroprusside is further elevated in a fur mutant background, indicating that additional regulators are implicated in this control process.