RESUMEN
Bacterial genes for molybdenum-containing and tungsten-containing enzymes are often differentially regulated depending on the metal availability in the environment. Here, we describe a new family of transcription factors with an unusual DNA-binding domain related to excisionases of bacteriophages. These transcription factors are associated with genes for various molybdate and tungstate-specific transporting systems as well as molybdo/tungsto-enzymes in a wide range of bacterial genomes. We used a combination of computational and experimental techniques to study a member of the TF family, named TaoR (for tungsten-containing aldehyde oxidoreductase regulator). In Desulfovibrio vulgaris Hildenborough, a model bacterium for sulfate reduction studies, TaoR activates expression of aldehyde oxidoreductase aor and represses tungsten-specific ABC-type transporter tupABC genes under tungsten-replete conditions. TaoR binding sites at aor promoter were identified by electrophoretic mobility shift assay and DNase I footprinting. We also reconstructed TaoR regulons in 45 Deltaproteobacteria by comparative genomics approach and predicted target genes for TaoR family members in other Proteobacteria and Firmicutes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/metabolismo , Desulfovibrio vulgaris/genética , Desulfovibrio vulgaris/metabolismo , Molibdeno/metabolismo , Factores de Transcripción/metabolismo , Compuestos de Tungsteno/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Transporte Biológico , Desulfovibrio vulgaris/aislamiento & purificación , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Familia de Multigenes , Regiones Promotoras Genéticas , Regulón , Factores de Transcripción/genéticaRESUMEN
Although the enzymes for dissimilatory sulfate reduction by microbes have been studied, the mechanisms for transcriptional regulation of the encoding genes remain unknown. In a number of bacteria the transcriptional regulator Rex has been shown to play a key role as a repressor of genes producing proteins involved in energy conversion. In the model sulfate-reducing microbe Desulfovibrio vulgaris Hildenborough, the gene DVU_0916 was observed to resemble other known Rex proteins. Therefore, the DVU_0916 protein has been predicted to be a transcriptional repressor of genes encoding proteins that function in the process of sulfate reduction in D. vulgaris Hildenborough. Examination of the deduced DVU_0916 protein identified two domains, one a winged helix DNA-binding domain common for transcription factors, and the other a Rossman fold that could potentially interact with pyridine nucleotides. A deletion of the putative rex gene was made in D. vulgaris Hildenborough, and transcript expression studies of sat, encoding sulfate adenylyl transferase, showed increased levels in the D. vulgaris Hildenborough Rex (RexDvH) mutant relative to the parental strain. The RexDvH-binding site upstream of sat was identified, confirming RexDvH to be a repressor of sat. We established in vitro that the presence of elevated NADH disrupted the interaction between RexDvH and DNA. Examination of the 5' transcriptional start site for the sat mRNA revealed two unique start sites, one for respiring cells that correlated with the RexDvH-binding site and a second for fermenting cells. Collectively, these data support the role of RexDvH as a transcription repressor for sat that senses the redox status of the cell.
Asunto(s)
Proteínas Bacterianas/metabolismo , Desulfovibrio vulgaris/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , NAD/metabolismo , Sulfato Adenililtransferasa/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Desulfovibrio vulgaris/genética , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/fisiología , Sulfato Adenililtransferasa/antagonistas & inhibidores , Sulfato Adenililtransferasa/genéticaRESUMEN
BltR is a MerR family transcriptional factor, experimentally characterized in Bacillus subtilis. It activates transcription of genes encoding multidrug transporter Blt and spermine/spermidine acetyltransferase BltD. Here we studied BltR dependent regulons in 25 bacterial genomes using the comparative genomic approach. The structure of the promoter regions of regulated genes is typical for MerR family activators: the binding sites are located in long spacers between promoter elements. Regulated genes are usually co-localized with regulator genes and are divergently transcribed with them. The studied transcriptional factors regulate the transcription of multidrug transporter and spermine/spermidine acetyltransferase genes. These transporters can be either secondary or ATP-dependent. The phylogenetic analysis demonstrated that their role as multidrug transporters is conserved.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Regulón/genética , Transactivadores/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Sitios de Unión/genética , Biología Computacional , Proteínas de Unión al ADN/genética , Familia de Multigenes/genética , Filogenia , Regiones Promotoras Genéticas/genética , Transactivadores/genéticaRESUMEN
BACKGROUND: Heavy metal resistance (HMR) in Eubacteria is regulated by a variety of systems including transcription factors from the MerR family (COG0789). The HMR systems are characterized by the complex signal structure (strong palindrome within a 19 or 20 bp promoter spacer), and usually consist of transporter and regulator genes. Some HMR regulons also include detoxification systems. The number of sequenced bacterial genomes is constantly increasing and even though HMR resistance regulons of the COG0789 type usually consist of few genes per genome, the computational analysis may contribute to the understanding of the cellular systems of metal detoxification. RESULTS: We studied the mercury (MerR), copper (CueR and HmrR), cadmium (CadR), lead (PbrR), and zinc (ZntR) resistance systems and demonstrated that combining protein sequence analysis and analysis of DNA regulatory signals it was possible to distinguish metal-dependent members of COG0789, assign specificity towards particular metals to uncharacterized loci, and find new genes involved in the metal resistance, in particular, multicopper oxidase and copper chaperones, candidate cytochromes from the copper regulon, new cadmium transporters and, possibly, glutathione-S-transferases. CONCLUSION: Our data indicate that the specificity of the COG0789 systems can be determined combining phylogenetic analysis and identification of DNA regulatory sites. Taking into account signal structure, we can adequately identify genes that are activated using the DNA bending-unbending mechanism. In the case of regulon members that do not reside in single loci, analysis of potential regulatory sites could be crucial for the correct annotation and prediction of the specificity.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Farmacorresistencia Bacteriana/genética , Genómica , Metales Pesados/metabolismo , Metales Pesados/farmacología , Bacterias/clasificación , Bacterias/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Filogenia , Elementos Reguladores de la Transcripción , Regulón , Análisis de Secuencia , Factores de Transcripción/genéticaRESUMEN
Bulbospinal muscular atrophy--a rare disease with X-linked recessive type of inheritance. It is caused by expansion of trinucleotide repetitions in the gene of androgenic receptor (AR). We elaborated a method of DNA-testing with usage of nonradioactive registration of mutant alleles of AR gene. DNA-diagnosis was performed in 16 patients with clinical pattern of bulbospinal muscular atrophy and diagnosis was confirmed in 11 patients. Carriage of mutant alleles was found in 7 women--relatives of the patients. Presymptomatic diagnosis revealed the presence of mutant alleles in 2 boys. Unstability of alleles of mutant AR gene was observed in one family: in sons there was more (upon 5) of CAG-triplets.
