RESUMEN
The Salmonella regulatory protein SlyA is implicated in virulence, survival in macrophages and resistance to oxidative stress and anti-microbial peptides. SlyA is a member of the MarR family of winged-helix transcription factors. Systematic mutational analysis of the SlyA operator sequence and of the predicted DNA-binding region of SlyA shows that no single base pair in the palindromic SlyA operator sequence is essential for DNA binding, and identifies amino acid residues required to allow SlyA to recognise DNA. Combining the structure-function studies described here and elsewhere with the structures of MarR family proteins suggests a possible model for regulation of SlyA binding to DNA.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN/metabolismo , Salmonella typhimurium/enzimología , Factores de Transcripción/metabolismo , Sitios de Unión , Análisis Mutacional de ADN , Modelos Biológicos , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
Dps proteins play a major role in the protection of bacterial DNA from damage by reactive oxygen species. Previous studies have implicated the extended lysine-containing N-terminal regions of Dps subunits in DNA binding, but this part of the structure has not previously been observed crystallographically. Here the structures of two Dps proteins (DpsA and DpsB) from Lactococcus lactis MG1363 reveal for the first time the presence of an N-terminal alpha helix that extends from the core of the Dps subunit. Consequently, the N-terminal helices are displayed in parallel pairs on the exterior of the dodecameric Dps assemblies. Both DpsA and DpsB bind DNA. Deletion of the DpsA N-terminal helix impaired DNA binding. The N-terminal Lys residues of Escherichia coli Dps have been implicated in DNA binding. Replacement of the lactococcal DpsA Lys residues 9, 15 and 16 by Glu did not inhibit DNA binding. However, DNA binding was inhibited by EDTA, suggesting a role for cations in DNA binding. In contrast to E. coli, Bacillus brevis and Mycobacterium smegmatis Dps:DNA complexes, in which DNA interacts with crystalline Dps phases, L. lactis DNA:Dps complexes appeared as non-crystalline aggregates of protein and DNA in electron micrographs.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Lactococcus lactis/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , ADN/ultraestructura , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Estructura Secundaria de Proteína , Soluciones/químicaRESUMEN
The SlyA protein of Salmonella enterica serovar Typhimurium is a member of the MarR family of transcription regulators and is required for virulence and survival in professional macrophages. Isolated SlyA protein was able to bind a specific DNA target without posttranslational modification. This suggested that SlyA might not be activated by directly sensing an external signal but rather that the intracellular concentration of SlyA is enhanced in appropriate environments through the action of other transcription factors. Analysis of slyA transcription reveals the presence of a promoter region located upstream of the previously recognized SlyA repressed promoter. The newly identified upstream promoter region did not respond to SlyA but was activated by Mg(II) starvation in a PhoP-dependent manner. We present here evidence for a direct link between two transcription factors (PhoP and SlyA) crucial for Salmonella virulence.
Asunto(s)
Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Hemolisinas/genética , Salmonella typhimurium/genética , Factores de Transcripción/genética , Toxinas Bacterianas/biosíntesis , Secuencia de Bases , Medios de Cultivo , Proteínas Hemolisinas/biosíntesis , Magnesio , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/patogenicidad , VirulenciaRESUMEN
The SlyA protein from Salmonella typhimurium is a transcription factor that contributes to virulence. It is shown that a slyA mutant is attenuated in the presence of murine macrophages compared with the parent strain. Moreover, after growth in minimal medium, survival of the slyA mutant was reduced. Altered levels of flagellin (fliC), PagC, IroN, and outer membrane proteins suggest that the slyA mutation affects the surface properties of Salmonella. The isolated SlyA protein is a cofactor-free homodimer that recognizes five sites within the promoter region of the slyA gene. One of these sites contained a near perfect inverted repeat TTAGCAAGCTAA. The other four sites contained related sequences. Occupation of the SlyA sites in the slyA promoter prevented open-complex formation, consistent with the pattern of slyA::lacZ expression parental and slyA mutant strains. By combining the footprinting data with potential SlyA binding sites recovered from a pool of random DNA sequences, a consensus was defined and used to probe the NIH Salmonella unfinished genomes data base. These searches revealed the presence of consensus SlyA sites upstream of omp, ispA, xseB, slyA, and a gene encoding a protein with homology to a hemagglutinin. Accordingly, transcription of an omp::lacZ fusion was reduced in a slyA mutant. Given the difficulties in obtaining a comprehensive picture of intracellular gene expression, the definition of the DNA sequence recognized by a transcription factor (SlyA) that is essential for survival in the macrophage environment should allow a complete regulon of genes with altered expression upon exposure to macrophages to be determined once the S. typhimurium genome annotation is complete.
Asunto(s)
Toxinas Bacterianas/genética , Proteínas Hemolisinas/genética , Proteínas de la Membrana , Regulón , Salmonella typhimurium/genética , Factores de Transcripción , Transcripción Genética , Animales , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , ADN Bacteriano/metabolismo , Electroforesis en Gel de Poliacrilamida , Flagelina/metabolismo , Proteínas Hemolisinas/metabolismo , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Regiones Promotoras GenéticasRESUMEN
The Escherichia coli hlyE gene (also known as clyA or sheA) codes for a novel pore-forming toxin. Previous work has shown that the global transcription factors FNR and CRP positively regulate hlyE expression by binding at the same site. Here in vivo transcription studies reveal that FNR occupies the hlyE promoter more frequently than CRP, providing a mechanism for the moderate upregulation of hlyE expression in response to two distinct environmental signals (oxygen and glucose starvation). It has been reported that H-NS interacts with two large regions of the hlyE promoter (PhlyE), one upstream of the -35 element and one downstream of the -10 element. Here we identify two high-affinity H-NS sites, H-NS I, located at the 3' end of the extended upstream footprint, and H-NS II, located at the 5' end of the extended downstream footprint. It is suggested that these high-affinity sites initiate the progressive formation of higher order complexes, allowing a range of H-NS-mediated regulatory effects at PhlyE. Finally, the identification of a SlyA binding site that overlaps the H-NS I site in PhlyE suggests a mechanism to explain how SlyA overproduction enhances hlyE expression by antagonizing the negative effects of H-NS.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Hemolisinas/metabolismo , Factores de Transcripción , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Sitios de Unión , Medios de Cultivo , Proteína Receptora de AMP Cíclico , Huella de ADN , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hierro-Azufre/metabolismo , Receptores de Superficie Celular/metabolismo , Salmonella/metabolismoRESUMEN
The Salmonella regulatory protein SlyA is implicated in virulence, survival in macrophages and resistance to oxidative stress and anti-microbial peptides. SlyA is a member of the MarR family of winged-helix transcription factors. Systematic mutational analysis of the SlyA operator sequence and of the predicted DNA-binding region of SlyA shows that no single base pair in the palindromic SlyA operator sequence is essential for DNA binding, and identifies amino acid residues required to allow SlyA to recognise DNA. Combining the structure-function studies described here and elsewhere with the structures of MarR family proteins suggests a possible model for regulation of SlyA binding to DNA (AU)
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