RESUMEN

Choline favors the pathogenesis of Pseudomonas aeruginosa because hemolytic phospholipase C and phosphorylcholine phosphatase (PchP) are synthesized as a consequence of its catabolism. The experiments performed here resulted in the identification of the factors that regulate both the catabolism of choline and the gene coding for PchP. We have also identified and characterized the promoter of the pchP gene, its transcriptional organization and the factors that affect its expression. Deletion analyses reveal that the region between -188 and -68 contains all controlling elements necessary for pchP expression: a hypothetical -12/-24 promoter element, a consensus sequence for the integration host factor (-141/-133), and a palindromic sequence resembling a binding site for a potential enhancer binding protein (-190/-174). Our data also demonstrate that choline catabolism and NtrC (nitrogen regulatory protein) are necessary for the full expression of pchP and is partially dependent on σ(54) factor.

Asunto(s)

Colina/metabolismo , Regulación Bacteriana de la Expresión Génica , Monoéster Fosfórico Hidrolasas/metabolismo , Pseudomonas aeruginosa/metabolismo , ARN Polimerasa Sigma 54/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Expresión Génica , Orden Génico , Genes Bacterianos , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/genética , Fosforilcolina , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/genética , ARN Polimerasa Sigma 54/genética , Eliminación de Secuencia , Factores de Transcripción/genética

RESUMEN

BACKGROUND: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. RESULTS: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. CONCLUSIONS: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon.

Asunto(s)

Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , ARN Polimerasa Sigma 54/metabolismo , Regulón , Xylella/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa Sigma 54/química , ARN Polimerasa Sigma 54/genética , Xylella/química , Xylella/genética

RESUMEN

Expression of the flagellar genes in Rhodobacter sphaeroides is dependent on one of the four sigma-54 factors present in this bacterium and on the enhancer binding proteins (EBPs) FleQ and FleT. These proteins, in contrast to other well-characterized EBPs, carry out activation as a hetero-oligomeric complex. To further characterize the molecular properties of this complex we mapped the binding sites or upstream activation sequences (UASs) of six different flagellar promoters. In most cases the UASs were identified at approximately 100 bp upstream from the promoter. However, the activity of the divergent promoters flhAp-flgAp, which are separated by only 53 bp, is mainly dependent on a UAS located approximately 200 bp downstream from each promoter. Interestingly, a significant amount of activation mediated by the upstream or contralateral UAS was also detected, suggesting that the architecture of this region is important for the correct regulation of these promoters. Sequence analysis of the regions carrying the potential FleQ/FleT binding sites revealed a conserved motif. In vivo footprinting experiments with the motAp promoter allowed us to identify a protected region that overlaps with this motif. These results allow us to propose a consensus sequence that represents the binding site of the FleQ/FleT activating complex.

Asunto(s)

Proteínas Bacterianas/metabolismo , Flagelos/genética , Regiones Promotoras Genéticas , ARN Polimerasa Sigma 54/metabolismo , Rhodobacter sphaeroides/metabolismo , Transactivadores/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Flagelos/química , Flagelos/metabolismo , Datos de Secuencia Molecular , Unión Proteica , ARN Polimerasa Sigma 54/genética , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/genética , Transactivadores/química , Transactivadores/genética , Activación Transcripcional

RESUMEN

Salmonella enterica polymyxin B (PM) resistance is modulated mainly by substitutions of the acyl chains and the phosphate groups on the lipid A moiety of lipopolysaccharide. These modifications are mediated by genes under the control of the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems. In this study, a deletion in the gene encoding the alternative sigma(54) factor, rpoN, was shown to increase PM resistance without affecting protamine sensitivity. The results presented here showed that the increased polymyxin resistance observed in the DeltarpoN mutant occurs through a PmrA/PhoP-independent pathway. Downregulation of one or more genes belonging to the RpoN regulon may provide an additional mechanism of defence against membrane-permeabilizing antimicrobial peptides that helps the pathogen to survive in different environments.

Asunto(s)

Proteínas Bacterianas/genética , Regulación hacia Abajo , Polimixina B/farmacología , ARN Polimerasa Sigma 54/genética , Salmonella typhimurium/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , ARN Polimerasa Sigma 54/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Eliminación de Secuencia

RESUMEN

The intracellular pathogen Brucella abortus has an alternative sigma factor sigma54 (RpoN) highly similar to Sinorhizobium meliloti NtrA. RpoN was described to be required for the transcription of a wide range of genes involved in diverse physiological functions including the regulation of virulence-related factors in both plants and animal pathogens. B. abortus rpoN gene restored the normal growth of an S. meliloti ntrA mutant in minimal media with succinic acid as a sole carbon source as well as the formation of functional nodules in alfalfa, thus revealing that the gene is functional. B. abortus rpoN mutant and B. abortus wild-type strain harboring a multicopy plasmid coding for a wild-type rpoN gene displayed reduced survival under stationary-phase conditions suggesting that expression of RpoN must be tightly regulated. Real-time PCR analysis revealed that B. abortus rpoN expression is downregulated during the stationary phase of growth. This regulation is absent in the rpoN mutant background, indicating that RpoN regulates its own expression. Intracellular multiplication in HeLa or J774 cells, and survival in BALB/c mice of the rpoN mutant, are not affected. However 2weeks postinfection survival of rpoN mutant complemented with a multicopy plasmid containing a wild-type rpoN gene is reduced, thus suggesting that overexpression of rpoN may misregulate the expression of genes involved in this stage of infection.

Asunto(s)

Proteínas Bacterianas/metabolismo , Brucella abortus/metabolismo , Brucelosis/microbiología , ARN Polimerasa Sigma 54/metabolismo , Sinorhizobium meliloti/metabolismo , Animales , Proteínas Bacterianas/genética , Brucella abortus/genética , Brucella abortus/crecimiento & desarrollo , Femenino , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Células HeLa , Humanos , Medicago sativa/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación , ARN Polimerasa Sigma 54/genética , Sinorhizobium meliloti/genética

RESUMEN

The phytopathogen Xylella fastidiosa produces long type IV pili and short type I pili involved in motility and adhesion. In this work, we have investigated the role of sigma factor sigma(54) (RpoN) in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from the non-pathogenic citrus strain J1a12, and microarray analyses of global gene expression comparing the wild type and rpoN mutant strains showed few genes exhibiting differential expression. In particular, gene pilA1 (XF2542), which encodes the structural pilin protein of type IV pili, showed decreased expression in the rpoN mutant, whereas two-fold higher expression of an operon encoding proteins of type I pili was detected, as confirmed by quantitative RT-PCR (qRT-PCR) analysis. The transcriptional start site of pilA1 was determined by primer extension, downstream of a sigma(54)-dependent promoter. Microarray and qRT-PCR data demonstrated that expression of only one of the five pilA paralogues, pilA1, was significantly reduced in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that sigma(54) differentially regulates genes involved in type IV and type I fimbrial biogenesis, and is involved in biofilm formation in X. fastidiosa.

Asunto(s)

Fimbrias Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , ARN Polimerasa Sigma 54/genética , ARN Polimerasa Sigma 54/metabolismo , Xylella/genética , Xylella/metabolismo , Secuencia de Aminoácidos , Adhesión Bacteriana/genética , Secuencia de Bases , Biopelículas/crecimiento & desarrollo , Citrus , Proteínas Fimbrias/genética , Fimbrias Bacterianas/ultraestructura , Eliminación de Gen , Perfilación de la Expresión Génica , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Sitio de Iniciación de la Transcripción , Xylella/crecimiento & desarrollo , Xylella/ultraestructura

RESUMEN

The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a -24/-12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.

Asunto(s)

Proteínas Bacterianas/genética , Herbaspirillum/genética , ARN Polimerasa Sigma 54/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Ensayo de Cambio de Movilidad Electroforética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Herbaspirillum/efectos de los fármacos , Herbaspirillum/metabolismo , Factores de Integración del Huésped/genética , Factores de Integración del Huésped/metabolismo , Operón Lac , Datos de Secuencia Molecular , Oxígeno/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Compuestos de Amonio Cuaternario/farmacología , ARN Polimerasa Sigma 54/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo