RESUMEN
The mechanisms responsible for activation of the MtrAB two-component regulatory signal transduction system, which includes sensor kinase MtrB and response regulator MtrA, are unknown. Here, we show that an MtrB-GFP fusion protein localized to the cell membrane, the septa, and the poles in Mycobacterium tuberculosis and Mycobacterium smegmatis. This localization was independent of MtrB phosphorylation status but dependent upon the assembly of FtsZ, the initiator of cell division. The M. smegmatis mtrB mutant was filamentous, defective for cell division, and contained lysozyme-sensitive cell walls. The mtrB phenotype was complemented by either production of MtrB protein competent for phosphorylation or overproduction of MtrA(Y102C) and MtrA(D13A) mutant proteins exhibiting altered phosphorylation potential, indicating that either MtrB phosphorylation or MtrB independent expression of MtrA regulon genes, including those involved in cell wall processing, are necessary for regulated cell division. In partial support of this observation, we found that the essential cell wall hydrolase ripA is an MtrA target and that the expression of bona fide MtrA targets ripA, fbpB, and dnaA were compromised in the mtrB mutant and partially rescued upon MtrA(Y102C) and MtrA(D13A) overproduction. MtrB septal assembly was compromised upon FtsZ depletion and exposure of cells to mitomycin C, a DNA damaging agent, which interferes with FtsZ ring assembly. Expression of MtrA targets was also compromised under the above conditions, indicating that MtrB septal localization and MtrA regulon expression are linked. We propose that MtrB septal association is a necessary feature of MtrB activation that promotes MtrA phosphorylation and MtrA regulon expression.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/genética , Regulón/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Western Blotting , División Celular , Membrana Celular/metabolismo , Pared Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente , Mutación , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Fosforilación , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Two component regulatory systems are used widely by bacteria to coordinate changes in global gene expression profiles in response to environmental signals. The SenX3-RegX3 two component system of Mycobacterium tuberculosis has previously been shown to play a role in virulence and phosphate-responsive control of gene expression. We demonstrate that expression of SenX3-RegX3 is controlled in response to growth conditions, although the absolute changes are small. Global gene expression profiling of a RegX3 deletion strain and wild-type strain in different culture conditions (static, microaerobic, anaerobic), as well as in an over-expressing strain identified a number of genes with changed expression patterns. Among those were genes previously identified as differentially regulated in aerobic culture, including ald (encoding alanine dehydrogenase) cyd,encoding a subunit of the cytochrome D ubiquinol oxidase, and gltA1, encoding a citrate synthase. Promoter activity in the upstream regions of both cydB and gltA1 was altered in the RegX3 deletion strain. DNA-binding assays confirmed that RegX3 binds to the promoter regions of ald, cydB and gltA1 in a phosphorylation-dependent manner. Taken together these data suggest a direct role for the SenX-RegX3 system in modulating expression of aerobic respiration, in addition to its role during phosphate limitation.
Asunto(s)
Proteínas Bacterianas/fisiología , Genes Bacterianos , Mycobacterium tuberculosis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Perfilación de la Expresión Génica , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Operón , Fosforilación , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , VirulenciaRESUMEN
We have previously shown that expression of chiZ (Rv2719c), encoding a cell wall hydrolase, is upregulated in response to DNA damaging agents and exposure to cephalexin. Furthermore, increased levels of ChiZ lead to decreased viability, loss of membrane integrity and defects in FtsZ-GFP localization and cell division. We now show that ChiZ N'-terminal 110 amino acid region, containing the cell wall hydrolase activity, is sufficient to modulate FtsZ-GFP localization. Further, we found that FtsZ-GFP rings are stabilized in a chiZ deletion strain indicating that ChiZ activity regulates FtsZ assembly. Overexpression of ftsZ did not reverse the reduction in viability caused by overproduction of ChiZ indicating that ChiZ neither interacts with nor directly influences FtsZ assembly. Bacterial two-hybrid assays revealed that ChiZ interacts with FtsI and FtsQ, two other septasomal proteins, but not with FtsZ. Finally, we show that ChiZ is not required for virulence of Mycobacterium tuberculosis in murine macrophages and mice. Our data suggest that optimal levels and activity of the cell wall hydrolase ChiZ are required for regulated cell division in mycobacteria.