RESUMEN
Rhodococcus erythropolis CCM2595 is able to efficiently utilize phenol and other aromatic compounds. We cloned and sequenced its complete gene cluster - catA, catB, catC, catR, pheR, pheA2, pheA1 - involved in the ortho-cleavage pathway of phenol. The activity of the key enzyme of the phenol degradation pathway, two-component phenol hydroxylase, was found to be induced by phenol. When both phenol and succinate were present in the medium, phenol hydroxylase activity decreased substantially. To analyze the regulation of phenol degradation at the transcriptional level, the transcriptional fusions of the divergently oriented promoters PpheA2 and PpheR with the gfpuv reporter gene were constructed. The promoters driving expression of the genes of the pheR-pheA2pheA1 cluster were localized by determining the respective transcriptional start points. Measurements of GFP fluorescence as well as quantitative RT-PCR revealed that expression of the phe genes is induced by phenol at the transcriptional level. The transcription of pheA2A1 and pheR was repressed by succinate, whereas no repression by glucose or glycerol was observed. Activation of the R. erythropolis CCM2595 pheA2 promoter by PheR, an AraC-type transcriptional regulator, was demonstrated by overexpression of the pheR gene. Analysis of the transcriptional regulation of two similar phe clusters from R. jostii RHA1 by various substrates showed that the type of carbon catabolite repression and the temporal transcriptional pattern during cultivation are different in each of the three phe clusters analyzed.
Asunto(s)
Proteínas Bacterianas/genética , Represión Catabólica , Fenol/metabolismo , Rhodococcus/metabolismo , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Regulación Bacteriana de la Expresión Génica , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Familia de Multigenes , Regiones Promotoras Genéticas , Rhodococcus/enzimología , Rhodococcus/genéticaRESUMEN
The effect of nitrogen regulation on the level of transcriptional control has been investigated in a variety of bacteria, such as Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, and Streptomyces coelicolor; however, until now there have been no data for mycobacteria. In this study, we found that the OmpR-type regulator protein GlnR controls nitrogen-dependent transcription regulation in Mycobacterium smegmatis. Based on RNA hybridization experiments with a wild-type strain and a corresponding mutant strain, real-time reverse transcription-PCR analyses, and DNA binding studies using cell extract and purified protein, the glnA (msmeg_4290) gene, which codes for glutamine synthetase, and the amtB (msmeg_2425) and amt1 (msmeg_6259) genes, which encode ammonium permeases, are controlled by GlnR. Furthermore, since glnK (msmeg_2426), encoding a PII-type signal transduction protein, and glnD (msmeg_2427), coding for a putative uridylyltransferase, are in an operon together with amtB, these genes are part of the GlnR regulon as well. The GlnR protein binds specifically to the corresponding promoter sequences and functions as an activator of transcription when cells are subjected to nitrogen starvation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium smegmatis/metabolismo , Nitrógeno/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Datos de Secuencia Molecular , Mutación , Mycobacterium smegmatis/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
We announce the completion of the genome sequence of a phenol derivative-degrading bacterium, Rhodococcus erythropolis strain CCM2595. This bacterium is interesting in the context of bioremediation for its capability to degrade phenol, catechol, resorcinol, hydroxybenzoate, hydroquinone, p-chlorophenol, p-nitrophenol, pyrimidines, and sterols.