RESUMO
Strain KBC1, an anaerobic bacterium, that dechlorinates tetrachloroethene (PCE) to trichloroethene was isolated. This strain also dechlorinated high concentrations of PCE at a temperature range of 10 to 40 degrees C and showed high oxygen tolerance. Based on the 16S rRNA gene sequence analysis, this microorganism was identified as a species of the genus Desulfitobacterium. Several species of this genus have been reported to be potent ortho-chlorophenol and PCE dechlorinators; however, the gene coding PCE-specific dehalogenase had not been cloned thus far. In this report, we identified a novel PCE reductive dehalogenase (PrdA) gene from the Desulfitobacterium sp. strain KBC1. These prd genes, including putative membrane anchor protein, were classified as novel type of PCE reductive dehalogenase (approximately 40% homology with the general PCE dehalogenase). It was revealed that the two open reading frames had been transcribed as identical mRNA and were induced strictly in the presence of PCE. This transcriptional regulation appeared to be controlled by the transcriptional activator located downstream of prdAB operon. According to the substrate utility of the strain KBC1 and phylogenetic analysis of PrdA, this microorganism may be expected to play the role of a primary dechlorinator of PCE in the environment.
Assuntos
Desulfitobacterium/genética , Oxirredutases/genética , Transcrição Gênica , Motivos de Aminoácidos , Sequência de Aminoácidos , Biodegradação Ambiental , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Desulfitobacterium/classificação , Desulfitobacterium/enzimologia , Desulfitobacterium/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Genes Reguladores/genética , Dados de Sequência Molecular , Oxirredutases/metabolismo , Oxigênio/toxicidade , Filogenia , Sinais Direcionadores de Proteínas , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Temperatura , Tetracloroetileno/metabolismo , Ativação Transcricional , Tricloroetileno/metabolismoRESUMO
Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance.
Assuntos
Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Glicerolfosfato Desidrogenase/genética , Solventes/farmacologia , Cicloexanos/farmacologia , Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Xilenos/farmacologiaRESUMO
We constructed a reporter system to detect a superoxide-generating methyl viologen using SoxRS of Escherichia coli and GFP of Aequorea victoria. E. coli carrying this plasmid exhibited strong fluorescence when grown in the presence of a superoxide-generating reagent methyl viologen. The fluorescence intensity observed in the stationary phase culture of the transformant increased in response to the methyl viologen concentration in a range of 0.01 microM to 10 microM.
Assuntos
Escherichia coli/genética , Paraquat/análise , Superóxidos/química , Escherichia coli/citologia , Proteínas de Escherichia coli/genética , Fluorescência , Genes Reporter , Proteínas de Fluorescência Verde/genética , Indicadores e Reagentes , Métodos , Transativadores/genética , Transdução GenéticaRESUMO
Transcriptional initiation sites of the ostA gene involved in organic solvent sensitivity in Escherichia coli were found by primer extension analysis. Two transcriptional initiation sites were newly identified at -133 and -48 nucleotides from the initiation codon of ostA, but the previously reported sigmaE-dependent one at -227 could not be detected. No heat-inducible expression of ostA was observed by Northern blotting analysis, indicating that the contribution of sigmaE-dependent transcription was very small if any. SigmaD-dependent promoter-like sequences were found just upstream of the newly identified transcriptional initiation sites by computer-aided analysis. Deletion analysis of ostA-lacZ fusions demonstrated that these two promoters contributed almost equally to the constitutive expression of the ostA gene.
