Isolation and Genetic Characterization of Phenol-Degradating Bacterium from a Salt Lake in Inner Mongolia

Salt lake environments are economically and ecologically important issues. Like any other ecosystems in the world, salt lake ecosystems are facing various kinds of pollutions. However, little information is available on the biodegradation of organic pollutants by halophilic microorganisms in such environments. In the present work we isolated a strain of phenol-degradating bacteria from a salt lake in Inner Mongolia. Sequence of 16S rRNA suggested that the strain belong toPropionibacteriumgenus. The strain can grow with phenol as a sole carbon source under 10% (w/v) salt concentration. PCR amplifications with the primers for multicomponent phenol hydroxylase or catechol 1, 2 dioxygenase gene were applied to genome DNA of the strain, suggesting that phenol might be transformed to catechol by multicomponent phenol hydroxylase and further degradated through ortho-ring cleavage pathway. When the primers for catechol 1, 2 dioxygenase were applied to the DNA obtained from the lake water, at least two types of catechol 1, 2 dioxygenase genes sequences were obtained, suggesting the presence of the bacteria with diverse aromatic compound-degradating genes.

An AraC/XylS Family Member at a High Level in a Hierarchy of Regulators for Phenol-Metabolizing Enzymes in Comamonas testosteroni R5

ABSTRACT Comamonas testosteroni strain R5 expresses a higher level of phenol-oxygenating activity than any other bacterial strain so far characterized. The expression of the operon encoding multicomponent phenol hydroxylase (mPH), which is responsible for the phenol-oxygenating activity, is controlled by two transcriptional regulators, PhcS and PhcR, in strain R5. In this study, we identified a third transcriptional regulator for the mPH operon (PhcT) that belongs to the AraC/XylS family. While the disruption of phcT in strain R5 significantly reduced the expression of the mPH operon, it did not eliminate the expression. However, the disruption of phcT in strain R5 increased the expression of phcR. The phenol-oxygenating activity was abolished by the disruption of phcR, indicating that PhcT alone was not sufficient to activate the expression of the mPH operon. The disruption of phcS has been shown in our previous study to confer the ability of strain R5 to express the mPH operon in the absence of the genuine substrate for mPH. PhcT was not involved in the gratuitous expression. Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.

PhcS Represses Gratuitous Expression of Phenol-Metabolizing Enzymes in Comamonas testosteroni R5

ABSTRACT We identified an open reading frame, designatedphcS, downstream of the transcriptional activator gene (phcR) for the expression of multicomponent phenol hydroxylase (mPH) in Comamonas testosteroni R5. The deduced product of phcS was homologous to AphS ofC. testosteroni TA441, which belongs to the GntR family of transcriptional regulators. The transformation of Pseudomonas aeruginosa PAO1c (phenol negative, catechol positive) with pROR502 containingphcR and the mPH genes conferred the ability to grow on phenol, while transformation with pROR504 containingphcS, phcR, and mPH genes did not confer this ability. The disruption of phcS in strain R5 had no effect on its phenol-oxygenating activity in a chemostat culture with phenol. The phenol-oxygenating activity was not expressed in strain R5 grown in a chemostat with acetate. In contrast, the phenol-oxygenating activity in the strain with a knockoutphcS gene when grown in a chemostat with acetate as the limiting growth factor was 66% of that obtained in phenol-grown cells of the strain with a knockout in the phcSgene. The disruption of phcS and/orphcR and the complementation in trans of these defects confirm that PhcS is a trans-acting repressor and that the unfavorable expression of mPH in thephcS knockout cells grown on acetate requires PhcR. These results show that the PhcS protein repressed the gratuitous expression of phenol-metabolizing enzymes in the absence of the genuine substrate and that strain R5 acted by an unknown mechanism in which the PhcS-mediated repression was overcome in the presence of the pathway substrate.