Cloning and sequencing of a phenol hydroxylase gene of Pseudomonas pseudoalcaligenes strain MH1: a bacterium able to mineralize various aromatic compounds.

Abstract

The phenol-degrading strain Pseudomonas pseudoalcaligenes MH1, identified by the rRNA approach, was isolated from wastewater enrichment culture. It utilized phenol up to 1.5 g/L as the sole source of carbon and energy. In addition, cresols (o-, m-, p-), 4-hydroxybenzoic acid, syringic acid, and vanillic acid were metabolized as sole substrates by phenol-grown cells of strain MH1. Using primers, designed on the basis of the sequence of the dmp operon of P. putida strain CF600, a gene encoding phenol hydroxylase, which catalyzes the hydroxylation of phenol to catechol, was detected in strain MH1. The whole phenol hydroxylase operon of strain MH1 was amplified in a polymerase chain reaction fragment of 5.207 kb that was cloned and sequenced. The total sequence revealed a cluster of six ATG starting open reading frames (ORFs). Analysis of the regulatory signals showed a putative promoter region, 40 bp upstream from the transcriptional start of ORF1, which have a strong homology to a set of positively controlled promoters. Comparison of the MH1 phenol hydroxylase gene sequence with those of other Pseudomonas strains revealed higher homology except in the 5' region. Thus, the deduced amino acid sequence of the first subunit of phenol hydroxylase of P. pseudoalcaligenes strain MH1 exhibited a difference at the N-terminal region (the first 10 amino acids) compared with that of known phenol hydroxylase of Pseudomonas strains.