Genes involved in anaerobic metabolism of phenol in the bacterium Thauera aromatica

Genes involved in the anaerobic metabolism of phenol in the denitrifying ba cterium Thauera aromatica have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphat e by an unknown phosphoryl donor ("phenylphosphate synthase") and subsequen t carboxylation of phenylphosphate to 4-hydroxybenzoate under release of ph osphate ("phenylphosphate carboxylase"). Both enzyme activities are strictl y phenol induced. Two-dimensional gel electrophoresis allowed identificatio n of several phenol-induced proteins. Based on N-terminal and internal amin o acid sequences of such proteins, degenerate oligonucleotides were designe d to identify the corresponding genes. A chromosomal DNA segment of about 1 4 kbp was sequenced which contained 10 genes transcribed in the same direct ion. These are organized in tno adjacent gene clusters and include the gene s coding for five identified phenol-induced proteins. Comparison with seque nces in the databases revealed the following similarities: the gene product s of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases, We propose that these ORFs are components of the phenylphosphate synthase system. Three ORF s showed similarity to the ubiD gene product, 3-octaprenyl-3-hydroxybenzoat e carboxy lyase; UbiD catalyzes the decarboxylation of a 3-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the ubiY ge ne product, an isoenzyme of UbiD, We propose that (some of) these four prot eins are involved in the carboxylation of phenylphosphate. A 700-bp PCR pro duct derived from one of these ORFs cross-hybridized with DNA from differen t Thauera and Azoarcus strains, even from those which have not been reporte d to grow with phenol. One ORF showed similarity to the mutT gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opp osite direction codes for a protein highly similar to the DmpR regulatory p rotein of Pseudomonas putida, DmpR controls transcription of the genes of a erobic phenol metabolism, suggesting a similar regulation of anaerobic phen ol metabolism by the putative regulator.