Genetic analysis of phenylalanine and tyrosine degradation by Burkholderia cepacia DBO1

The metabolism of the two aromatic amino acids phenylalanine and tyrosine are related in most bacterial species. Phenylalanine and tyrosine are synthesized from chorismate through phenylpyruvate and p-hydroxyphenylpyruvate respectively. The canonical pathway for the catabolism of phenylalanine proceeds through initial hydroxylation of phenylalanine to tyrosine. However, the catabolism of phenylalanine is not well understood in some microorganisms and recently one alternate pathway for phenylalanine degradation involving phenylacetate as an intermediate was described. Burkholderia cepacia DBO1 utilizes separate catabolic pathways for the utilization of phenylalanine and tyrosine. In order to clarify phenylalanine degradation in B. cepacia DBO1 we made extensive use of plasposon-based random insertional mutagenesis to generate mutants in the catabolic pathways of interest. Four mutant strains were obtained that could not grow on tyrosine and ten strains were obtained that that could not grow on phenylalanine. Interestingly, all the tyrosine catabolism mutant strains grew on phenylalanine, indicating that phenylalanine is not degraded through tyrosine. Sequence analysis of the phenylalanine mutant strains showed that the transposon insertions fall into three groups: an operon encoding phenylacetate degradation, a gene encoding a regulator, and a gene encoding pyruvate oxidoreductase. These data, combined with HPLC metabolite accumulation studies indicate that degradation of phenylalanine proceeds through phenylacetate. The enzymatic function of the proteins predicted to be involved in the catabolic pathway were examined through suitable biochemical techniques (enzymes assays and metabolite identification). RT-PCR experiments were used to examine the regulation of the genes involved in phenylalanine degradation in B. cepacia DBO1.