Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in theecologically important marine Roseobacter lineage

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the alpha -proteobacteria was investigated, Initi al screens suggested that isolates in the Roseobacter lineage can degrade a romatic compounds via the beta -ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates wer e screened for the presence of protocatechuate 3,4-dioxgenase, a key enzyme in the beta -ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, b enzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuat e, and coumarate), Four of the Roseobacter group isolates had inducible pro tocatechuate 3,4-dioxygenase activity in cell extracts when grown on p-hydr oxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates, Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coil to yield dioxygenase activity. Additional genes involved in the protocatechuate bra nch of the beta -ketoadipate pathway (pcaC, pcaQ, and pobA) were found to c luster with pcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the two Roseobacter group iso lates than between genes from either Roseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH succes sfully amplified a fragment of pcaH from two additional Roseobacter group i solates, and Southern hybridization indicated the presence of pcaH in the r emaining two isolates, This evidence of protocatechuate 3,4-dioxygenase and the beta -ketoadipate pathway was found in all six Roseobacter isolates, s uggesting widespread abilities to degrade aromatic compounds in this marine lineage.