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

Abstract

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the alpha-proteobacteria was investigated. Initial screens suggested that isolates in the Roseobacter lineage can degrade aromatic compounds via the beta-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, 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, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3, 4-dioxygenase activity in cell extracts when grown on p-hydroxybenzoate. 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 coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the beta-ketoadipate pathway (pcaC, pcaQ, and pobA) were found to cluster with pcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the two Roseobacter group isolates than between genes from either Roseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additional Roseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3, 4-dioxygenase and the beta-ketoadipate pathway was found in all six Roseobacter isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.

FIG. 1

Schematic of the protocatechuate branch of the β-ketoadipate pathway in some prokaryotes (20). Gene designations are shown in italics. CoA, coenzyme A.

FIG. 2

Restriction map of the chromosomal pca regions from S. stellata E-37 (A) and Y3F (B). The locations of genes and their transcriptional directions are shown relative to selected restriction endonuclease recognition sites. Horizontal lines indicate the DNA regions contained on recombinant plasmids, whose designations are shown above the corresponding line.

FIG. 3

Protein sequence alignments of PcaH. Aligned residues that are identical or similar are shown with black backgrounds or boxed, respectively. Residues presumed to be involved in substrate specificity are indicated by a dot, and those demonstrated to be involved in catalysis and Fe²⁺ binding in P. putida are indicated by an asterisk (31). Gray regions underscored with arrows indicate residues used for the design of the P34OID degenerate PCR primers, with identical and similar residues shaded gray and boxed, respectively. Alignments are shown for P. putida ATCC 23975 (L14836), Acinetobacter sp. strain ADP1 (M33798), B. cepacia DB01 (M30799), R. opacus 1CP (AF003947), and Streptomyces sp. strain 2056 (AF019386). Alignments were conducted using the PILEUP program of the Genetics Computer Group package.

FIG. 4

Phylogenetic tree of PcaGH protein sequences. The tree is based on the deduced amino acids encoded by the pcaGH genes and is unrooted, with CatA from Acinetobacter sp. strain ADP1 (Z36909) as the outgroup. Bootstrap values greater than 50% are indicated at branch nodes. The scale bar indicates the amount of genetic change measured as the number of amino acid substitutions per site.

FIG. 5

Organization of gene clusters for protocatechuate metabolism in selected bacteria. Arrows indicate the direction of transcription. Bold double lines indicate genes separated by <10 kbp. The information was compiled from the following sources: Acinetobacter strain ADP1 (12), P. putida (7, 9, 20, 39), Pseudomonas sp. strain HR199 (32), R. opacus 1CP (7), A. tumefaciens A348 (39), and S. stellata E-37 and isolate Y3F (this study).