Novel organization of the genes for phthalate degradation from Burkholderia cepacia DBO1

Abstract

Burkholderia cepacia DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthalate degradation to two separate regions on the cosmid clones. Analysis of the nucleotide sequence of these two regions showed that the genes for phthalate degradation are arranged in at least three transcriptional units. The gene for phthalate dioxygenase reductase (ophA1) is present by itself, while the genes for an inactive transporter (ophD) and 4,5-dihydroxyphthalate decarboxylase (ophC) are linked and the genes for phthalate dioxygenase oxygenase (ophA2) and cis-phthalate dihydrodiol dehydrogenase (ophB) are linked. ophA1 and ophDC are adjacent to each other but are transcribed in opposite directions, while ophA2B is located 4 kb away. The genes for the oxygenase and reductase components of phthalate dioxygenase are located approximately 7 kb away from each other. The gene for the putative phthalate permease contains a frameshift mutation in contrast to genes for other permeases. Strains deleted for ophD are able to transport phthalate into the cell at rates equivalent to that of the wild-type organism, showing that this gene is not required for growth on phthalate.

FIG. 1

Catabolic pathway for the metabolism of phthalate to protocatechuate by B. cepacia DBO1. The genes for each enzyme have been given the designation oph for o-phthalate degradation.

FIG. 2

Complementation tests were done to determine the locations of the genes for phthalate degradation from B. cepacia DBO1. Abbreviations: PCA, protocatechuate; DHP, 4,5-dihydroxyphthalate; DHDP, cis-phthalate dihydrodiol; B, BamHI; E, EcoRI; H, HindIII; M, MluI; N, NspV; S, SphI; St, SstI. Arrows indicate the direction of transcription of the lac promoter on the vector. The gray bars under the consensus map indicate other regions described in the text: the 3.2-kb SphI fragment on the left is pGJZ1305, and the 1.6-kb EcoRI fragment on the right is pGJZ1303.

FIG. 3

Different organization of the genes involved in phthalate degradation from B. cepacia DBO1, P. putida NMH102-2 (66), and C. testosteroni M4-1 (51). Designations: ophA1 and pht2, genes coding for phthalate dioxygenase reductase; ophA2 and pht3, genes coding for phthalate dioxygenase; ophB, pht4, and phtC, genes coding for 4,5-dihydro-4,5-dihydroxyphthalate dehydrogenase; ophC, pht5, and phtD, genes coding for 4,5-dihydroxyphthalate decarboxylase; pht3 and phtR, genes coding for putative phthalate transporter; orf1 and ophD, genes coding for products that show homology to the putative phthalate transporter. Abbreviations: B, BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; N, NspV; S, SacI; Sm, SmaI; Sp, SphI. The dashed lines representing phtC and half of phtR indicate that the sequences for these have not been determined.

FIG. 4

SDS-PAGE of an in vitro transcription-translation of a clone containing orf1 and ophD. Lane 1, pTRC99A control; lane 2, pGJZ1321. The numbers on the left are the running positions of the molecular weight markers (from top to bottom: ovalbumin, carbonic anhydrase, β-lactoglobulin, and lysozyme; in thousands). The arrows on the right indicate the two translated proteins, Orf1 and OphD.

FIG. 5

Uptake of phthalate by B. cepacia DBO1 (wild type) grown on phthalate (■) and p-hydroxybenzoate (▴) and B. cepacia DBO304 (ophD mutant) grown on phthalate (•). Error bars represent the standard deviation from three independent assays.

FIG. 6

Dendrograms showing the relationship of OphA1, OphA2, and OphB to similar proteins. Reference strains include B. cepacia DBO1 (this work), P. putida NMH102-2 (66), Flavobacterium sp. strain ATCC 39823 (50), P. pseudoalcaligenes POB310 (22), Pseudomonas sp. strain ATCC 19151 (11), C. testosteroni T2 (42), Alcaligenes sp. strain BR60 (58), P. pseudoalcaligenes KF707 (31), P. putida F1 (88), Pseudomonas sp. strain D12 (25), and A. calcoaceticus BD413 (61).

FIG. 7

Conserved regions in PDR, PDO, and related proteins. The consensus shown below the aligned sequences is where all sequences are identical. An asterisk indicates those positions where all but one sequence is identical. A period indicates a gap in the aligned sequences. The number before each sequence is the distance from the N-terminal end of the protein.