Differential expression of two catechol 1,2-dioxygenases in Burkholderia sp. strain TH2

Abstract

Burkholderia sp. strain TH2, a 2-chlorobenzoate (2CB)-degrading bacterium, metabolizes benzoate (BA) and 2CB via catechol. Two different gene clusters for the catechol ortho-cleavage pathway (cat1 and cat2) were cloned from TH2 and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that while both catechol dioxygenases (CatA1 and CatA2) were produced in BA-grown cells, CatA1 was undetectable when strain TH2 was grown on 2CB or cis,cis-muconate (CCM), an intermediate of catechol degradation. However, production of CatA1 during growth on 2CB or CCM was observed when cat2 genes were disrupted. The difference in the production of CatA1 and CatA2 was apparently due to a difference in inducer recognition by the regulators of the gene clusters. The inducer of CatA1 was found to be BA, not 2CB, by using a 2-halobenzoate dioxygenase gene (cbd) disruptant, which is incapable of transforming (chloro)benzoate. It was also found that CCM or its metabolite acts as an inducer for CatA2. When cat2 genes were disrupted, the growth rate in 2CB culture was reduced while that in BA culture was not. These results suggest that although cat2 genes are not indispensable for growth of TH2 on 2CB, they are advantageous.

FIG. 1.

Schematic representation of the ben-cat1 and cat2 genes. The open arrows indicate locations and directions of transcription of ORFs. IS1599 is indicated by a gray box. The orientation of the open arrow in IS1599 indicates the direction of transcription of the ORF within it. The thick solid line above the gene map indicates the DNA fragment from plasmids pBB311, pBP144, pCASL3, pPCA2, and pCAD that were sequenced in both strands. The open box indicates the omega cassette used for gene disruption. Abbreviations for restriction endonucleases: A, ApaI; B, BamHI; ET22, EcoT22I; EV, EcoRV; H, HindIII; N, NotI; Na, NaeI; Nc, NcoI; P, PstI; S, SalI, Sc, SacI; Sm, SmaI; Sp, SphI; Xb, XbaI; X, XhoI. aa, amino acids.

FIG. 2.

(A) SDS-PAGE analysis of strain TH2, TCD1, and TCD2 proteins induced by different substrates. (B) CatA1 protein detected by Western blot analysis with a polyclonal rabbit antiserum raised against synthetic peptides (see Materials and Methods). The approximate molecular masses of marker proteins (kilodaltons) are indicated on the left. CatA positions are indicated by the arrows.

FIG. 3.

(A) SDS-PAGE analysis of strain TD2 proteins induced by different substrates. (B) CatA1 protein detected by Western blot analysis with a polyclonal rabbit antiserum raised against synthetic peptides (see Materials and Methods). Approximate molecular masses of marker proteins (kilodaltons) are indicated on the left. The CatA1 position is indicated by the arrows.

FIG. 4.

(A) SDS-PAGE analysis of strain TH2, TCD1, and TCD2 proteins induced during growth on CCM. (B) CatA1 protein detected by Western blot analysis with a polyclonal rabbit antiserum raised against synthetic peptides (see Materials and Methods). Approximate molecular masses of marker proteins (kilodaltons) are indicated on the left. CatA positions are indicated by the arrows.

FIG. 5.

Oxidation products of BA resulting from IPTG-induced expression of the xylXYZ genes on the TOL plasmid in E. coli BL21 (DE3). Data were obtained by HPLC analysis at A₂₃₀ of BA subjected to conversion by E. coli BL21(DE3)p16TLXYZ grown on M9 medium containing 2 mM BA and 10 mM glucose. Data shown are for samples taken at the indicated times.

FIG. 6.

Amount of DHB remaining in reaction mixtures of strains TH2, TCD1, and TBD1 provided with BA. DHB was monitored by HPLC analysis. The amount of DHB was determined by measuring the area under the DHB peak.