Cloning, Expression, and Nucleotide Sequence of the Pseudomonas aeruginosa 142 ohb Genes Coding for Oxygenolytic ortho Dehalogenation of Halobenzoates

Abstract

We have cloned and characterized novel oxygenolyticortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degradingPseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5αF′(pOD22) and DH5αF′(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISPOHBstructural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (β-ISP) and 48,243 Da (α-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized inE. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components provided by the heterologous hosts. ISPOHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. TheohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohbgenes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. Theohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.

Construction and Characterization of Two Recombinant Bacteria That Grow on ortho - and para -Substituted Chlorobiphenyls

“…terminal component, iron-sulfur protein (ISP OHB ), of P. aeruginosa 142 ortho-halobenzoate 1,2-dioxygenase, and the putative transcriptional regulatory gene ohbR (44). Several transformants were selected on L agar-tetracycline and replica plated on K1 agar-tetracycline with 2-CBA as a sole carbon source and with L agar-tetracycline as a control.…”

“…Electrotransformation. Competent cells of E. coli and strain VP44 were prepared (11) and transformed with plasmid DNA as described elsewhere (44). Transformants of VP44 were selected on L agar-tetracycline (10 g/ml) and replica plated on K1 plates with CBAs and CBs as a growth substrate.…”

“…Analyses. CBAs were analyzed by high-pressure liquid chromatography as described elsewhere (44). PCBs were quantified by gas chromatographic analysis using an electron capture detector (ECD) as described elsewhere (34).…”

“…Metabolism by bph pathway enzymes of the congeners targeted at the aerobic step of a two-phase PCB bioremediation scheme produces the respective ortho-, para-, and ortho-plus para-CBAs. Hence, we have cloned oxygenolytic ortho-dechlorination ohb (44) and hydrolytic para-dechlorination fcb (43) genes into PCB-cometabolizing Comamonas testosteroni VP44 (31). The data presented here demonstrate that introducing these dehalogenase genes resulted in highly efficient recombinant pathways, enabling the recombinant variants to grow on, dechlorinate, and completely mineralize ortho-and para-substituted monochlorobiphenyls.…”

“…On the other hand, a number of CBA-degrading bacteria that remove chlorine prior to oxidation of the aromatic ring, funneling the resulting nonchlorinated benzoate or catechol into central metabolic pathways, have been isolated (13,14,35,42,46,50,51). While no dehalogenation of chloropentadiene has been documented so far (9), several CBA dehalogenation genes have been cloned and characterized (18,33,37,38,39,43,44), and these are potentially useful for constructing organisms that would grow on PCBs.…”