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

Abstract

ABSTRACT We have cloned and characterized novel oxygenolytic ortho -dehalogenation ( ohb ) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas 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 ISP OHB structural 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 in E. 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 ISP OHB utilized electron transfer components provided by the heterologous hosts. ISP OHB 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. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli . The ohb genes are flanked by an IS 1396 -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. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.