Cloning, expression, and nucleotide sequence of the Pseudomonas aeruginosa142 ohb genes coding for oxygenolytic ortho dehalogenation of halobenzoates

We have cloned and characterized novel oxygenolytic ortho-dehalogenation (o hb) genes from X-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA )-degrading Pseudomonas aeruginosa 142, Among 3,700 Escherichia coil recomb inants, two clones, DH5 alpha F'(pOD22) and: DH5 alpha F'(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclon e 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 ISPOHB structural genes ohbA a nd ohbB, which encode polypeptides with molecular masses of 20,253 Da (beta -ISP) and 48,243 Da (alpha-ISP), respectively, were identified; these prote ins 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-diox ygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components pro vided by the heterologous hosts; ISPOHB formed a new phylogenetic cluster t hat includes aromatic oxygenases featuring atypical structural-functional o rganization and is distant from the other members of the family of primary aromatic oxygenases, A putative IclR-type regulatory gene (ohbR) was locate d upstream of the ohbAB genes, An open reading frame (ohbC) of unknown func tion 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 ac cord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da tran sposase 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 reg ion is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 a nd 5984 to 5997.