Integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry and molecular cloning for the identification and functional characterization of mobile ortho-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2

Protein mass spectrometry and molecular cloning techniques were used to ide ntify and characterize mobile. o-halobenzoate oxygenase genes in Pseudomona s aeruginosa strain JB2 and Pseudomonas huttiensis strain D1. Proteins indu ced in strains JB2 and DI by growth on 2-chlorobenzoate (2-CBa) were extrac ted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by matrix-assisted laser desorption ionization-time of flight mas s spectrometry. Two bands gave significant matches to OhbB and OhbA, which have been reported to be the a and P subunits, respectively, of an ortho-1, 2-halobenzoate dioxygenase of P. aeruginosa strain 142 (T. V. Tsoi, E. G. P lotnikova, J. R. Cole, W. F. Guerin, N1. Bagdasarian, and J. N1. Ticdje, Ap pl. Environ. Microbiol. 65:2151-2162, 1999). PCR and Southern hybridization experiments confirmed that ohbAB were present in strain JB2 and were trans ferred from strain JB2 to strain Dl. While the sequences of ohbA from strai ns JB2, D1, and 142 were identical, the sequences of AM from strains JB2 an d DI were identical to each other but differed slightly from that of strain 142. PCR analyses and Southern hybridization analyses indicated that ohbAB were conserved in strains JB2 and DI and in strain 142 but that the region s adjoining these genes were divergent. Expression of ohbAB in Escherichia coli resulted in conversion of o-chlorobenzoates to the corresponding (chlo ro)catechols with the following apparent affinity: 2-CBa approximate to 2,5 -dichlorobenzoate > 2,3,5-trichlorobenzoate > 2,4-dichlorobenzoate. The act ivity of OhbAB(JB2) appeared to differ from that reported for Ohb-AB(142) p rimarily in that a chlorine in the para position posed a greater impediment to catalysis with the former. Hybridization analysis of spontaneous 2-CBa- mutants of strains JB2 and D1 verified that ohbAB were lost along with the genes, suggesting that all of the genes may be contained in the same mobil e element. Strains JB2 and 142 originated from California and Russia, respe ctively. Thus, ohbAB and/or the mobile element on which they are carried ma y have a global distribution.