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
Wj. Hickey et G. Sabat, 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, APPL ENVIR, 67(12), 2001, pp. 5648-5655
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.