Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867.
Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions. One set of the enzymes is constitutive whereas the other is strictly inducible. To date, only the gene encoding the constitutively-expressed gentisate dioxygenase had been cloned and characterized. A mutant strain of P25X, designated G56, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate. The proteome profiles of P. alcaligenes P25X and mutant G56 cells grown in the presence and absence of gentisate were compared after two-dimensional polyacrylamide gel electrophoresis. Eight distinctive protein spots (designated M1-M8) which were observed only in induced cells of strain G56 but absent in noninduced cells were further analyzed by matrix-assisted laser desorption/ionization-time of flight, quadrupole-TOF and N-terminal sequencing. Of the 15 proteins (including seven up-regulated) examined, 13 showed sequence similarities to proteins with assigned functions in other microorganisms. The identification of protein M5 which showed high homology to a gentisate dioxygenase from Ralstonia sp. U2 indicated the putative function of this protein being consistent with the inducible gentisate 1,2-dioxygenase in P. alcaligenes. In addition, the induction of stress proteins and other adaptation phenomena were also observed.
Zhao
Bing
B
Programme in Environmental Microbiology, Department of Microbiology, Faculty of Medicine, National University of Singapore, Block MD4, 5 Science Drive 2, Singapore 117597, Republic of Singapore.
Yeo
Chew Chieng
CC
Lee
Chee Chow
CC
Geng
Anli
A
Chew
Fook Tim
FT
Poh
Chit Laa
CL
D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D019992:Databases as Topic; D015180:Electrophoresis, Gel, Two-Dimensional; D005841:Gentisates / Q000494:pharmacology*; D006838:Hydrocarbons / Q000737:chemistry; D013058:Mass Spectrometry; D008954:Models, Biological; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D044202:Pseudomonas alcaligenes / Q000187:drug effects / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015854:Up-Regulation
D001426:Bacterial Proteins; D005841:Gentisates; D006838:Hydrocarbons; D010455:Peptides; D020543:Proteome