SEQUENCES AND EXPRESSION OF PYRUVATE-DEHYDROGENASE GENES FROM PSEUDOMONAS-AERUGINOSA

A mutant of Pseudomonas aeruginosa, OT2100, which appeared to be defec tive in the production of the fluorescent yellow-green siderophore pyo verdine had been isolated previously following transposon mutagenesis (T. R. Merriman and I. L. Lament, Gene 126:17-23, 1993). DNA from eith er side of the transposon insertion site was cloned, and the sequence was determined. The mutated gene had strong identity,vith the dihydrol ipoamide acetyltransferase (E2) components of pyruvate dehydrogenase ( PDH) from other bacterial species. Enzyme assays revealed that the mut ant was defective in the E2 subunit of PDH, preventing assembly of a f unctional complex. PDH activity in OT2100 cell extracts was restored w hen extract from an El mutant was added. On the basis of this evidence , OT2100 was identified as an aceB or E2 mutant. A second gene, aceA, which is likely to encode the El component of PDH, was identified upst ream from aceB. Transcriptional analysis revealed that aceA and aceB a re expressed as a 5-kb polycistronic transcript from a promoter upstre am of aceA. An intergenic region of 146 bp was located between aceA an d aceB, and a 2-kb aceB transcript that originated from a promoter in the intergenic region was identified. DNA fragments upstream of aceA a nd aceB were shown to have promoter activities in P. aeruginosa, altho ugh only the aceA promoter was active in Escherichia coli. It is likel y that the apparent pyoverdine-deficient phenotype of mutant OT2100 is a consequence of acidification of the growth medium due to accumulati on of pyruvic acid in the absence of functional PDH.