OSMOPROTECTANT-DEPENDENT EXPRESSION OF PLCH, ENCODING THE HEMOLYTIC PHOSPHOLIPASE-C, IS SUBJECT TO NOVEL CATABOLITE REPRESSION CONTROL IN PSEUDOMONAS-AERUGINOSA PAO1

Expression of the hemolytic phospholipase C (PlcH) of Pseudomonas aeru ginosa is induced under phosphate starvation conditions or in the pres ence of the osmoprotectants choline and glycine betaine. Because choli ne and glycine betaine may serve as carbon and energy sources in addit ion to conferring osmoprotection to P. aeruginosa, it seemed possible that induction of plcH is subject to catabolite repression control (CR C) by tricarboxylic cycle intermediates such as succinate. Total phosp holipase (PLC) activity in osmoprotectant-induced cultures of P. aerug inosa PAO1 supplemented with 20 mM succinate was three- to fourfold lo wer than the levels in cultures supplemented with the non-catabolite r epressive substrate lactate. Analyses of osmoprotectant-dependent plcH expression in a derivative of strain PAO1 containing a plcH::lacZ ope ron fusion showed that (i) succinate prevented induction of plcH expre ssion by osmoprotectants; and (ii) addition of succinate reduced or sh ut down further expression of plcH in osmoprotectant-induced bacteria, while cultures supplemented with lactate had little or no change in p lcH expression. RNase protection analysis confirmed that repression of plcH occurs at the transcriptional level. However, a P. aeruginosa mu tant decoupled in CRC exhibited a phenotype similar to that of the wil d-type strain (PAO1) with respect to succinate-dependent repression of plcH expression. Osmoprotectant-induced total PLC activities, levels of expression of plcH measured with the same plcH::lacZ fusion, and le vels of plcH transcription in a CRC deficient strain reflected those s een in strain PAO1. This indicates that CRC of plcH functions by a dis tinct mechanism which differs from that regulating the glucose or mann itol catabolic pathway. A strain carrying a mutation in vfr, which enc odes the Escherichia coli Crp homolog in P. aeruginosa, still exhibite d a wild-type phenotype with respect to osmoprotectant-dependent expre ssion and CRC of plcH. These data indicate that there is a novel CRC s ystem that regulates the expression of plcH in P. aeruginosa.