Osmoprotectant-dependent expression of plcH, encoding the hemolytic phospholipase C, is subject to novel catabolite repression control in Pseudomonas aeruginosa PAO1

Abstract

Expression of the hemolytic phospholipase C (PlcH) of Pseudomonas aeruginosa is induced under phosphate starvation conditions or in the presence of the osmoprotectants choline and glycine betaine. Because choline and glycine betaine may serve as carbon and energy sources in addition to conferring osmoprotection to P. aeruginosa, it seemed possible that induction of plcH is subject to catabolite repression control (CRC) by tricarboxylic cycle intermediates such as succinate. Total phospholipase (PLC) activity in osmoprotectant-induced cultures of P. aeruginosa PAO1 supplemented with 20 mM succinate was three- to fourfold lower than the levels in cultures supplemented with the non-catabolite-repressive substrate lactate. Analyses of osmoprotectant-dependent plcH expression in a derivative of strain PAO1 containing a plcH::lacZ operon fusion showed that (i) succinate prevented induction of plcH expression by osmoprotectants; and (ii) addition of succinate reduced or shut down further expression of plcH in osmoprotectant-induced bacteria, while cultures supplemented with lactate had little or no change in plcH expression. RNase protection analysis confirmed that repression of plcH occurs at the transcriptional level. However, a P. aeruginosa mutant decoupled in CRC exhibited a phenotype similar to that of the wild-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 levels of plcH transcription in a CRC-deficient strain reflected those seen in strain PAO1. This indicates that CRC of plcH functions by a distinct mechanism which differs from that regulating the glucose or mannitol catabolic pathway. A strain carrying a mutation in vfr, which encodes the Escherichia coli Crp homolog in P. aeruginosa, still exhibited a wild-type phenotype with respect to osmoprotectant-dependent expression and CRC of plcH. These data indicate that there is a novel CRC system that regulates the expression of plcH in P. aeruginosa.