MOLECULAR CHARACTERIZATION OF MUTANTS AFFECTED IN THE OSMOPROTECTANT-DEPENDENT INDUCTION OF PHOSPHOLIPASE-C IN PSEUDOMONAS-AERUGINOSA PAO1

Production of the two phospholipases C (PLCs) in Pseudomonas aeruginos a PAO1 is induced under conditions of phosphate limitation, or by the osmoprotectants choline or glycine betaine. Tn5 mutagenesis was perfor med on strain PAO1 to isolate mutants deficient in choline-dependent i nduction of PLC. Two mutants, Tn5T1 and Tn5G19, were identified which produce decreased levels of PLC in phosphate-replete media supplemente d with choline. A total of 136 and 496 bp of flanking DNA from Tn5G19 and Tn5T1 was cloned by an inverse polymerase chain reaction (PCR) and sequenced. The DNA flanking the Tn5T1 insertion contains an open read ing frame predicted to encode a peptide that is approx. 60% identical to the N-terminus of a previously identified protein (P35) of unknown function from Escherichia coli. The P35 gene, which is located in the nusA-infB operon in E. coli, was designated orp (osmoprotectant regula tor of PLC). Haemolytic titres, total PlcH protein and beta-galactosid ase activity expressed from a chromosomally inserted plcH-lacZ operon fusion were reduced in strain Tn5T1 in comparison with the parental st rain (PAO1) carrying the same fusion. However, this mutant expressed s everal-fold higher levels of plcH message than strain PAO1 in the pres ence of choline, while the phosphate-starvation-dependent transcript o f plcH could not be detected in this mutant. The defects in Tn5T1 are complemented by a DNA fragment, isolated from a genomic library of PAO 1, that carries the orp gene. The deduced amino acid sequence of the D NA fragment cloned from Tn5G19 exhibits 84% identity with the betB gen e product of E. coli that has betaine aldehyde dehydrogenase activity. This enzyme catalyses the conversion of betaine aldehyde to glycine-b etaine. Unlike the parental strain, the Tn5G19 mutant could not utiliz e choline as a sole carbon, nitrogen and energy source, and it was def icient in betaine aldehyde dehydrogenase activity. Also, consistent wi th a disruption of betB in Tn5G19, choline inhibited growth of this st rain in media containing 0.7 M NaCl, while glycine-betaine restores gr owth to wild-type levels. The defects in Tn5G19 are complemented by a DNA fragment from PAO1 that carries the betB gene. The orp gene is loc ated between 0.6 to 6.6 min while betB is located between 10.5 to 12.5 min on the chromosome of PAO1.