Identification of an Escherichia coli pepA homolog and its involvement in suppression of the algB phenotype in mucoid Pseudomonas aeruginosa

Strains of Pseudomonas aeruginosa isolated from the respiratory tracts of p atients with cystic fibrosis often display a mucoid morphology due to high levels of expression of the exopolysaccharide alginate. The response regula tor AlgB is required for full transcription of the alginate biosynthetic op eron. Repeated attempts to demonstrate a direct interaction between AlgB an d the promoter region of algD, the first gene in the alginate operon, have thus far been unsuccessful. The possibility that AlgB exerts its effect on algD indirectly exists. To identify putative genes under the control of Alg B which affect algD transcription, transposon mutagenesis of nonmucoid algB derivatives of the mucoid strain FRD1 was employed, Of approximately 3,000 transposon mutants screened, 6 were found to display phenotypes which were mucoid relative to the phenotype of the parental algB strain. The phenotyp es of these mutants ranged from being only slightly mucoid to being indisti nguishable from that of the original FRD1 strain, One of the particularly m ucoid transposon mutants was chosen for further study. This strain was foun d to be disrupted in a previously uncharacterized open reading frame with 5 6% amino acid identity to PepA of Escherichia coli. PepA is classified as a leucine aminopeptidase, and homologs have been detected in a number of bac terial, plant, and animal species. This novel gene has been designated phpA (P. aeruginosa homolog of pepA). The insertional inactivation of phpA was found to correlate,vith the mucoid phenotype and an increase in algD transc ription in the algB strain. Expression of phpA from an ectopic chromosomal locus compensated for the transposon insertion in the native phpA gene, res toring algD transcription to levels similar to those observed in the parent al algB strain. While phpA expression did not appear to be under the contro l of AlgB at the transcriptional level, this study demonstrates that loss o f phpA in an algB genetic background had a positive effect on alginate expr ession and, more specifically, on transcription of the alginate biosyntheti c operon.