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

Abstract

Strains of Pseudomonas aeruginosa isolated from the respiratory tracts of patients with cystic fibrosis often display a mucoid morphology due to high levels of expression of the exopolysaccharide alginate. The response regulator AlgB is required for full transcription of the alginate biosynthetic operon. Repeated attempts to demonstrate a direct interaction between AlgB and 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 AlgB 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 phenotypes of these mutants ranged from being only slightly mucoid to being indistinguishable from that of the original FRD1 strain. One of the particularly mucoid transposon mutants was chosen for further study. This strain was found to be disrupted in a previously uncharacterized open reading frame with 56% amino acid identity to PepA of Escherichia coli. PepA is classified as a leucine aminopeptidase, and homologs have been detected in a number of bacterial, 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 with the mucoid phenotype and an increase in algD transcription in the algB strain. Expression of phpA from an ectopic chromosomal locus compensated for the transposon insertion in the native phpA gene, restoring algD transcription to levels similar to those observed in the parental algB strain. While phpA expression did not appear to be under the control of AlgB at the transcriptional level, this study demonstrates that loss of phpA in an algB genetic background had a positive effect on alginate expression and, more specifically, on transcription of the alginate biosynthetic operon.