AlgR functions in algC expression and virulence in Pseudomonas syringae pv. syringae

Abstract

Pseudomonas syringae pv. syringae strain FF5 is a phytopathogen associated with a rapid dieback on ornamental pear trees. P. syringae and the human pathogen Pseudomonas aeruginosa produce the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. In P. aeruginosa, the response regulator AlgR (AlgR1) is required for transcription of algC and algD, which encode key enzymes in the alginate biosynthetic pathway. In P. syringae FF5, however, algR is not required for the activation of algD. Interestingly, algR mutants of P. syringae remain nonmucoid, indicating an undefined role for this response regulator in alginate biosynthesis. In the current study, the algC promoter region was cloned from P. syringae pv. syringae strain FF5, and sequence analysis of the algC promoter indicated the presence of potential binding sites for AlgR and sigma(54), the alternative sigma factor encoded by rpoN. The algC promoter from P. syringae FF5 (PsalgC) was cloned upstream of a promoterless glucuronidase gene (uidA), and the PsalgC-uidA transcriptional fusion was used to monitor algC expression in strains FF5.32 (algR mutant of P. syringae FF5) and PG4180.K2 (rpoN mutant of P. syringae pv. glycinea PG4180). Expression of the PsalgC-uidA fusion was fourfold lower in both the algR and rpoN mutants as compared to respective wild-type strains, indicating that both AlgR and sigma(54) are required for full activation of algC transcription in P. syringae pv. syringae. AlgR from P. syringae was successfully overproduced in Escherichia coli as a C-terminal translational fusion to the maltose-binding protein (MBP). Gel shift experiments indicated that MBP-AlgR binds strongly to the algC promoter region. Biological assays demonstrated that the algR mutant was significantly impaired in both pathogenicity and epiphytic fitness as compared to the wild-type strain. These results, along with the gene expression studies, indicate that AlgR has a positive role in the activation of algC in P. syringae and contributes to both virulence and epiphytic fitness. Furthermore, the symptoms observed with wild-type P. syringae FF5 suggest that this strain can move systemically in leaf tissue, and that a functional copy of algR is required for systemic movement.