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

Abstract

Pseudomonas syringaepv. syringae strain FF5 is a phytopathogen associated with a rapid dieback on ornamental pear trees.P. syringaeand the human pathogenPseudomonas aeruginosaproduce the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. InP. aeruginosa, the response regulator AlgR (AlgR1) is required for transcription ofalgCandalgD, which encode key enzymes in the alginate biosynthetic pathway. InP. syringaeFF5, however,algRis not required for the activation ofalgD. Interestingly,algRmutants ofP. syringaeremain nonmucoid, indicating an undefined role for this response regulator in alginate biosynthesis. In the current study, thealgCpromoter region was cloned fromP. syringaepv. syringae strain FF5, and sequence analysis of thealgCpromoter indicated the presence of potential binding sites for AlgR andσ54, the alternative sigma factor encoded byrpoN. ThealgCpromoter fromP. syringaeFF5 (PsalgC) was cloned upstream of a promoterless glucuronidase gene (uidA), and thePsalgC–uidAtranscriptional fusion was used to monitoralgCexpression in strains FF5.32 (algRmutant ofP. syringaeFF5) and PG4180.K2 (rpoNmutant ofP. syringaepv. glycinea PG4180). Expression of thePsalgC–uidAfusion was fourfold lower in both thealgRandrpoNmutants as compared to respective wild-type strains, indicating that both AlgR andσ54are required for full activation ofalgCtranscription inP. syringaepv. syringae. AlgR fromP. syringaewas successfully overproduced inEscherichia colias a C-terminal translational fusion to the maltose-binding protein (MBP). Gel shift experiments indicated that MBP–AlgR binds strongly to thealgCpromoter region. Biological assays demonstrated that thealgRmutant 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 ofalgCinP. syringaeand contributes to both virulence and epiphytic fitness. Furthermore, the symptoms observed with wild-typeP. syringaeFF5 suggest that this strain can move systemically in leaf tissue, and that a functional copy ofalgRis required for systemic movement.