Characterization of the alginate biosynthetic gene cluster in Pseudomonas syringae pv. syringae

Abstract

Alginate, a copolymer of D-mannuronic acid and L-guluronic acid, is produced by a variety of pseudomonads, including Pseudomonas syringae. Alginate biosynthesis has been most extensively studied in P. aeruginosa, and a number of structural and regulatory genes from this species have been cloned and characterized. In the present study, an alginate-defective (Alg ؊ ) mutant of P. syringae pv. syringae FF5 was shown to contain a Tn5 insertion in algL, a gene encoding alginate lyase. A cosmid clone designated pSK2 restored alginate production to the algL mutant and was shown to contain homologs of algD, alg8, alg44, algG, algX (alg60), algL, algF, and algA. The order and arrangement of the structural gene cluster were virtually identical to those previously described for P. aeruginosa. Complementation analyses, however, indicated that the structural gene clusters in P. aeruginosa and P. syringae were not functionally interchangeable when expressed from their native promoters. A region upstream of the algD gene in P. syringae pv. syringae was shown to activate the transcription of a promoterless glucuronidase (uidA) gene and indicated that transcription initiated upstream of algD as described for P. aeruginosa. Transcription of the algD promoter from P. syringae FF5 was significantly higher at 32°C than at 18 or 26°C and was stimulated when copper sulfate or sodium chloride was added to the medium. Alginate gene expression was also stimulated by the addition of the nonionic solute sorbitol, indicating that osmolarity is a signal for algD expression in P. syringae FF5.The phytopathogenic bacterium Pseudomonas syringae produces two well-characterized extracellular polysaccharide (EPS) molecules: levan, a polymer of fructofuranan, and alginate, a copolymer of O-acetylated ␤-1,4-linked D-mannuronic acid and its C-5 epimer, L-guluronic acid (21, 29). Possible roles for the EPS molecules produced by P. syringae are varied and include avoidance of host plant cell recognition, resistance of bacterial cells to desiccation, and enhancement of epiphytic fitness (33, 41). Furthermore, alginate has been implicated in a symptom known as water soaking, where the intercellular tissues of infected plants become filled with water (20, 29). However, a role for alginate in the symptomology or virulence of P. syringae has not been proven.Alginate biosynthesis has been extensively studied in Pseudomonas aeruginosa, where it functions as a major virulence factor in strains infecting the lungs of cystic fibrosis patients (56). In P. aeruginosa, genes that encode the biosynthesis and regulation of alginate map to four chromosomal locations. With the exception of algC, which is located at 10 min, most of the structural genes are located at 34 min. The regulatory genes map at 10 and 13 min, and the loci responsible for the genotypic switch to alginate production are located at 68 min (46). Most of the structural genes for alginate biosynthesis are clustered within an 18-kb region in the P. aeruginosa chromosome (16). Structural genes that have...

The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000

“…Among plant pathogenic pseudomonads, alginate production has been linked to epiphytic fitness and the production of watersoaked lesions (53). All of the genes required for alginate biosynthesis in P. aeruginosa are present in DC3000, although some of the alginate regulatory genes are absent, indicating that the regulation of alginate biosynthesis differs between P. aeruginosa and P. syringae, consistent with previous reports (54). Three genes encoding levansucrases, required for the biosynthesis of the polysaccharide levan, were also identified in DC3000.…”

Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana

“…g. Strain did not elicit tissue collapse (an HR) when inoculated into tobacco leaves at 5 ¥ 10 genes known to be involved in P. syringae pathogenesis. These include hrp/hrc genes (Galan and Collmer, 1999), genes encoding putative effectors of the hrp/hrc type III secretion system located both within the conserved effector locus (CEL) flanking the hrp/hrc cluster (Alfano et al, 2000) and elsewhere in the genome (virPphA, avrPpiB and avrPphD; Cournoyer et al, 1995;Jackson et al, 1999;Arnold et al, 2001a), biosynthetic genes for the P. syringae phytotoxin coronatine (cfl, cfa; Bender et al, 1999) and algA, which directs the synthesis of the exopolysaccharide alginate (Peñaloza-Vázquez et al, 1997). Additionally, several ipx fusions are to novel genes that would be predicted to be involved in virulence, including a gene encoding a putative pectin lyase (ipx31), which may be involved in degrading plant cell walls.…”

The Pseudomonas syringae Genome Encodes a Combined Mannuronan C-5-epimerase and O-Acetylhydrolase, Which Strongly Enhances the Predicted Gel-forming Properties of Alginates

“…In Azotobacter vinelandii, which expresses extracellular epimerases, this mechanism controls the degree of epimerization (11,14). In bacteria a periplasmic ME is encoded by algG, which is found in the alginate biosynthetic gene cluster (15)(16)(17). Previous studies demonstrated that epimerase-defective algG mutants of Pseudomonas aeruginosa or Pseudomonas fluorescens produce pure polymannuronic acid, which suggests that algG is the sole ME in these bacteria (15,18).…”