Loss of wbpL disrupts O‐polysaccharide synthesis and impairs virulence of plant‐associated Pseudomonas strains

Abstract

Despite its importance for membrane stability and pathogenicity of mammalian pathogens, functions of the O-polysaccharide (OPS) of lipopolysaccharide (LPS) remain unclear in plant-associated bacteria. Genetic information about OPS biosynthesis in these bacteria is largely missing. Genome analysis of various plant-associated Pseudomonas strains revealed that one of the two known OPS biosynthesis clusters from Pseudomonas aeruginosa PAO1, the common polysaccharide antigen (CPA) gene cluster, is only conserved in some strains of the Pseudomonas fluorescens group. For the O-specific antigen (OSA) biosynthesis cluster, the putative genomic position could be identified, but orthologues of most functional important OSA biosynthesis enzymes could not be detected. Nevertheless, orthologues of the glycosyltransferase WbpL, required for initiation of CPA and OSA synthesis in P. aeruginosa PAO1, could be identified in the analysed Pseudomonas genomes. Knockout mutations of wbpL orthologues in Pseudomonas syringae pv. tomato DC3000 (Pst) and Pseudomonas cichorii ATCC10857/DSM50259 (Pci) resulted in strains lacking the OPS. Infection experiments of Arabidopsis thaliana plants revealed a reduced entry into the leaf apoplast after spray inoculation and a reduced apoplastic amplification of Pst ∆wbpL. Stab and spray inoculation of lettuce (Lactuca sativa) leaves with Pci ∆wbpL causes reduced infection symptoms compared to the wild-type strain. Furthermore, swarming motility was reduced in ∆wbpL mutants of Pst and Pci. This might be a possible reason for reduced bacterial titres after surface inoculation and reduced bacterial amplification in the plant. Our results imply that the presence of lipopolysaccharide OPS is required for efficient host colonization and full virulence of plant-pathogenic Pseudomonas bacteria.

Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the Pseudomonas syringae species complex

“…Identifying the CPA locus in P. syringae has been challenging due to the lack of similarity to wzm/wzt/wbpX orthologues (Kutschera et al, 2019a). Previous work Fig.…”

“…Identifying the CPA locus in P. syringae has been challenging due to the lack of similarity to wzm / wzt / wbpX orthologues (Kutschera et al ., 2019a). Previous work identified five R‐LPS mutants by Tn5 mutagenesis that had inserts in a region with homology to the CPA biosynthetic pathway from P. aeruginosa PAO1 (Mesarich et al ., 2017).…”

“…What has been demonstrated recently within P. syringae is the depth and complexity of both the killing and sensitivity spectra generated from a wide range of isolates (Baltrus et al, 2019). While it is clear that LPS is a receptor for some tailocins ( Kutschera et al, 2019a;Kandal et al, 2020), a link between the sensitivity to tailocins and the structural variation in LPS has not been demonstrated until recently. Carim et al (2020) have shown within the genus Pseudomonas that OSA composition and display are the most important contributors to tailocin sensitivity.…”

“…Similarly, the structures of OPS in plant‐pathogenic bacteria such as P. syringae are highly variable at the pathovar level (Ovod et al ., 1997a). For P. syringae , the OPS appears to be comprised of the CPA (Mesarich et al ., 2017; Kutschera et al ., 2019a) with a backbone consisting of a tri‐ or tetra‐saccharide repeating unit, comprising either l ‐ or d ‐Rhamnose (Rha), or a combination of the two, with various linkages. These are decorated with side chains consisting of different sugars such as N ‐acetyl‐glucosamine (GlcNAc), fucose (Fuc), Rha, and N ‐acetyl‐fucosamine (FucNAc) (Ovod et al ., 1997a; Zdorovenko and Zdorovenko, 2010).…”

“…Furthermore, LPS was not observed in these mutants, suggesting CPA is the sole OPS in Psa (Hockett et al ., 2017). Other recent papers have shown a relationship between OSA and tailocin sensitivity among the Pseudomonads (Carim et al ., 2020), directly identified components of CPA biosynthesis in P. syringae through gene knockouts (Kutschera et al ., 2019a) and increased resistance to tailocins (Kandel et al ., 2020).…”