Role for Rhizobium rhizogenes K84 Cell Envelope Polysaccharides in Surface Interactions

Abstract

dRhizobium rhizogenes strain K84 is a commercial biocontrol agent used worldwide to control crown gall disease. The organism binds tightly to polypropylene substrate and efficiently colonizes root surfaces as complex, multilayered biofilms. A genetic screen identified two mutants in which these surface interactions were affected. One of these mutants failed to attach and form biofilms on the abiotic surface although, interestingly, it exhibited normal biofilm formation on the biological root tip surface. This mutant is disrupted in a wcbD ortholog gene, which is part of a large locus predicted to encode functions for the biosynthesis and export of a group II capsular polysaccharide (CPS). Expression of a functional copy of wcbD in the mutant background restored the ability of the bacteria to attach and form normal biofilms on the abiotic surface. The second identified mutant attached and formed visibly denser biofilms on both abiotic and root tip surfaces. This mutant is disrupted in the rkpK gene, which is predicted to encode a UDP-glucose 6-dehydrogenase required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis in rhizobia. The rkpK mutant from strain K84 was deficient in O-antigen synthesis and exclusively produced rough LPS. We also show that strain K84 does not synthesize the KPS typical of some other rhizobia strains. In addition, we identified a putative type II CPS, distinct from KPS, that mediates cell-surface interactions, and we show that O antigen of strain K84 is necessary for normal cell-cell interactions in the biofilms.

Plasmid curing and the loss of grip – The 65-kb replicon of Phaeobacter inhibens DSM 17395 is required for biofilm formation, motility and the colonization of marine algae

“…The putative biofilm-plasmid contains, for example, the complete rhamnose operon [13] and an operon with a type I secretion polysaccharide export system (YP 006564766, YP 006564765; [24]) followed by three genes for the conversion of d-mannose-1-phosphate into GDP-l-fucose (YP 006564764 to YP 006564762). In rhizobia, where biofilm formation is essential for the efficient colonization of root surfaces and nodulation [6,11], a homolog of the Phaeobacter UDP-glucose 6-dehydrogenase (YP 006564778) is required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis, as recently shown by knock-out analyses in Rhizobium rhizogenes K84 [1].…”

Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.)

“…Similar rkp-2 regions are found in the genomes of other Rhizobium and Agrobacterium strains. In R. leguminosarum and R. rhizogenes , rkpK homologues have been implicated in the biosynthesis of CPS, EPS or LPS [88,89]. The sinorhizobial rkp-3 region contains genes involved in KPS polymerization and export, and also strain-specific genes that determine its sugar composition [79].…”

“…A cluster of 19 genes located in the CIAT 899 megaplasmid encode functions putatively related to CPS biosynthesis. Recently, an orthologous syntenic cluster of R. rhizogenes K84 was postulated to be involved in the biosynthesis of a CPS different from the typical high molecular weight sinorhizobial KPS [89]. The putative K84 polysaccharide seems to be required for normal attachment and biofilm formation on an abiotic hydrophobic surface, but not on tomato roots [89].…”

Sinorhizobium fredii HH103 syrM inactivation affects the expression of a large number of genes, impairs nodulation with soybean and extends the host‐range to Lotus japonicus

“…For EPS quantification, bacterial cultures were grown on YM for 96 h at 28 C (OD 600 = 1.2-1.3) under shaking conditions. Cells were removed by centrifugation (20,000 g, 15 min) and total carbohydrate amounts of the EPS-containing supernatants were determined using the anthrone-sulphuric acid method, which measures the total reducing sugar content in a given sample, as previously described (Abarca-Grau et al, 2012). Four independent experiments in duplicate were carried out.…”