Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Abstract

Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGR⍀fixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed Rhizobium spp. are unique soil bacteria that have found an ecological niche within cortical cells of legume roots. Rhizobia infect highly organized root nodules, where they reduce atmospheric nitrogen to ammonia, which is then exported to the plant cells in the form of amides or ureides. Nodulation (nod) genes control the biosynthesis of lipo-chito-oligosaccharides (Nod factors) that are required for nodule initiation (6,40). Genes required for the conversion of nitrogen into ammonia can be divided into two classes: the nif genes, which encode nitrogenase (22,54), and the fix genes, which are involved in electron transport and membrane synthesis, etc. fix genes have many and varied functions (13, 45).Nineteen nod boxes (conserved cis-acting regulatory sequences located in the 5Ј-end-flanking region of nodD-regulated, flavonoid-inducible genes) are present on the symbiotic plasmid pNGR234a of the broad-host-range Rhizobium sp. strain NGR234 (17,28). Knockout mutation of the open reading frame that is 800 bp downstream of NB6 (17) provokes a Nod ϩ Fix Ϫ phenotype on Vigna unguiculata that results from nodules with defective peribacteroid membranes. Preliminary observations suggested that the mutant is unable to synthesize a polysaccharide component, shown here to be an O-antigen polysaccharide, that is a prominent product of apigenin-induced vegetative cells and bacteroids induced by the wild-type strain (16,24).Lipopolysaccharides (LPSs) are major structural and antigenic components of the rhizobial outer membrane (9,26,41,51). Their normal expression and structures are essential for successful symbiotic infection (8,15,26,31,33,34,49,58). In the present study, we show that the flavonoid-inducible polysaccharide is a rhamnan O antigen, covalently linked to a structurally modified core oligosaccharide lipid A (thus, an LPS). Isolation and analysis of this unique O antigen shows that it consists of a trisaccharide repeat of L-rhamnose, having

Flavonoid-Inducible Modifications to Rhamnan O Antigens Are Necessary for Rhizobium sp. Strain NGR234-Legume Symbioses

“…2, aqueous phase, lane 1), in agreement with earlier studies on the structure of NGR234 LPS produced under noninducing conditions (23). The rhamnan was still produced by the knockouts in rmlC, y4gM, y4hA, and noeE, whereas deletion of rmlB to wbgA and mutation in fixF abolished rhamnan synthesis, as reported earlier (35,48). The presence of rhamnan in the rmlC knockout extractions (Fig.…”

“…Earlier DNA sequence comparisons (19) as well as recent (September 2005) BLAST (2) searches suggest functions for many of the 12 ORFs that lie between nod boxes NB5 and NB7 ( Table 1). Six of the ORFs probably take part in rhamnose synthesis and production of the flavonoid-inducible rhamnan O antigen (35,48). Further analysis of rhamnan O antigen production by these ORFs was undertaken, and the role of this new LPS structure was tested in a variety of NGR234 legume hosts.…”

“…All mutants constructed using the omega antibiotic resistance cassettes are collectively called knockout mutants. Two other previously described mutants (NGR⍀fixF and NGR⌬noeE) were also included in the analysis (25,48). To test the role of each gene, rhizobia were extracted with hot phenol-water, and the cell-associated polysaccharides were separated by PAGE.…”

“…The proteins encoded by the rmlB-wbgA and fixF genes are necessary for the synthesis of a new, rhamnose-rich, O antigen (35,48). The short intergenic spacing between the rmlB, rmlD, rmlA, and wbgA open reading frames (ORFs) suggests that they form a small operon (Fig.…”

Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria

“…These changes have included differences in the DOC-PAGE banding pattern of the LPS, the production of a secondary rhamnan O-chain in the case of Rhizobium sp. NGR234 (44,45), the glycosyl composition and O-acetylation changes in Rhizobium leguminosarum LPS O-chain polysaccharide (43), and an increase in long chain fatty acylation of the lipid A of R. leguminosarum LPS (43). The changes reported for R. leguminosarum and Rhizobium sp.…”