K. Dale Noel

The presence of a novel type of surface polysaccharide in Rhizobium meliloti requires a new fatty acid synthase-like gene cluster involved in symbiotic nodule development

Bacterial exopolysaccharide (EPS) and lipopolysaccharide (LPS) molecules have been shown to play important roles in plant‐bacterium interactions. Here we have demonstrated that the fix‐23 loci, which compensate for exo mutations during symbiotic nodule development, are involved in the production of a novel polysaccharide that is rich in 3‐deoxy‐D manno‐2‐octulosonic acid (Kdo) but is not the classical LPS. This molecule is likely to be a surface antigen since antiserum to whole Rhizobium meliloti cells reacts strongly with it, and since mutations in fix‐23 result in an inability to produce this polysaccharide and to bind bacteriophage 16‐3. It is likely that this Kdo‐rich polysaccharide is analogous to certain Escherichia coli K‐antigens which are anchored to the membrane via a phospholipid moiety. DNA sequence analysis of one gene cluster of this region revealed that the predicted protein products of six genes exhibit a high degree of homology and similar organization to those of the rat fatty acid synthase multifunctional enzyme domains.

Rhizobial purine and pyrimidine auxotrophs: nutrient supplementation, genetic analysis, and the symbiotic requirement for the novo purine biosynthesis

Previously described Rhizobium leguminosarum bv. phaseoli mutants elicit nodules on bean without infection thread formation. These mutants were shown to be purine or, in one case, pyrimidine auxotrophs. Each of the seven purine auxotrophs grew normally when supplied the penultimate precursor of inosine, 5-aminoimidazole-4-carboxamide riboside. Four seemed blocked early in the purine pathway, because they were also thiamine auxotrophs. Reversion analysis and genetic complementation using cloned wild-type DNA showed that in each mutant a single mutation was responsible for both the symbiotic defect and purine or pyrimidine auxotrophy. The mutations were mapped to five dispersed chromosomal locations. The previously reported weak Calcofluor staining of these mutants on minimal agar appeared to be caused by partial growth on contaminating nutrients in the agar, rather than deficient exopolysaccharide production. Nodulation by the mutants was not enhanced by supplying purine or pyrimidine compounds exogenously. Furthermore, with or without added purine, the purine auxotrophs grew in the root environment as well as the wild type. However, nodulation by the purine auxotrophs was enhanced greatly in the presence of 5-aminoimidazole-4-carboxamide riboside. The results suggest that undiminished metabolic flow through de novo purine biosynthesis, or a particular intermediate in the pathway, is essential in early symbiotic interactions.

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

Rhizobium etli CE3 bacteroids were isolated from Phaseolus vulgaris root nodules. The lipopolysaccharide (LPS) from the bacteroids was purified and compared with the LPS from laboratory-cultured R. etli CE3 and from cultures grown in the presence of anthocyanin. Comparisons were made of the O-chain polysaccharide, the core oligosaccharide, and the lipid A. Although LPS from CE3 bacteria and bacteroids are structurally similar, it was found that bacteroid LPS had specific modifications to both the O-chain polysaccharide and lipid A portions of their LPS. Cultures grown with anthocyanin contained modifications only to the O-chain polysaccharide. The changes to the O-chain polysaccharide consisted of the addition of a single methyl group to the 2-position of a fucosyl residue in one of the five O-chain trisaccharide repeat units. This same change occurred for bacteria grown in the presence of anthocyanin. This methylation change correlated with the inability of bacteroid LPS and LPS from anthocyanin-containing cultures to bind the monoclonal antibody JIM28. The core oligosaccharide region of bacteroid LPS and from anthocyanin-grown cultures was identical to that of LPS from normal laboratory-cultured CE3. The lipid A from bacteroids consisted exclusively of a tetraacylated species compared with the presence of both tetra- and pentaacylated lipid A from laboratory cultures. Growth in the presence of anthocyanin did not affect the lipid A structure. Purified bacteroids that could resume growth were also found to be more sensitive to the cationic peptides, poly-l-lysine, polymyxin-B, and melittin.

2-O-Methylation of Fucosyl Residues of a Rhizobial Lipopolysaccharide Is Increased in Response to Host Exudate and Is Eliminated in a Symbiotically Defective Mutant

ABSTRACT When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395α395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395α395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps+ strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.

Role of BacA in Lipopolysaccharide Synthesis, Peptide Transport, and Nodulation by Rhizobium sp. Strain NGR234

BacA of Sinorhizobium meliloti plays an essential role in the establishment of nitrogen-fixing symbioses with Medicago plants, where it is involved in peptide import and in the addition of very-long-chain fatty acids (VLCFA) to lipid A of lipopolysaccharide (LPS). We investigated the role of BacA in Rhizobium species strain NGR234 by mutating the bacA gene. In the NGR234 bacA mutant, peptide import was impaired, but no effect on VLCFA addition was observed. More importantly, the symbiotic ability of the mutant was comparable to that of the wild type for a variety of legume species. Concurrently, an acpXL mutant of NGR234 was created and assayed. In rhizobia, AcpXL is a dedicated acyl carrier protein necessary for the addition of VLCFA to lipid A. LPS extracted from the NGR234 mutant lacked VLCFA, and this mutant was severely impaired in the ability to form functional nodules with the majority of legumes tested. Our work demonstrates the importance of VLCFA in the NGR234-legume symbiosis and also shows that the necessity of BacA for bacteroid differentiation is restricted to specific legume-Rhizobium interactions.

Mapping of complementation groups within a Rhizobium leguminosarum CFN42 chromosomal region required for lipopolysaccharide synthesis

SummaryA major genetic region specifying portions of the carbohydrate structure of Rhizobium leguminosarum CFN42 lipopolysaccharide was analyzed by Tn5 mutagenesis, constructing deletions in cloned DNA, restriction mapping, and complementation analysis. Mutations affecting lipopolysaccharide synthesis were arranged in nine complementation groups spanning 18 kb of DNA. One mutation resulted in O-polysaccharide-containing lipopolysaccharide having a slightly increased mobility in gel electrophoresis. This mutation did not affect the symbiosis with bean plants. The other mutations eliminated the 0-polysaccharide-containing lipopolysaccharide and resulted in strains defective in eliciting bean nodule development.

Antigenic Change in the Lipopolysaccharide of Rhizobium etli CFN42 Induced by Exudates of Phaseolus vulgaris

Growth of Rhizobium etli CE3 in the presence of exudates from Phaseolus vulgaris resulted in a modified lipopolysaccharide (LPS) that no longer reacted with monoclonal antibody JIM28. However, the overall LPS structure appeared not to be greatly altered, as revealed by unchanged mobility in gel electrophoresis and partial or unaltered reactivity with other antibodies. Activity that triggered LPS antigenic conversion was exuded from both seeds and roots, but reactivity with one of the antibodies indicated that the resulting alterations were not identical. Antibody binding to the LPS decreased as a function of the concentration of exudate present during growth of the bacteria. The antigenic change did not occur if purified LPS or nongrowing bacteria were incubated with the exudates. Exudate-induced LPS modification did not require the Sym plasmid.

Genetic Locus Required for Antigenic Maturation of Rhizobium etli CE3 Lipopolysaccharide

Rhizobium etli modifies lipopolysaccharide (LPS) structure in response to environmental signals, such as low pH and anthocyanins. These LPS modifications result in the loss of reactivity with certain monoclonal antibodies. The same antibodies fail to recognize previously isolated R. etli mutant strain CE367, even in the absence of such environmental cues. Chemical analysis of the LPS in strain CE367 demonstrated that it lacked the terminal sugar of the wild-type O antigen, 2,3,4-tri-O-methylfucose. A 3-kb stretch of DNA, designated as lpe3, restored wild-type antigenicity when transferred into CE367. From the sequence of this DNA, five open reading frames were postulated. Site-directed mutagenesis and complementation analysis suggested that the genes were organized in at least two transcriptional units, both of which were required for the production of LPS reactive with the diagnostic antibodies. Growth in anthocyanins or at low pH did not alter the specific expression of gusA from the transposon insertion of mutant CE367, nor did the presence of multiple copies of lpe3 situated behind a strong, constitutive promoter prevent epitope changes induced by these environmental cues. Mutations of the lpe genes did not prevent normal nodule development on Phaseolus vulgaris and had very little effect on the occupation of nodules in competition with the wild-type strain.

Compounds Exuded by Phaseolus vulgaris That Induce a Modification of Rhizobium etli Lipopolysaccharide

Exudates released from germinating seeds and roots of a black-seeded bean (Phaseolus vulgaris cv. Midnight Black Turtle Soup) induce an antigenic change in the lipopolysaccharide (LPS) of Rhizobium etli CE3. By spectroscopic analyses and chromatographic comparisons with derived standards, the chemical structures of the aglycone portions of the major inducing molecules from seed exudate were deduced, and they were identified as delphinidin, cyanidin, petunidin, and malvidin. These anthocyanidins were present in seed exudate mainly as glycosides, the chief inducer being delphinidin 3-glucoside. Also present were 3-glucosides of petunidin and malvidin and glycosides of cyanidin and delphinidin. Seed exudate from a bean variety deficient in anthocyanins did not induce the LPS conversion. The ability of root exudate to induce an antigenic change in the LPS was due to compounds other than anthocyanins.

The structures of the lipopolysaccharide core components from rhizobium leguminosarum biovar phaseoli CE3 and two of its symbiotic mutants, CE109 and CE309

The structures for the core regions of the lipopolysaccharides (LPSs) from R. leguminosarum bv. phaseoli CE3 and two symbiotic mutants were determined by g.l.c.-m.s., proton nuclear magnetic resonance spectroscopy (n.m.r.), fast-atom-bombardment mass spectrometry (f.a.b.-m.s.), and by comparison with known structures from the LPS of R. leguminosarum bv. trifolii ANU843. The core oligosaccharides were separated into two components, P2-2 and P2-3, by gel-filtration chromatography using Bio-Gel P2. The P2-2 oligosaccharide from CE3 is a tetrasaccharide consisting of 3-deoxy-D-manno-2-octulosonic acid (Kdo), mannose, galactose and galacturonic acid. The mannosyl residue is alpha-linked to O-4 of Kdo, and the galactosyl and galactosyluronic residues are alpha-linked to O-4 and O-6, respectively, of the mannosyl residue. The P2-2 oligosaccharide from mutant CE109 is missing the galactosyluronic residue, while that from mutant CE309 is missing both the galactosyl and galactosyluronic residues. The P2-3 oligosaccharide from CE3 LPS is a trisaccharide consisting of two galactosyluronic residues alpha-linked to the O-4 and O-7 of Kdo. Fraction P2-3 from mutant CE309 has the same structure as CE3 P2-3. Fraction P2-3 from mutant CE109 contains galacturonic acid and Kdo, but its structure differs from that of CE3 P2-3.