Arlette Savagnac

The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N‐acylation of Nod factors by different fatty acids

Rhizobia synthesize mono‐N‐acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation (nod) genes, including the nodABC genes present in all rhizobial species. The N‐acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti, an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (ω‐1) hydroxylated fatty acid, whereas in Rhizobium tropici, a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non‐polar deletion of nodA, and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N‐acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.

Structure of the Mesorhizobium huakuii and Rhizobium galegae Nod factors: a cluster of phylogenetically related legumes are nodulated by rhizobia producing Nod factors with alpha,beta-unsaturated N-acyl substitutions.

Rhizobia are symbiotic bacteria that synthesize lipochitooligosaccharide Nod factors (NFs), which act as signal molecules in the nodulation of specific legume hosts. Based on the structure of their N‐acyl chain, NFs can be classified into two categories: (i) those that are acylated with fatty acids from the general lipid metabolism; and (ii) those (= αU‐NFs) that are acylated by specific α,β‐unsaturated fatty acids (containing carbonyl‐conjugated unsaturation(s)). Previous work has described how rhizobia that nodulate legumes of the Trifolieae and Vicieae tribes produce αU‐NFs. Here, we have studied the structure of NFs from two rhizobial species that nodulate important genera of the Galegeae tribe, related to Trifolieae and Vicieae. Three strains of Mesorhizobium huakuii, symbionts of Astragalus sinicus, produced as major NFs, pentameric lipochitooligosaccharides O‐sulphated and partially N‐glycolylated at the reducing end and N‐acylated, at the non‐reducing end, by a C18:4 fatty acid. Two strains of Rhizobium galegae, symbionts of Galega sp., produced as major NFs, tetrameric O‐carbamoylated NFs that could be O‐acetylated on the glucosamine residue next to the non‐reducing terminal glucosamine and were N‐acylated by C18 and C20 α,β‐unsaturated fatty acids. These results suggest that legumes nodulated by rhizobia synthesizing αU‐NFs constitute a phylogenetic cluster in the Galegoid phylum.

Rhizobium fredii synthesizes an array of lipooligosaccharides, including a novel compound with glucose inserted into the backbone of the molecule

Flavonoid cues from the plant host cause symbiotic, nitrogen‐fixing rhizobia to synthesize lipochitooligosaccharides (LCOs), which act as return signals to initiate the nodulation process. Rhizobium fredii strain USDA257 is known to produce four LCOs, all substituted with vaccenic acid (C18:1). We show here that a mutant strain can replace vaccenic acid with a C16:0 side chain, and that strain USDA191 synthesizes additional LCOs that differ from one another in the length and unsaturation of their fatty acyl substituents. USDA191 and 257DH4 both produce a novel LCO with glucose substituted for in the backbone of the molecule.

Rhizobium sp. BR816 produces a complex mixture of known and novel lipochitooligosaccharide molecules.

Rhizobial lipochitooligosaccharide (LCO) signal molecules induce various plant responses, leading to nodule development. We report here the LCO structures of the broadhost range strain Rhizobium sp. BR816. The LCOs produced are all pentamers, carrying common C18:1 or C18:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing/terminal residue. A second acetyl group can be present on the penultimate N-acetylglucosamine from the nonreducing terminus. Two novel characteristics were observed: the reducing/terminal residue can be a glucosaminitol (open structure) and the degree of acetylation of this glucosaminitol or of the reducing residue can vary.

Structure determination of mycolic acids by using charge remote fragmentation

The collision-induced remote site fragmentation process of closed-shell ions, such as carboxylate anions, is a very potent analytical tool for the structural determination of fatty acids. This leads to an easy location of branch points, double bonds, cyclopropane rings and other functional groups. Although corynomycolic acid mixtures from Corynebacterium diphtheriae can be directly analyzed by negative-ion fast atom bombardment combined with collisionally activated decomposition spectra, mycolic acid mixtures from mycobacteria need a preliminary chemical cleavage. They are oxidized to beta-keto esters and then submitted to a retro-Claisen reaction. The resulting fatty acids were then converted into pentafluorobenzyl derivatives and introduced directly into a high pressure ion source working in the negative ion mode. The resulting gas phase carboxylate anions are activated to decompose by collision with helium atoms. When applied to M3-mycolic acids from Mycobacterium fallax, this method allows for the characterization of a new tri-unsaturated mycolic acid, which has the middle and the remote double bonds separated by two methylene groups.

Branched fatty acids from mycobacterium aurum

New methyl-branched fatty acids were isolated from the lipids of Mycobacterium aurum, belonging to both saturated and non-saturated series. The most abundant component of the former series was identified as a C22-mycosanoic acid (2-L, 4-L-dimethyleicosanoic acid). The unsaturated fraction contained a mixture of 2-L, 4-L-dimethyl-11-eicosenoic acid and 2-L, 4-L-dimethyl-14-eicosenoic acid. The biosynthetic precursors of these, according to the hypothesis of elongation by propionate units, were found in the non-branched hexadecenoic fraction. The lipidic fraction containing mycosanoic acid was a partially acylated oligosaccharide devoid of sulfate or phosphate groups.

Rhizobium Nod Factor Structure and the Phylogeny of Temperate Legumes

The family Leguminosae is one of the largest families of flowering plants, including more than 18,000 species (Young, Johnston, 1989). Their symbiotic bacterial partners are very diverse and do not form a discrete clade. Although they are collectively referred to as rhizobia, they are now classified in distinct Rhizobium, Bradyrhizobium, Azorhizobium and Sinorhizobium (Young, Haukka, 1996). Some rhizobia are indeed more closely related to nonsymbiotic bacteria than they are to other rhizobia. However, all rhizobial species possess common nodulation (nod) genes, the nodABC genes, and consequently produce nodulation signals, the Nod factors, that belong to the same chemical family: they are chitin oligomers of 4 or 5 glucosamine residues that are mono-N-acylated at the terminal non-reducing end. The common chitin oligomer core can be substituted at both ends of the molecule, and each rhizobial species or biovar produces a set of major Nod factors with characteristic substitutions (Denarie et al., 1996). These substitutions are under the control of specific nod genes. In the case of Sinorhizobium meliloti, it has been shown that the three substitutions are essential for effective infection of the host plant, alfalfa. The structure of major Nod factors has now been determined for most rhizobial species and their comparison indicates that Nod factor structure is related to rhizobial host range (Denarie et al., 1996).

Nod Factors from Rhizobium Fredii USDA257

Rhizobium fredii is a fast-growing Rhizobium isolated from soybean nodules (1). It is able to infect and nodulate “ ancestral” strain of soybean (Glycine soja) together with a strain originated from China (Glycine max cv. Peking). In contrast with Bradyrhizobium species it does not produce an effective symbiosis with cultivated soybean US strains (such as McCall).

Structural Characterization and Biological Activity of the Lipochitooligosaccharidic Signals of Rhizobium huakuii

Rhizobium huakuii is the symbiont of Astragalus sinicus which is a legume of great agricultural importance in China and Japan. The symbiotic relationship between R. huakuii and A. sinicus is reported to be very specific. This suggests that R. huakuii strains produce Nod factors whose structure and specificity differ from those already known.

Flavonoid-inducible regions in the symbiotic plasmid of Rhizobium etli

Control of gene expression by compounds present in root exudates is an important aspect for the ecology of rizospheric bacteria. Flavonoid compounds are particularly relevant for interactions between bacteria of the genus Rhizobium and leguminous plants. Data from different laboratories indicate that flavonoids are responsible for the induction of genes involved in the nodulation process (reviewed in ref. 1). Flavonoids also influence a variety of processes including the determination of efficiency of nodulation (2), competitivity (3), chemotaxis (4) and resistance to plant phytoalexins (5). Thus, the identification and analysis of bacterial genomic regions controlled by root flavonoids is an important step for the understanding of the molecular aspects that culminate in the establishment of a nitrogen -fixing symbiosis.