S. Jabbouri

Keys to Symbiotic Harmony

At least three different sets of symbiotic signals (here, they are compared to locks and keys) are exchanged between legumes and rhizobia during nodule development. Flavonoids, the first of these, emanate from the plant and interact with rhizobial NodD proteins that serve as both environmental

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10–9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.

Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host‐specificity gene

Rhizobia secrete specific lipo‐chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N‐acetyl‐d‐glucosamine of LCOs is substituted at C6 with 2‐O‐methyl‐l‐fucose which can be acetylated or sulphated. We identified a flavonoid‐inducible locus on the symbiotic plasmid pNGR234a that contains a new nodulation gene, noeEwhich is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non‐sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum. Furthermore, mutation of noeE (NGRΔnoeE ) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus. The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRΔnoeE leads to the production of a mixture of LCOs that are sulphated on C6 of the reducing terminus and sulphated on the 2‐O‐methylfucose residue. Together, these findings show that noeE is a host‐specificity gene which probably encodes a fucose‐specific sulphotransferase.

nodD2 of Rhizobium sp. NGR234 is involved in the repression of the nodABC operon.

Transcriptional regulators of the lysR family largely control the expression of bacterial symbiotic genes. Rhizobium sp. NGR234 contains at least four members of this family: two resemble nodD, while two others are more closely related to syrM. Part of the extremely broad host range of NGR234 can be attributed to nodD1, although the second gene shares a high degree of DNA sequence homology with nodD2 of R. fredii USDA191. A nodD2 mutant of NGR234 was constructed by insertional mutagenesis. This mutant (NGRΩnodD2) was deficient in nitrogen fixation on Vigna unguiculata and induced pseudonodules on Tephrosia vogelii. Several other host plants were tested, but no correlation could be drawn between the phenotype and nodule morphology. Moreover, nodD2 has a negative effect on the production of Nod factors: mutation of this gene results in a fivefold increase in Nod factor production. Surprisingly, while the structure of Nod factors from free‐living cultures of NGRΩnodD2 remained unchanged, those from V. unguiculata nodules induced by the same strain are non‐fucosylated and have a lower degree of oligomerization. In other words, developmental regulation of Nod factor production is also abolished in this mutant. Competitive RNA hybridizations, gene fusions and mobility shift assays confirmed that nodD2 downregulates expression of the nodABC operon.

SyrM1 of Rhizobium sp. NGR234 Activates Transcription of Symbiotic Loci and Controls the Level of Sulfated Nod Factors

One or more transcriptional regulators of the LysR class control transcription of rhizobial nodulation genes. In Rhizobium sp. NGR234, two copies of nodD (nodD1 and nodD2) are present on the symbiotic plasmid pNGR234a. The complete sequence of pNGR234a revealed two additional nodD homologues, syrM1 and syrM2. Competitive RNA hybridization analyses involving a mutant of syrM1 (NGRΔsyrM1) showed that a number of symbiotic genes (e.g., nolXBTUVW) are expressed in an syrM1-dependent manner. Assays in which regions upstream of nolB and nolW were fused to promotorless lacZ confirmed that SyrM1 is required for their late induction. Mutation of syrM1 also drastically reduced production of sulfated Nod factors as shown by reverse phase-thin layer chromatography (RP-TLC). SyrM1 controls sulfation of Nod factors via one of the two chromosomal nodPQ loci. It thus seems likely that syrM1 of NGR234 encodes a transcriptional activator that regulates the expression of genes involved in both the early and late stages of i...

Differentiation of O-Acetyl and O-Carbamoyl esters of N-Acetyl-Glucosamine by decomposition of their oxonium ions. application to the structure of the nonreducing terminal residue of Nod factors

Nod factors are substituted N-acyl chito-oligomers secreted by plant symbiotic bacteria of the Rhizobium family. Substitutions on the oligosaccharide core specify their recognition by host plants. A method using tandem mass spectrometry is proposed to locate the O-acetyl and O-carbamoyl substituents on the nonreducing terminal residue of the chito-oligomers. As model compounds, all the positional isomers of monoacetyl and monocarbamoyl esters of 1-O-methyl-N-acetyl-α-D-glucosamine were synthesized. Oxonium ions (MH − CH3OH)+ were generated by liquid secondary ion mass spectrometry (LSIMS) and their decomposition was recorded on a tandem magnetic instrument. Large differences were observed in the relative abundances of ions resulting from elimination of water and of the O-ester substituent from metastable oxonium ions. Deuterium exchange reactions indicated parallel elimination pathways involving either exchangeable or carbon-linked hydrogens. The intensity ratios of some of the ions generated by collisions with helium atoms allowed the isomers to be distinguished. The main dissociation routes were identified. Metastable and collision-induced decomposition of the B1 ions from Nod factors of Sinorhizobium meliloti and Azorhizobium caulinodans resembled that of the 6-O-substituted N-acetylglucosamine models. Decomposition of the B1 ion from Mesorhizobium loti and Rhizobium etli Nod factors, was similar to that of 3-O-carbamoyl N-acetyl-glucosamine and different to that of the 4-O isomer. 6-O- and 3-O-carbamoylation specified by the nodU and nolO genes, respectively, of Rhizobium. sp. NGR234 were confirmed.

Region II of Rhizobium sp. NGR234 Inhibits Nodulation of Medicago sativa by R. meliloti nodIJ and nodQ1 Mutants

Rhizobium sp. NGR234 contains a plasmid-borne locus that hybridized strongly to region II of R. meliloti. Surprisingly, NGR region II completely inhibited nodulation of Medicago sativa when conjugated into either R. meliloti nodIJ or nodQ1 region II mutants. Further characterization showed that region II of NGR234 contains three putative coding open reading frames (ORFs), which are homologous to, respectively, hypothetical protein A of R. leguminosarum, hypothetical protein C of R. leguminosarum, and ORF2 of Agrobacterium tumefaciens insertion sequence (IS) 66, as well as an ORF of unknown function located downstream of nodQ1 of R. meliloti. A site-directed mutation in the IS66 homologue improved nodulation efficiency on some NGR234 hosts, but the structure and composition of the Nod-factor family produced by the mutant were unchanged. The quantity of Nod factors secreted was reduced by two-thirds, however. Insertions or deletions of the genes encoding either the hypothetical protein C or the IS66 homolog...

Bioconversion of triterpenes by mycobacteria. Structure and conformation of the products of degradation of 7,11-dioxodihydrolanosterol by Mycobacterium phlei

While mycobacteria are unable to degrade lanosterol and dihydrolanosterol, principal components of wool fat, we observed the transformation of some of their autoxidation products by Mycobacterium phlei. By analogy with the mechanism of degradation of cholesterol, this difference was assumed to be due to the requirement for the presence of an enone group before the side-chain can be degraded. This paper reports the spectroscopic determination of the structure of the major metabolites of 7,11-dioxodihydrolanosterol. The side-chain is degraded from eight carbon atoms to three, the terminal carbon atom being oxidized to a primary alcohol or a methyl ester. The tetracyclic skeleton can undergo regioselective oxidation–reduction modifications at the 3- and 7-position. Their conformational analysis, carried out by 2D-NMR methods, indicates a chair form for ring A of 3β-hydroxy derivatives, while it is highly deformed for 3-keto compounds as predicted formerly by Mislow for this lanostane series.