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.

Unusual methyl-branched alpha,beta-unsaturated acyl chain substitutions in the Nod Factors of an arctic rhizobium, Mesorhizobium sp. strain N33 (Oxytropis arctobia).

Mesorhizobium sp. strain N33 (Oxytropis arctobia), a rhizobial strain isolated in arctic Canada, is able to fix nitrogen at very low temperatures in association with a few arctic legume species belonging to the genera Astragalus, Onobrychis, and Oxytropis. Using mass spectrometry and nuclear magnetic resonance spectroscopy, we have determined the structure of N33 Nod factors, which are major determinants of nodulation. They are pentameric lipochito-oligosaccharides 6-O sulfated at the reducing end and exhibit other original substitutions: 6-O acetylation of the glucosamine residue next to the nonreducing terminal glucosamine and N acylation of the nonreducing terminal glucosamine by methyl-branched acyl chains of the iso series, some of which are alpha,beta unsaturated. These unusual substitutions may contribute to the peculiar host range of N33. Analysis of N33 whole-cell fatty acids indicated that synthesis of the methyl-branched fatty acids depended on the induction of bacteria by plant flavonoids, suggesting a specific role for these fatty acids in the signaling process between the plant and the bacteria. Synthesis of the methyl-branched alpha,beta-unsaturated fatty acids required a functional nodE gene.

Isolation of oligopeptides from the water-soluble extract of goat cheese and their identification by mass spectrometry.

A procedure for the separation and identification of small peptides from the water-soluble fraction of a goat cheese was developed. The water-soluble extract was ultrafiltered (1000 Da membrane cutoff), and peptides were isolated by sequential chromatography: size exclusion chromatography (HPLC-grade water), anion exchange chromatography (phosphate buffer gradient), and semipreparative reverse-phase high-performance liquid chromatography (water/acetonitrile gradient). The fractions obtained were analyzed by combined mass spectrometry methods including electrospray ionization, liquid secondary ionization, and tandem mass spectrometry to identify and to confirm the sequences of 28 tri- to octapeptides naturally appearing in goat cheese during ripening. Among these peptides, 26 are produced by degradation of caseins but do not correspond to the known specific cleavages due to chymosin. Only low correlation was found between hydrophobicity of peptides and HPLC elution time with acetonitrile gradient.

Bradyrhizobium sp. Strains that nodulate the leguminous tree Acacia albida produce fucosylated and partially sulfated nod factors.

We determined the structures of Nod factors produced by six different Bradyrhizobium sp. strains nodulating the legume tree Acacia albida (syn. Faidherbia albida). Compounds from all strains were found to be similar, i.e., O-carbamoylated and substituted by an often sulfated methyl fucose and different from compounds produced by Rhizobium-Mesorhizobium-Sinorhizobium strains nodulating other species of the Acaciae tribe.

Nod factor requirements for efficient stem and root nodulation of the tropical legume Sesbania rostrata.

Azorhizobium caulinodans ORS571 synthesizes mainly pentameric Nod factors with a household fatty acid, an N-methyl, and a 6-O-carbamoyl group at the nonreducing-terminal residue and with a d-arabinosyl, an l-fucosyl group, or both at the reducing-terminal residue. Nodulation on Sesbania rostrata was carried out with a set of bacterial mutants that produce well characterized Nod factor populations. Purified Nod factors were tested for their capacity to induce root hair formation and for their stability in an in vitro degradation assay with extracts of uninfected adventitious rootlets. The glycosylations increased synergistically the nodulation efficiency and the capacity to induce root hairs, and they protected the Nod factor against degradation. The d-arabinosyl group was more important than the l-fucosyl group for nodulation efficiency. Replacement of the 6-O-l-fucosyl group by a 6-O-sulfate ester did not affect Nod factor stability, but reduced nodulation efficiency, indicating that the l-fucosyl group may play a role in recognition. The 6-O-carbamoyl group contributes to nodulation efficiency, biological activity, and protection, but could be replaced by a 6-O-acetyl group for root nodulation. The results demonstrate that none of the studied substitutions is strictly required for triggering normal nodule formation. However, the nodulation efficiency was greatly determined by the synergistic presence of substitutions. Within the range tested, fluctuations of Nod factor amounts had little impact on the symbiotic phenotype.

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-alpha-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.

N-deacetylation of Sinorhizobium meliloti Nod factors increases their stability in the Medicago sativa rhizosphere and decreases their biological activity.

Nod factors excreted by rhizobia are signal molecules that consist of a chitin oligomer backbone linked with a fatty acid at the nonreducing end. Modifications of the Nod factor structures influence their stability in the rhizosphere and their biological activity. To test the function of N-acetyl groups in Nod factors, NodSm-IV(C16:2,S) from Sinorhizobium meliloti was enzymatically N-deacetylated in vitro with purified chitin deacetylase from Colletotrichum lindemuthianum. A family of partially and completely deacetylated derivatives was produced and purified. The most abundant chemical structures identified by mass spectrometry were GlcN(C16:2)-GlcNAc-GlcNH2-GlcNAc(OH)(S), GlcN(C16,2)-GlcNAc-GlcNH2-GlcNH2(OH)(S), and GlcN(C16:2)-GlcNH2-GlcNH2-GlcNH2(OH)(S). In contrast to NodSm-IV(C16:2,S), the purified N-deacetylated derivatives were stable in the rhizosphere of Medicago sativa, indicating that the N-acetyl groups make the carbohydrate moiety of Nod factors accessible for glycosyl hydrolases of the host plant. The N-deacetylated derivatives displayed only a low level of activity in inducing root hair deformation. Furthermore, the N-deacetylated molecules were not able to stimulate Nod factor degradation by M. sativa roots, a response elicited by active Nod factors. These data show that N-acetyl groups of Nod factors are required for biological activity.

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 (= alphaU-NFs) that are acylated by specific alpha,beta-unsaturated fatty acids (containing carbonyl-conjugated unsaturation(s)). Previous work has described how rhizobia that nodulate legumes of the Trifolieae and Vicieae tribes produce alphaU-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 alpha,beta-unsaturated fatty acids. These results suggest that legumes nodulated by rhizobia synthesizing alphaU-NFs constitute a phylogenetic cluster in the Galegoid phylum.

Phaseolus vulgaris recognizes Azorhizobium caulinodans Nod factors with a variety of chemical substituents.

Phaseolus vulgaris is a promiscuous host plant that can be nodulated by many different rhizobia representing a wide spectrum of Nod factors. In this study, we introduced the Rhizobium tropici CFN299 Nod factor sulfation genes nodHPQ into Azorhizobium caulinodans. The A. caulinodans transconjugants produce Nod factors that are mostly if not all sulfated and often with an arabinosyl residue as the reducing end glycosylation. Using A. caulinodans mutant strains, affected in reducing end decorations, and their respective transconjugants in a bean nodulation assay, we demonstrated that bean nodule induction efficiency, in decreasing order, is modulated by the Nod factor reducing end decorations fucose, arabinose or sulfate, and hydrogen.

[Nod factors, chemical signal exchange between bacteria and leguminous plants in nitrogen fixing symbiosis]. / Les facteurs Nod, éléments de reconnaissance entre bactéries et plantes légumineuses lors de leur symbiose fixatrice d'azote.

The early steps of the nitrogen-fixing symbiosis between plant legumes and soil bacteria (rhizobium) are mediated by an exchange of chemical signals between the two partners. Upon gene activation by plant root secretions (flavonoids), bacteria synthesize lipochitooligomers (called Nod Factors, NFs) that induce root hair deformations, cortical cell divisions, allow bacterial entry and produce nodule organogenesis at nano to picomole concentrations. Substitutions occurring on the lipochitooligosaccharide core are essential for recognition and activity. Biosynthesis of these molecules is now fully dissected, by looking at the structural changes in NFs induced by gene mutation or gene transfers. From the biodiversity studies of NFs, it appears that their structures belong with the phylogenetic evolution of plants, rather than that of bacteria, suggesting a coevolution of symbiotic bacteria with their plant receptors. Some preliminary and indirect observations indicate that similar molecules seem to exist in non-legumes plants, in batrachians and fishes beeing possibly involved in their embryogenesis, but they are probably at at a so low concentration that all attempts to detect them directly fail up to now.

Nodule-inducing activity of synthetic Sinorhizobium meliloti nodulation factors and related lipo-chitooligosaccharides on alfalfa. Importance of the acyl chain structure.

Sinorhizobium meliloti nodulation factors (NFs) elicit a number of symbiotic responses in alfalfa (Medicago sativa) roots. Using a semiquantitative nodulation assay, we have shown that chemically synthesized NFs trigger nodule formation in the same range of concentrations (down to 10(-10) M) as natural NFs. The absence of O-sulfate or O-acetate substitutions resulted in a decrease in morphogenic activity of more than 100-fold and approximately 10-fold, respectively. To address the question of the influence of the structure of the N-acyl chain, we synthesized a series of sulfated tetrameric lipo-chitooligosaccharides (LCOs) having fatty acids of different lengths and with unsaturations either conjugated to the carbonyl group (2E) or located in the middle of the chain (9Z). A nonacylated, sulfated chitin tetramer was unable to elicit nodule formation. Acylation with short (C8) chains rendered the LCO active at 10(-7) M. The optimal chain length was C16, with the C16-LCO being more than 10-fold more active than the C12- and C18-LCOs. Unsaturations were important, and the diunsaturated 2E,9Z LCO was more active than the monounsaturated LCOs. We discuss different hypotheses for the role of the acyl chain in NF perception.

The nolL gene from Rhizobium etli determines nodulation efficiency by mediating the acetylation of the fucosyl residue in the nodulation factor.

The nodulation factors (Nod factors) of Rhizobium etli and R. loti carry a 4-O-acetyl-L-fucosyl group at the reducing end. It has been claimed, based on sequence analysis, that NolL from R. loti participates in the 4-O-acetylation of the fucosyl residue of the Nod factors, as an acetyl-transferase (D. B. Scott, C. A. Young, J. M. Collins-Emerson, E. A. Terzaghi, E. S. Rockman, P. A. Lewis, and C. E. Pankhurst. Mol. Plant-Microbe Interact. 9:187-197, 1996). Further support for this hypothesis was obtained by studying the production of Nod factors in an R. etli nolL::Km mutant. Chromatographic and mass spectrometry analysis of the Nod factors produced by this strain showed that they lack the acetyl-fucosyl substituent, having a fucosyl group instead. Acetyl-fucosylation was restored upon complementation with a wild-type nolL gene. These results indicate that the nolL gene determines 4-O-acetylation of the fucosyl residue in Nod factors. Analysis of the predicted NolL polypeptide suggests a transmembranal location and that it belongs to the family of integral membrane transacylases (J. M. Slauch, A. A. Lee, M. J. Mahan, and J. J. Mekalanos. J. Bacteriol. 178:5904-5909, 1996). NolL from R. loti was also proposed to function as a transporter; our results show that NolL does not determine a differential secretion of Nod factors from the cell. We also performed plant assays that indicate that acetylation of the fucose conditions efficient nodulation by R. etli of some Phaseolus vulgaris cultivars, as well as of an alternate host (Vigna umbellata).

Carbamoylation of azorhizobial Nod factors is mediated by NodU.

Lipochitooligosaccharides (LCOs) synthesized by Azorhizobium caulinodans ORS571 are substituted at the nonreducing-terminal residue with a 6-O-carbamoyl group. LCO biosynthesis in A. caulinodans is dependent on the nodABCSUIJZnoeC operon. Until now, the role of the nodulation protein NodU in the synthesis of azorhizobial LCOs remained unclear. Based on sequence similarities and structural analysis of LCOs produced by a nodU mutant, a complemented nodU mutant, and Escherichia coli DH5 alpha expressing the nodABCSU genes, NodU was shown to be involved in the carbamoylation step.

nolO and noeI (HsnIII) of Rhizobium sp. NGR234 are involved in 3-O-carbamoylation and 2-O-methylation of Nod factors.

Loci unique to specific rhizobia direct the adjunction of special groups to the core lipo-oligosaccharide Nod factors. Host-specificity of nodulation (Hsn) genes are thus essential for interaction with certain legumes. Rhizobium sp. NGR234, which can nodulate >110 genera of legumes, possesses three hsn loci and secretes a large family of Nod factors carrying specific substituents. Among them are 3-O (or 4-O)- and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O (and 3-O)-acetyl substituents. The hsnIII locus comprises a nod box promoter followed by the genes nodABCIJnolOnoeI. Complementation and mutation analyses show that the disruption of any one of nodIJ, nolO, or noeI has no effect on nodulation. Conjugation of nolO into Rhizobium fredii extends the host range of the recipient to the non-hosts Calopogonium caeruleum and Lablab purpureus, however. Chemical analyses of the Nod factors produced by the NodI, NolO, and NoeI mutants show that the nolO and noeI gene products are required for 3 (or 4)-O-carbamoylation of the nonreducing terminus and for 2-O-methylation of the fucosyl group, respectively. Confirmation that NolO is a carbamoyltransferase was obtained from analysis of the Nod factors produced by R. fredii containing nolO; all are carbamoylated at O-3 (or O-4) on the nonreducing terminus. Since mutation of both nolO and nodU fails to completely abolish production of monocarbamoylated NodNGR factors, it is clear that a third carbamoyltransferase must exist. Nevertheless, the specificities of the two known enzymes are clearly different. NodU is only able to transfer carbamate to O-6 while NolO is specific for O-3 (or O-4) of NodNGR factors.

Combined mass spectrometric methods for the characterization of human hemoglobin variants localized within alpha T9 peptide: identification of Hb Villeurbanne alpha 89 (FG1) His-->Tyr.

Mutation-induced amino acid exchanges occurring on the large T9 peptide of the alpha-chain of human hemoglobin (residues 62-90) are difficult to identify. Despite their high m/z value (around m/z 3000), collision-induced dissociation spectra of liquid secondary ion mass spectrometrically generated protonated alpha T9 peptides were performed successfully. In parallel electrospray mass spectrometry (MS) was used both to measure the molecular mass of the intact proteins and to determine the number of protonatable sites in the alpha T9 peptides. Peptide ladder sequencing using carboxypeptidase digestions and analysis of the truncated peptides by matrix-assisted laser desorption ionization time-of-flight MS confirmed the interpretation. This set of methods allowed the characterization of three hemoglobin variants, with amino acid exchanges located in the alpha T9 part of the sequence. Two of them, Hb Aztec [alpha 76(EF5) Met-->Thr] and Hb M-Iwate [alpha 87(F8) His-->Tyr] were already known. The third [alpha 89(FG1) His-->Tyr] was novel and named Hb Villeurbanne.

Rhizobium sp. strain NGR234 NodZ protein is a fucosyltransferase.

Rhizobium sp. strain NGR234 produces a large family of lipochitooligosaccharide Nod factors carrying specific substituents. Among them are 3-O- (or 4-O-) and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O-acetyl substitutions. Investigations on the genetic control of host specificity revealed a number of loci which directly affect Nod factor structure. Here we show that insertion and frameshift mutations in the nodZ gene abolish fucosylation of Nod factors. In vitro assays using GDP-L-fucose as the fucose donor show that fucosyltransferase activity is associated with the nodZ gene product (NodZ). NodZ is located in the soluble protein fraction of NGR234 cells. Together with extra copies of the nodD1 gene, the nodZ gene and its associated nod box were introduced into ANU265, which is NGR234 cured of the symbiotic plasmid. Crude extracts of this transconjugant possess fucosyltransferase activity. Fusion of a His6 tag to the NodZ protein expressed in Escherichia coli yielded a protein able to fucosylate both nonfucosylated NodNGR factors and oligomers of chitin. NodZ is inactive on monomeric N-acetyl-D-glucosamine and on desulfated Rhizobium meliloti Nod factors. Kinetic analyses showed that the NodZ protein is more active on oligomers of chitin than on nonfucosylated NodNGR factors. Pentameric chitin is the preferred substrate. These data suggest that fucosylation occurs before acylation of the Nod factors.

Nod factors of Azorhizobium caulinodans strain ORS571 can be glycosylated with an arabinosyl group, a fucosyl group, or both.

In addition to the previously described arabinosylated Nod factors, Azorhizobium caulinodans can also produce fucosylated Nod factors and Nod factors that are both arabinosylated and fucosylated. The presence of a plasmid carrying extra copies of a subset of nod genes as well as bacterial growth conditions influence the relative proportion of carbamoylated, fucosylated, and arabinosylated Nod factors. By using a root hair formation assay, we demonstrate that the Nod factor glycosylations are important for biological activity on Sesbania rostrata 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, noeE, which 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 (NGRdelta noeE) 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 NGRdelta noeE 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.

Sinorhizobium teranga bv. acaciae ORS1073 and Rhizobium sp. strain ORS1001, two distantly related Acacia-nodulating strains, produce similar Nod factors that are O carbamoylated, N methylated, and mainly sulfated.

We have determined the structures of Nod factors produced by strains representative of Sinorhizobium teranga bv. acaciae and the so-called cluster U from the Rhizobium loti branch, two genetically different symbionts of particular Acacia species. Compounds from both strains were found to be similar, i.e., mainly sulfated, O carbamoylated, and N methylated, indicating a close relationship between host specificity and Nod factor structure, regardless of the taxonomy of the bacterial symbiont.

Mycobacterium bovis BCG genes involved in the biosynthesis of cyclopropyl keto- and hydroxy-mycolic acids.

The resurgence of tuberculosis and the emergence of multidrug-resistant mycobacteria necessitate the development of new antituberculosis drugs. The biosynthesis of mycolic acids, essential elements of the mycobacterial envelope, is a good target for chemotherapy. Species of the Mycobacterium tuberculosis complex synthesize oxygenated mycolic acids with keto and methoxy functions. In contrast, the fast-growing Mycobacterium smegmatis synthesizes oxygenated mycolic acids with an epoxy function. We describe the isolation and sequencing of a cluster of four genes from Mycobacterium bovis bacillus Calmette-Guerin (BCG), coding for methyl transferases, and which, when transferred into M. smegmatis, allow the synthesis of ketomycolic acid, in addition to an as yet undescribed mycolic acid, hydroxymycolic acid. These oxygenated mycolic acids, unlike the regular mycolic acids of M. smegmatis, and similar to the mycolic acids of M. bovis, are highly cyclopropanated. Furthermore, there is a perfect match between the structures of the keto- and the hydroxy-mycolic acids. We propose a biosynthetic model in which there is a direct relationship between these two types of mycolic acid.