Jean-Claude Promé

Identification et caractérisation des précurseurs biologiques des carraghénanes par spectroscopie de r.m.n.-13C

Abstract Carrageenans from the following red algae, Eucheuma spinosum , Eucheuma cottonii (Solieriaceae), Chondrus crispus gametophytes, and Gigartina stellata (Gigartinaceae) have been fractionated in order to obtain enriched fractions of carrageenan precursor(s) (μ- and ν-carrageenans). I.r. and 13 C-n.m.r. spectroscopy have been used to identify structures present in these fractions and in total extracts from Ahnfeltia concinna and Ahnfeltia gigartinoides (Phyllophoraceae) and both methods are compared. ν-Carrageenan (precursor of ι-carrageenan) was the only precursor that could be detected in E. spinosum , E. cottonii , and the two Ahnfeltia species. E. cottonii fraction was also enriched in ι-carrageenan; this structure was partially methylated at O-6 of the d -galactose residue (m.s. determination). μ-Carrageenan (precursor of κ-carrageenan) was identified as the only precursor in the gametophyte fraction of C. crispus . Both structures μ and ν were present in G. stellata . 13 C-N.m.r. spectroscopy is shown to be a good method of characterizing these structures, giving more accurate results than i.r. spectroscopy.

Symbiotic and Taxonomic Diversity of Rhizobia Isolated from Acacia tortilis subsp. raddiana in Africa

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana from various sites in the North and South of Sahara was analyzed for their diversity at both taxonomic and symbiotic levels. On the basis of whole cell protein (SDS-PAGE) and 16S rDNA sequence analysis, most of the strains were found to belong to the Sinorhizobium and Mesorhizobium genera where they may represent several different genospecies. Despite their chromosomal diversity, most A. tortilis Mesorhizobium and Sinorhizobium symbionts exhibited very similar symbiotic characters. Nodulation tests showed that the strains belong to the Acacia-Leucaena-Prosopis nodulation group, although mainly forming non-fixing nodules on species other than A. tortilis. Most of the strains tested responded similarly to flavonoid nod gene inducers, as estimated by using heterologous nodA-lacZ fusions. Thin layer chromatography analysis of the Nod factors synthesized by overproducing strains showed that most of the strains exhibited similar profiles. The structures of Nod factors produced by four different Sinorhizobium sp. strains were determined and found to be similar to other Acacia-Prosopis-Leucaena nodulating rhizobia of the Sinorhizobium-Mesorhizobium-Rhizobium branch. They are chitopentamers, N-methylated and N-acylated by common fatty acids at the terminal non reducing sugar. The molecules can also be 6-O sulfated at the reducing end and carbamoylated at the non reducing end. The phylogenetic analysis of available NodA sequences, including new sequences from A. tortilis strains, confirmed the clustering of the NodA sequences of members of the Acacia-Prosopis-Leucaena nodulation group.

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, anl-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 thel-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.

First observation of non-covalent complexes for a tannin–protein interaction model investigated by electrospray ionisation mass spectroscopy

Abstract Non-covalent complexes for a tannin–protein interaction model have been analyzed by mass spectrometry. The model is the polyphenol penta- O -galloyl- d -glucopyranose (PGG), a representative member of the hydrolysable tannin family and the nonapeptide hormone bradykinin (BDK). This is the first observation by electrospray ionisation mass spectrometry of non-covalent complexes for a tannin–protein interaction model. The technique should prove to be a powerful tool of investigation in this field.

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.

The pivotal role of tandem mass spectrometry in structural determinations of Nod factors produced by Rhizobia: Nod factors produced by wild-type strains of Mesorhizobium huakii and Rhizobium sp. mus10

Abstract Nod factors are signalling molecules secreted by bacteria of the Rhizobium family that plays an important role in initiating the early events of the nitrogen-fixing symbiosis between Rhizobia and legumes. They possess a lipochitooligomeric structure decorated by several functional groups. The structural details are essential for their specific recognition by plants and for switching on a genetic mechanism leading to the formation of nodules. Mass spectrometry is now able to fully characterise these molecules, even within mixtures and on minute amounts. It opens the possibility to study Nod factors from wild-type low-producing strains. It was demonstrated that Nod factors from the wild-type strain of Mesorhizobium huakii are almost the same as those produced by a genetically engineered over-producing strain. Rhizobium sp. mus10 is an Indian strain that nodulates the plant Sesbania. It was found that several Nod factors produced by this strain are identical to those produced by African strains that nodulate Sesbania species. However, two other Nod factors possessed new structural features. This synthetic possibility was probably acquired by the Rhizobium sp. mus10 strain by evolution in a different environment.

Hb Neuilly-Sur-Marne, a New Human Hemoglobin Variant with Ser-Asp-Leu Inserted between α86(F7) Leu and α87(F8) His: Characterization by High-Energy Collision-Induced Dissociation Liquid Secondary Ion Mass Spectrometry and Low Energy Collision-Induced Dissociation Tandem Mass Spectrometry in An Ion Trap Fitted with a Nanospray Ionization Source:

Hemoglobin (Hb) Neuilly-sur-Marne is a new α-chain variant found during a systematic screening. Electrospray mass measurements showed the presence of an abnormal α-chain displaying a shift of +315 u relative to the normal value. Tryptic cleavage of this chain and molecular weight determination of the peptides indicated that the 315 u shift was located into the αT-9 peptide, the molecular weight of which is higher than 3000 Da. High-energy collision spectra of MH+ ions generated by liquid secondary ion mass spectrometry from the normal and abnormal αT-9 afforded mainly amino-terminal containing ions. They indicated that these two peptides have an identical amino acid sequence from their 1st to 25th residues, the mass increase being thus located beyond this point. Too few ions were formed to establish reliably the sequence forward. It was hypothesized that this mass shift could result from a repeated sequence since the sum of the mass of the three residues—leucine, serine and aspartic acid—preceding position 25 is exactly 315 u. To get sequence information above position 25, decomposition of multicharged species was attempted. An ion trap fitted with a nanospray ionization source was used. It produced mainly triply- and quadruply-charged ions. Decomposition of the triply-charged ion afforded a series of singly-charged Y-ions in the expected region, giving a readily interpretable sequence. It confirmed the insertion of a Ser-Asp-Leu sequence above position 25. Surprisingly, decomposition of the quadruply-charged molecular ion gave too few ions to provide sequence information in the expected region. Spectra were dominated by some multicharged Y ions arising from cleavages close to the amino end. Tandem mass spectrometry experiments were performed on the abundant Y303+ ion and produced again a singly-charged Y ion series in the suitable domain which confirmed the above result. In Hb Neuilly-sur Marne this insertion of the Ser-Asp-Leu residues. between positions α-86 and α-87 is very likely due to a slipped strand mispairing mechanism.

Structural Determination of Unsaturated Long Chain Fatty Acids from Mycobacteria by Capillary Gas Chromatography and Collision Activation Dissociation Mass Spectrometry

Mycobacteria synthesize considerable quantities of a wide variety of fatty acids. In the mycolic acid series, for example, fatty acids containing over 80 carbon atoms have been detected. They represent the major lipid components of the cell walls of these organisms, and many studies have been devoted to their characterization. The shorter chain fatty acids (below C40) have also been studied extensively, especially the branched chain series.1 In fact mycobacteria produce highly complex mixtures of fatty acids. In Mycobacterium phlei, for example, more than 40 different fatty acids from C12 to C36 have been detected by gas chromatography-mass spectrometry (GC-MS).2

Early Events in the Azorhizobium Caulinodans—Sesbania Rostrata Symbiosis

The symbiotic interaction between the tropical leguminous plant Sesbania rostrata and the bacterium Azorhizobium caulinodans strain ORS571 is in many aspects a unique one (De Bruijn, 1989). In contrast to most legumes that form nitrogen-fixing nodules only on the roots, S. rostrata can be nodulated on the roots and the stem. The aerial nodules are formed at predetermined sites that are dormant root primordia located in vertical rows along the stem. These primordia develop into roots when immersed in water and/or into nodules upon infection with A. caulinodans. Root and stem nodule organogenesis are very similar and are intermediate between the indeterminate and determinate types of nodule development (Ndoye et al., 1994). Nodulation of S. rostrata stems is relatively unaffected by combined nitrogen and can be very extensive resulting in an extremely high fixation rate per plant. The closest relative of A. caulinodans is the diazotroph Xanthobacter, and A. caulinodans is unique amongst rhizobia in being capable of both symbiotic and free-living nitrogen fixation, the latter with assimilation of the formed ammonium for growth.