Signalling events elicited in plants by defined oligosaccharide structures.

Developments during the past year have confirmed that several classes of oligosaccharides are able to activate the plant cell machinery, leading either to defence reactions or to plant developmental processes. Both fungal and plant cell walls contain molecules that elicit plant defence reactions; however, most of the studies focus on the activities induced by lipochito-oligomers (LCOs, or Nod factors) produced by bacteria which trigger plant infection and nodule formation (organogenesis). LCOs can be described as growth regulators of plants in general as they also induce protoplast cell divisions of tobacco plant (a nonlegume) at femtomolar concentrations. Recognition of the appropriate symbiotic bacteria by legumes is mediated by the structure of Nod factors, but the structural determinants involved in this recognition process are not always clearly understood. Although specific substitutions of the oligochitin backbone by several chemical groups are involved, it seems that some host range variations of the bacteria can only be explained by small variations in the hydrophobic balance between both ends of the molecule.

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.

Conversion of sterols and triterpenes by mycobacteria. II. Transformation of 7-oxygenated sterols into androstane derivatives via a 7-deoxygenation.

The bioconversion of 7-oxygenated sterols by Mycobacterium aurum was studied in a preliminary investigation of the microbial conversion of wool wax. 7-Oxocholesterol was found to be transformed mainly into 3,17-dioxygenated androstane derivatives. 7 xi-Hydroxylated sterols were formed in an initial reduction step, and the C-7 hydroxyl group was then eliminated in a dehydration reaction. This was thought to take place during the isomerisation of cholest-4-en-3-one to cholest-5-en-3-one. Deuterium labelling experiments showed that this elimination proceeded faster for the C-7 alpha isomer, although it was not stereospecific. The C-7 alpha and C-7 beta-hydroxy isomers were weakly interconverted via the 7-oxo derivatives. Cholest-4-en-3-one, cholest-1,4-dien-3-one and cholest-4,6-dien-3-one all lost their side chains following a hydrogenation/dehydrogenation reaction. The resulting 3,17-dioxoandrostene or 3,17-androstadiene derivatives were mainly hydrogenated into 5 alpha-androstane-3,17-dione and 5 alpha-androstane-3 beta-ol-17-one. Elimination of the 3 beta-hydroxyl groups giving cholesta-3,5-dien-7-one, and subsequent microbial degradation of the side chain was not observed to any significant extent. The convergence of the bioconversion pathways of cholesterol and the 7-oxygenated cholesterols enabled crude, partially auto-oxidised cholesterol to be used as a substrate for the production of 3,17-dioxygenated androstane derivatives by M. aurum.

Conversion of sterols and triterpenes by mycobacteria. I Formation of progesterone and 1-dehydroprogesterone from Mycobacterium aurum, strain A+.

Side-chain degradation of sterols by bacteria is known to proceed via oxidation of a terminal methyl group followed by a succession of beta-oxidative steps. By this pathway, the pregnane backbone is not produced. However, examination of cholesterol degradation products using a strain of Mycobacterium aurum shows that progesterone and 1-dehydroprogesterone are present at low levels. These pregnane derivatives were identified by gas-liquid chromatography combined with mass spectrometry. This indicates that an alternative pathway for sterol side-chain degradation occurs in bacteria, which could be of great interest for the biological production of corticosteroid precursors.

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.

Biological activity of Rhizobium sp. NGR234 Nod-factors on Macroptilium atropurpureum.

The broad host range of Rhizobium sp. NGR234 is based mainly on its ability to secrete a family of lipooligosaccharide Nod factors. To monitor Nod-factor purification, we used the small seeded legume Macroptilium atropurpureum, which responds evenly and consistently to Nod factors. At concentrations between approximately equal to 10(-11) M and 10(-9) M, this response takes the form of deformation of the root hairs. Higher concentrations (approximately equal to 10(-9) to 10(-7) M), provoked profound "shepherd's crook" type curling of the root hairs. Similar concentrations of Nod factors of Bradyrhizobium japonicum, Rhizobium leguminosarum, and R. meliloti also provoked marked curling of the root hairs, but the latter two species are unable to nodulate Macroptilium. On the other hand, plant hormones, hormone-like substances, inhibitors of hormone action, as well as substituents of Nod factors were without effect in this bioassay. We thus conclude that only Nod factors are capable of inducing shepherd's crook type curling of Macroptilium root hairs. Perturbations in the auxin-cytokinin balance induced "pseudo" nodulation on M. atropurpureum, as did NodNGR factors at concentrations between 10(-7) and 10(-6) M. Concomitant inoculation of Macroptilium with a NodABC- mutant of NGR234 and sulfated NodNGR factors (NodNGR[S]) gave rise to plants that slowly greened, showing that the NodNGR factors permitted entry of the Nod- mutant into the roots.

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 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.

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).

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.

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.

Rhizobium tropici nodulation factor sulfation is limited by the quantity of activated form of sulfate.

Rhizobium tropici is a broad host-range symbiont of Phaseolus vulgaris. This bacterium produces a mixture of sulfated and non-sulfated N-methylated pentameric nodulation (Nod) factors. To understand the genetic bases of the partial sulfation of R. tropici Nod factors, which might be involved in the broad host-range of this species, we introduced in R. tropici CFN299 the recombinant plasmid pGMI515 carrying a set of nodulation (nod) genes of R. meliloti, including those involved in the sulfation of R. meliloti Nod factors. The CFN299 (pGMI515) transconjugant produced only sulfated Nod factors, but approximately half of them were no more N-methylated. Mutations in R. meliloti nodH gene did not decrease the Nod factor sulfation whereas inactivation of the nodPQ genes restored the production of a mixture of sulfated and non-sulfated molecules. These results suggest that the limiting step in R. tropici Nod factor sulfation is the production of activated sulfate donors. Mutations in the R. meliloti nodFEG and nodH genes did not change the N-methylation pattern, whereas mutations in nodPQ increased the degree of N-methylation, suggesting a metabolic link between sulfation and methylation of R. tropici Nod factors.

Purification and characterization of the 210-amino acid recombinant basic fibroblast growth factor form (FGF-2).

Four forms of basic fibroblast-growth factor (bFGF or FGF-2) using one AUG (155 amino acids) and three upstream CUG (210, 201 and 196 amino acids) start codons, were synthesized through an alternative use of initiation codons. The 210-amino acid form of FGF-2 (210FGF-2) was expressed in a plasmid vector under the control of a bacteriophage T7 RNA polymerase promoter system in Escherichia coli. Characterization of the purified protein was performed by electrospray mass spectrometry and Edman degradation. The recombinant 210FGF-2 produced in E. coli had a mitogenic activity similar to the 146-amino acid form extracted from tissues.

Class I and IVa beta-tubulin isotypes expressed in adult mouse brain are glutamylated.

Several types of post-translational modifications contribute to the high level of tubulin heterogeneity in the brain. An important modification is glutamylation of the major brain-specific isotypes, such as class Ia/b of alpha-tubulin and classes II and III of beta-tubulin. Here we describe experiments to determine if additional, minor tubulin isotypes, expressed in adult mouse brain, could also be glutamylated. Purified tubulin from adult mouse brain was cleaved with thermolysin. Proteolytically released carboxy-terminal peptides of both alpha- and beta-tubulin were isolated by sequential anion exchange and reverse-phase column-chromatography. Anionic peptides were then characterized by amino acid sequencing and mass spectrometry. We show that brain-specific class IVa and constitutive class I beta-tubulin isotypes can be glutamylated, at Glu434 and Glu441, respectively.

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. USADA191 and 257DH4 both produce a novel LCO with glucose substituted for N-acetyl-D-glucosamine in the backbone of the molecule.

Use of combined mass spectrometry methods for the characterization of a new variant of human hemoglobin: The double mutant hemoglobin villeparisis ß77(EF1) His → Tyr, ß 80 (EF4) Asn → Ser.

Hemoglobin Villeparisis was found during a systematic measurement of glycated hemoglobin. Electrospray mass spectra of the globin indicate an apparently unchanged molecular weight within the error range (0.01%). The tryptic digest of the ß chain shows a chromatographically abnormal ßT-9 peptide. The mass-to-charge ratio value of its [M+H](+) ion, as measured by liquid secondary ionization mass spectrometry, is one mass unit lower than that of the normal ßT-9. However, the electrospray mass spectrum of this peptide exhibits mainly a doubly charged ion, whereas the normal ßT-9 gives a triply charged ion. None of the allowed single amino acid substitutions for a 1-u shift down (Glu → Gln, Asp → Asn, or Asn → Ile) can explain the suppression of one protonation site. This can be due only to the replacement of the internal histidine by a nonbasic residue. Thus at least two amino acid exchanges occur within the same peptide: one involves the internal histidine, and the sum of the mass shifts is -1 u. Consideration of the ßT-9 sequence and taking account for the genetic code rules, the only possibility was (11)His → Tyr (+26 mass shift) associated with (14)Asn → Ser (-27 mass shift). This conclusion was consistent with the tandem mass spectrum of the [M+H](+) ion and was further confirmed by chemical microsequencing.

Fast atom bombardment mass spectrometry of Mycobacterial glycopeptidolipid antigens: Structural characterization by charge remote fragmentation.

Mycobacteria contain species- and type-specific antigens. Among them, glycopeptidolipids are present in medically relevant organisms belonging to Mycobacterium avium or M. fortuitum complexes. Fast-a tom bombardment mass spectrometry of glycopeptidolipids has proven to be difficult. In this article the cationization method with a metanitrobenzyl alcohol matrix, doped with sodium iodide, is described for analyzing these molecules. The molecular weight of the intact glycopeptidolipids was successfully determined and, using mass-analyzed ion kinetic energy spectrometry, the complete sequences of the peptide and saccharide moieties were elucidated. Moreover, the two structural variants present in these molecules were clearly differentiated. Application of the method showed that the same structural variant occurs in the glycopeptidolipids from two serologically related species of the M. fortuitum complex.

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.

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.

Isolation and structural characterization of a new non-phosphorylated lipoamino acid from Mycobacterium phlei.

A new non-phosphorylated lipoamino acid was extracted from Mycobacterium phlei, strain IST. It is particularly sensitive to alkaline hydrolysis, and contains a lysine residue joined to a 1,2-diglyceride via an ester linkage. The FAB-positive mass spectrum shows the presence of various molecular species of which the most abundant contains a palmitic and a tuberculostearic acid residue. An analogue of this lipid was synthesized, 1,2-dipalmitoyl 3-lysyl glycerol. Both its chromatographic behavior (TLC), and the decomposition pathways of the MH+ ions, studied by FAB MS and MIKE spectroscopy, were identical to the natural product.