Differentiation of O-acetyl and O-carbamoyl

Abstract

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.

Explanatory multivariate analysis of ToF-SIMS spectra for the discrimination of bacterial isolates

“…A possible biochemical origin for this signal is the oxonium ion from O-acetylated and O-carbamoylated N-acetylglucosamine, a collision induced dissociation fragment of which has been reportedly detected at m/z 84, in plant symbiotic bacteria. 31 Another candidate is the protonated pseudo-molecular ion of tetrahydropyridine, which like 1-pyrroline has also been detected in the head space of K. pneumoniae and C. freundii cultures. 28 The other likely origins are the amino acids glutamine, lysine or methylated proline.…”

“…The latter is a common intermediate of arginine and pyrimidine biosynthesis, 48 but is not specific to K. pneumoniae. Two other candidates for the peak at m/z 140 that are more likely are the fragment ion of 3-O-carbamoyl-1-O-methyl N-acetylglucosamine (oxonium ion fragments have been detected in plant symbiotic bacteria 31 ), and the sodium adduct of glycine betaine (betaines are known to serve as organic osmolytes, substances synthesised or taken up from the environment by cells for protection against osmotic stress, drought, high salinity or high temperature, and have been specifically observed in K. pneumoniae 49 ).…”

Acacia senegal and Prosopis chilensis-nodulating rhizobia Sinorhizobium arboris HAMBI 2361 and S. kostiense HAMBI 2362 produce tetra- and pentameric LCOs that are N-methylated, O-6-carbamoylated and partially sulfated

“…14;15 In a specific extension of the MS/MS approach, Treilhou et al studied the fragmentation patterns of fragment ions generated from N-acetylglucosamine esters and from LCOs, concentrating on ions containing the nonreducing terminal residues of LCOs. 16 They observed characteristic fragmentation patterns and product ion intensity ratios depending on the location of substituents on the nonreducing GlcNAc residue. They also noticed a characteristic ratio of product ions depending on the presence and absence of an N-methyl group.…”