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Characterization of a glutamate racemase from an uncultivated bacterium

ABSTRACT: Project description: In this study, we have characterized with distinct biochemical assays the activity of a predicted glutamate racemase from an uncultivated bacterium (Candidatus Saccharimonas aalborgenesis), classified taxonomically within the Candidate Phyla Radiation (CPR). This racemase was produced in a Salmonella auxotrophic mutant defective in its endogenous glutamate racemase. Despite the low identity of the predicated racemase from this uncultivated bacterium compared to that of Salmonella (MurI), ~32.0%, it exhibited high specificity for glutamate as substrate in in vitro racemization assays among the rest of canonical amino acids. Moreover, the expression of this novel racemase complemented the D-Glu auxotrophy of the host bacterium used as chassis (Salmonella). Besides these physiological analyses, the study also characterized the composition and structure of the peptidoglycan produced by bacteria expressing each of the two glutamate racemases, exogenous and endogenous. Data processing protocol: Three biological replicates of two Salmonella strains expressing either the predicted glutamate racemase from the uncultivated bacterium Candidatus Saccharimonas aalborgenesis (Delta-murI-CPR, R1-R3) or the endogenous glutamate racemase (MurI) (Delta-murI-SAL, R1-R3) were grown in the nutrient rich medium LB to reach the exponential phase. At this time, bacteria were collected by centrifugation, washed and lysed by boiling in a 4% SDS-containing buffer. From this material, peptidoglycan was purified by standard protocol. Briefly, after high-speed centrifugation, the pellet containing peptidoglycan as the only SDS-insoluble material was subjected to successive enzymatic treatments with alpha-amylase, pronase (as protease) and, muramidase to cleave the glycan chains and to yield individual uncross-linked and cross-linked muropeptides. The muropeptide mixtures were analysed by LC-MS/MS to obtain a list of parental masses representing the complexity of the sample, i.e. the diversity of co-existing muropeptide classes. The parental masses were assigned as "muropeptides" when a mass of 204.3, corresponding to the N-acetyl-glucosamine molecule, was detected in the fragmentation spectra. The complete lists of parental masses were examined using FreeStyle (Thermo) software and compared among samples (biological replicas and control-problem pairs) by statistical parameters using XCMS software.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Salmonella Enterica Subsp. Enterica Serovar Typhimurium (ncbitaxon:90371)

SUBMITTER: Francisco Garcia del Portillo

PROVIDER: MSV000093018 | MassIVE | Tue Oct 03 07:54:00 BST 2023

REPOSITORIES: MassIVE

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Characterization of a glutamate racemase from an uncultivated bacterium

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