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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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Project description:The control of amino acid synthesis and transport in bacteria has been well-investigated at the transcriptional level. The discovery of a small Hfq-dependent regulatory RNA, GcvB, added another layer of gene expression control at the post-transcriptional level. GcvB RNA has been shown to directly regulate multiple ABC transporters for amino acids in E. coli and Salmonella using a highly conserved G/U-rich domain, R1. To identify additional GcvB targets, we have combined a sRNA pulse-expression and microarray analysis of whole transcriptome changes with biocomputational target searches for C/A- rich target sites in Salmonella. Moreover, we have included GcvB mutant RNAs in our microarray approach providing a new target search approach by inactivating conserved domains or target interaction sites. This dual approach revealed further amino acid transporters and, in addition, genes involved in amino acid metabolism as consensus R1-dependent GcvB targets. Moreover, GcvB RNA seems to bind with at least two binding sites to an R1-independent target, the glycine transporter cycA. Using GFP reporter gene fusions we have now validated 21 GcvB targets which is best to our knowledge with ~1% of all Salmonella protein coding genes, the largest bacterial sRNA-controlled regulon. Intriguingly, GcvB rewires many primary control circuits and, thus, constitutes an important metabolic knot. To predict direct GcvB targets with better confidence, we expanded the sRNA pulse-expression approach to assay effects of several versions of GcvB sRNA. We cloned GcvB wild-type and mutants RNAs deleted for the conserved regions R1 or R2 (Fig. 1A) under control of an arabinose-inducible PBAD promoter, yielding plasmid pBAD-GcvB (pKP1-1), pBAD-GcvB delta R1 (pKP2-6) and pBAD-GcvB delta R2 (pKP30-1). For confirmation of inducible expression, Salmonella wild-type carrying pBAD control vector (pKP8-35), and Salmonella ΔgcvB carrying either control vector (Ctr) or the above GcvB expression plasmids were grown to mid-exponential phase (OD600 of 1) and treated with L-arabinose for up to 15 min. We used whole-genome S. typhimurium microarrays to determine relative mRNA expression changes at 10 min of induction, comparing the mRNA profiles of the pBAD-GcvB, pBAD-GcvB delta R1 or pBAD-GcvB delta R2 strains to that of the delta gcvB deletion mutant strain carrying the control vector. Microarrays used in this study were produced by in-situ synthesis as 8x15k multipack format from Agilent Technologies. Each microarray comprises 13268 60-mer S. typhimurium strain SL1344 specific oligonucleotides supplemented with 319 60-mer S. enterica subsp. serovar Typhimurium 14028S specific oligonucleotides, 360 60-mer S. typhimurium LT2 specific oligonucleotides and 360 60-mer oligonucleotides specific for 149 Salmonella sRNAs. The experimental design involves the use of Salmonella enterica serovar Typhimurium genomic DNA as the co-hybridized control for one channel on all microarrays. Two independent biological eperiments were analyzed.

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Characterization of a glutamate racemase from an uncultivated bacterium

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