Dataset Information

Spectral library based analysis of arginine phosphorylations in Staphylococcus aureus

ABSTRACT: Reversible protein phosphorylation is one of the major mechanisms in the regulation of protein expression and protein activity, controlling physiological functions of the important human pathogen Staphylococcus aureus. Phosphorylations at serine, threonine and tyrosine are known to influence for example protein activity in central metabolic pathways and the more energy-rich phosphorylations at histidine, aspartate or cysteine can be found as part of two component system sensor domains or mediating bacterial virulence. In addition to these well-known phosphorylations, the phosphorylation at arginine residues plays an essential role. Hence, the deletion mutant S. aureus COL ΔptpB (protein tyrosine phosphatase B) was studied since the protein PtpB is assumed to be an arginine phosphatase. A gel-free approach was applied to analyse the changes in the phosphoproteome of the deletion mutant ΔptpB and the wild type in growing cells, thereby focusing on the occurrence of phosphorylation on arginine residues. In order to enhance the reliability of identified phosphorylation sites at arginine residues, a subset of arginine phosphorylated peptides was chemically synthesised. Combined spectral libraries based on phosphoenriched samples, synthetic arginine phosphorylated peptides and classical proteome samples provide a sophisticated tool for the analysis of arginine phosphorylations. This way, 212 proteins phosphorylated on serine, threonine, tyrosine or arginine residues were identified within the mutant ∆ptpB and 102 in wild type samples. Among them, 207 arginine phosphosites were identified exclusively within the mutant ∆ptpB, widely distributed along the whole bacterial metabolism. This identification of putative targets of PtpB allows further investigation of the physiological relevance of arginine phosphorylations and provides the basis for reliable quantification of arginine phosphorylations in bacteria.

INSTRUMENT(S): LTQ Orbitrap Velos, LTQ Orbitrap Elite

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Sandra Maass

LAB HEAD: Dörte Becher

PROVIDER: PXD007167 | Pride | 2017-12-06

REPOSITORIES: Pride

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Publications

Spectral Library Based Analysis of Arginine Phosphorylations in <i>Staphylococcus aureus</i>.

Junker Sabryna S Maaβ Sandra S Otto Andreas A Michalik Stephan S Morgenroth Friedrich F Gerth Ulf U Hecker Michael M Becher Dörte D

Molecular & cellular proteomics : MCP 20171128 2

Reversible protein phosphorylation is one of the major mechanisms in the regulation of protein expression and protein activity, controlling physiological functions of the important human pathogen <i>Staphylococcus aureus</i> Phosphorylations at serine, threonine and tyrosine are known to influence for example protein activity in central metabolic pathways and the more energy-rich phosphorylations at histidine, aspartate or cysteine can be found as part of two component system sensor domains or m ...[more]

PMID: 29183913

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Project description:Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologs of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in ?-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1? mutant of a guanidinobutyrase (EC.3.5.3.7), an enzyme not previously demonstrated in fungi. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. The goal of the present study was to characterize arginine catabolism in K. lactis. To this end, CAR1, CAR2 and PRO3 orthologs in K. lactis were identified and functionally analysed by deletion, expression in S. cerevisiae and enzyme activity assays. Since deletion of the arginase gene in K. lactis was found not to completely abolish growth on arginine as a sole nitrogen source, the alternative pathway for arginine catabolism operating in this yeast was studied by a combination of transcriptome analysis, 13C and 15N isotope-based flux analysis and enzyme activity assays in cell extracts. To investigate arginine metabolism in the arginase-negative K. lactis strain, strains GG1632 (Klku80? KlCAR1 reference strain) and IMS0367 (Klcar1? Arg+) were grown in aerobic bioreactor batch cultures on glucose chemically defined medium with arginine as sole nitrogen source. RNA sequencing of samples taken during the exponential phase of growth on glucose-arginine media of the reference strain G1631 and the arginase less strain IMS0367 were compared resulting in the characterization of a new function.

Dataset Information

Spectral library based analysis of arginine phosphorylations in Staphylococcus aureus

Publications

Spectral Library Based Analysis of Arginine Phosphorylations in <i>Staphylococcus aureus</i>.

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