Succinylome-controlled antibiotic resistance in MRSA - Global proteome

ABSTRACT: A sucC and a sucD mutant from the Nebraska Transposon Mutant Library (NTML, Strain NE569 and NE1770) were found to be susceptible to cefoxitin and oxacillin when other mutations in TCA cycle genes did not affect their resistance to these antibiotics. The SucC strain (Strain NE569) accumulated Succinyl CoA, which is known to result in higher succinylation rate. We compared the proteome of the sucC mutant in exponential and stationary stages to the one of the WT strain JE2. The global proteome of the following strains were analyzed by TMT-labelled proteomics: Early exponential JE2 (3Hrs), Exponential JE2 (4Hrs), Exponential SucC (4Hrs), Stationary JE2 (10Hrs), Stationary SucC (12Hrs). For each condition, the bacterial pellet was collected and washed twice with PBS. The proteins were extracted by bead beating, 1000 ug of proteins were reduced with DTT, alkylated with IAA and digested with 1:50 (trypsin:protein, w:w) for 3 Hr at 37 C. 100ug of peptides were labelled with TMT11Plex reagents. The samples were pooled and enriched for Succinyl-lysine containing peptides using the PTMScan Succinyl-Lysine Motif Enrichment Kit following the supplier's instructions. The flow through was cleaned-up by C18 solid phase extraction and fractionated into 12 fractions as previously described (PMID: 21500348). A sample (5 ul) of 0.1 ug/ul of peptides from each fraction was analyzed by reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Waters nanoEquity UPLC system coupled with a QExactive HF-X mass spectrometer.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Staphylococcus Aureus (ncbitaxon:1280)

SUBMITTER: James P. O'Gara   Geremy Clair  

PROVIDER: MSV000086976 | MassIVE | Tue Mar 02 14:33:00 GMT 2021

SECONDARY ACCESSION(S): PXD024473

REPOSITORIES: MassIVE