Trapping and Identification of Cellular Substrates of the Staphylococcus aureus ClpC Chaperone

• Published in: Journal of Bacteriology 2013-10, Vol.195 (19), p.4506-4516
• Main Authors: Graham, Justin W, Lei, Mei G, Lee, Chia Y
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Chromatography; gel electrophoresis; Gene expression; Gene Expression Regulation, Bacterial - physiology; genes; Gram-positive bacteria; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; liquid chromatography; Mass spectrometry; mutation; protein-protein interactions; Proteins; Proteomics; quality control; Sequence Alignment; Staphylococcus aureus; Staphylococcus aureus - genetics; Staphylococcus aureus - metabolism; Staphylococcus infections; stress response; Substrates; tandem mass spectrometry; transcription factors; two hybrid system techniques
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/JB.00758-13

ClpC is an ATP-dependent Hsp100/Clp chaperone involved in protein quality control in low-GC Gram-positive bacteria. Previously, we found that ClpC affected the expression of a large number of genes, including capsule genes in Staphylococcus aureus. Here we constructed a His-tagged ClpC variant (ClpCtrap) with mutations within the Walker B motifs to identify the direct substrates of ClpC by copurification with ClpCtrap followed by gel electrophoresis combined with liquid chromatography-tandem mass spectrometry proteomics. We identified a total of 103 proteins that are potential substrates of ClpC in strain Newman. The direct protein-protein interaction of ClpC with a subset of the captured proteins was verified in a bacterial two-hybrid system. The captured proteins could be grouped into various functional categories, but most were related to proteins involved in the stress response. Several known ClpC substrates were captured, including ClpP, TrfA/MecA, ClpB, DnaK, DnaJ, GroL, RecA, and CodY, supporting the validity of our approach. Our results also revealed many new ClpC substrates, including AgrA, CcpA, RsbW, MurG, FtsA, SrtA, Rex, Atl, ClfA, and SbcC. Analysis of capsule production showed that three of the captured proteins, which were not previously known to be transcriptional regulators, did affect capsule production.