Summary of Study ST001174
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000786. The data can be accessed directly via it's Project DOI: 10.21228/M83398 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001174 |
| Study Title | Role of ClpCP in respiratory and fermentative growth |
| Study Summary | To determine metabolite concentrations and differences at the 48 hour time point for WT, ClpC mutant, srrAB mutant, and ClpC:srrAB double mutant |
| Institute | Montana State University |
| Department | Chemistry and Biochemistry |
| Laboratory | Copie Lab |
| Last Name | Eilers |
| First Name | Brian |
| Address | 103 Chemistry and Biochemistry Bldg, RM 144, Valerie Copie Lab, Bozeman, Montana, 59717, USA |
| brian.eilers@montana.edu | |
| Phone | 4069945116 |
| Submit Date | 2019-04-24 |
| Num Groups | 4 (WT, ClpC, srrAB, and ClpC:srrAB) |
| Total Subjects | 4 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | 1r |
| Analysis Type Detail | NMR |
| Release Date | 2019-05-15 |
| Release Version | 1 |
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Project:
| Project ID: | PR000786 |
| Project DOI: | doi: 10.21228/M83398 |
| Project Title: | The ClpCP complex modulates respiratory, but not fermentative, metabolism in Staphylococcus aureus and is regulated in a SrrAB-dependent manner. |
| Project Summary: | The staphylococcal respiratory regulator (SrrAB) modulates energy metabolism in Staphylococcus aureus. Studies have suggested that regulated protein catabolism facilitates energy homeostasis. Regulated proteolysis in S. aureus is achieved through protein complexes composed of a peptidase (ClpQ or ClpP) in association with an AAA+ family ATPase (typically ClpC or ClpX). In the current report, we tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy homeostasis in S. aureus. Strains deficient in one or more Clp complexes are attenuated for growth in the presence of puromycin, which causes enrichment of misfolded proteins. A ΔsrrAB strain had increased sensitivity to puromycin. Epistasis experiments suggested that the puromycin sensitivity phenotype of the ΔsrrAB strain was a result of decreased ClpC activity. Consistent with this, transcriptional activity of clpC was decreased in the ΔsrrAB mutant and overexpression of clpC suppressed the puromycin sensitivity of the ΔsrrAB strain. Genetic studies suggested that phosphorylated SrrA is required to influence puromycin resistance. ClpC positively influenced respiration in association with ClpP. ClpP was also required for optimal fermentative growth, whereas ClpC was dispensable. Metabolomics studies demonstrated that intracellular metabolic profiles of the ΔclpC and ΔsrrAB mutants are distinct from the wild type strain, supporting the notion that both ClpC and SrrAB affect central metabolism. We propose a model wherein SrrAB regulates energy homeostasis, in part, via modulation of regulated proteolysis. |
| Institute: | Montana State University |
| Department: | Chemistry and Biochemistry |
| Laboratory: | Copie Lab |
| Last Name: | Eilers |
| First Name: | Brian |
| Address: | 103 Chemistry and Biochemistry Bldg, RM 144, Valerie Copie Lab, Bozeman, Montana, 59717, USA |
| Email: | brian.eilers@montana.edu |
| Phone: | 4069945116 |
| Publications: | Journal of Bacteriology (accepted with revisions) |
Subject:
| Subject ID: | SU001240 |
| Subject Type: | Bacteria |
| Subject Species: | Staphylococcus aureus |
| Taxonomy ID: | 1280 |
Factors:
Subject type: Bacteria; Subject species: Staphylococcus aureus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA081503 | ClpC_3 | ClpC deletion mutant |
| SA081504 | ClpC_4 | ClpC deletion mutant |
| SA081505 | ClpC_2 | ClpC deletion mutant |
| SA081506 | ClpC_1 | ClpC deletion mutant |
| SA081507 | ClpC-SrrAB_1 | ClpC-SrrAB double deletion mutant |
| SA081508 | ClpC-SrrAB_2 | ClpC-SrrAB double deletion mutant |
| SA081509 | ClpC-SrrAB_4 | ClpC-SrrAB double deletion mutant |
| SA081510 | ClpC-SrrAB_3 | ClpC-SrrAB double deletion mutant |
| SA081511 | SrrAB_4 | SrrAB deletion mutant |
| SA081512 | SrrAB_1 | SrrAB deletion mutant |
| SA081513 | SrrAB_3 | SrrAB deletion mutant |
| SA081514 | SrrAB_2 | SrrAB deletion mutant |
| SA081515 | WT_2 | wild type |
| SA081516 | WT_3 | wild type |
| SA081517 | WT_1 | wild type |
| SA081518 | WT_4 | wild type |
| Showing results 1 to 16 of 16 |
Collection:
| Collection ID: | CO001234 |
| Collection Summary: | Aerobic growth for all strains was performed on four biological replicates for each cell group. Overnight cultures diluted 1:1000 were used to inoculate 25 mL of fresh TSB in a 250 mL flask with 220 rpm agitation at 37° C. Aliquots of 10 mL were collected at 48 hrs , centrifuged at 5000 rpm for 5 minutes, then rinsed once with 1 mL of 1X PBS, and centrifuged at 5000 rpm for 5 minutes. The supernatant was discarded, and cell pellets were frozen at -80 °C until further use. |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR001255 |
| Treatment Summary: | deletion mutants were compared to the wild-type after 48 hours of growth |
Sample Preparation:
| Sampleprep ID: | SP001248 |
| Sampleprep Summary: | Frozen cell pellets were resuspended in 1 mL of a 2:1 methanol/chloroform mixture and transferred to FastPrep lysis B matrix tubes (MP Biomedicals). Cells were lysed using the FastPrep-24 5G instrument and designated S. aureus settings (2 cycles at a speed of 6.0 m/s for 40 s)]. 300 μL of each layer of a 1:1 aqueous chloroform solution was added to each cell lysate. The tubes were vortexed, placed at -20 °C for 20 min, and centrifuged at 14,000 g for 10 minutes. 800 μL of the aqueous phase was transferred to microfuge tubes and placed in a Speedvac (no heat, manual run, volatile solvent) to dry overnight. Samples were resuspended in 600 μL of NMR buffer [0.25 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), 0.4 mM imidazole, 25 mM phosphate buffer, 90% H2O/10% D2O] and transferred to 5 mm Bruker NMR tubes. |
Analysis:
| Analysis ID: | AN001949 |
| Analysis Type: | NMR |
| Operator Name: | Brian Tripet |
| Num Factors: | 4 |
| Num Metabolites: | 38 |
| Units: | Attomoles/CFU |
NMR:
| NMR ID: | NM000148 |
| Analysis ID: | AN001949 |
| Instrument Name: | Bruker 600-MHz AVANCE III solution NMR spectrometer |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| NMR Comments: | equipped with an automatic SampleJet sample loading system, as well as a 5 mm triple resonance (1H, 15N, 13C) liquid-helium-cooled TCI probe (cryoprobe) and Topspin software (Bruker version 3.2). |
| Spectrometer Frequency: | 1H Larmor frequency |
| NMR Probe: | cryoprobe |
| NMR Solvent: | 10%D20 |
| NMR Tube Size: | 5mm |
| Shimming Method: | topshim |
| Pulse Sequence: | zgesgp |
| Water Suppression: | qfil |
