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
Email	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

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.

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