#METABOLOMICS WORKBENCH copielab_20190424_120010 DATATRACK_ID:1710 STUDY_ID:ST001174 ANALYSIS_ID:AN001949 PROJECT_ID:PR000786
VERSION             	1
CREATED_ON             	April 25, 2019, 3:56 pm
#PROJECT
PR:PROJECT_TITLE                 	The ClpCP complex modulates respiratory, but not fermentative, metabolism in
PR:PROJECT_TITLE                 	Staphylococcus aureus and is regulated in a SrrAB-dependent manner.
PR:PROJECT_SUMMARY               	The staphylococcal respiratory regulator (SrrAB) modulates energy metabolism in
PR:PROJECT_SUMMARY               	Staphylococcus aureus. Studies have suggested that regulated protein catabolism
PR:PROJECT_SUMMARY               	facilitates energy homeostasis. Regulated proteolysis in S. aureus is achieved
PR:PROJECT_SUMMARY               	through protein complexes composed of a peptidase (ClpQ or ClpP) in association
PR:PROJECT_SUMMARY               	with an AAA+ family ATPase (typically ClpC or ClpX). In the current report, we
PR:PROJECT_SUMMARY               	tested the hypothesis that SrrAB regulates a Clp complex to facilitate energy
PR:PROJECT_SUMMARY               	homeostasis in S. aureus. Strains deficient in one or more Clp complexes are
PR:PROJECT_SUMMARY               	attenuated for growth in the presence of puromycin, which causes enrichment of
PR:PROJECT_SUMMARY               	misfolded proteins. A ΔsrrAB strain had increased sensitivity to puromycin.
PR:PROJECT_SUMMARY               	Epistasis experiments suggested that the puromycin sensitivity phenotype of the
PR:PROJECT_SUMMARY               	ΔsrrAB strain was a result of decreased ClpC activity. Consistent with this,
PR:PROJECT_SUMMARY               	transcriptional activity of clpC was decreased in the ΔsrrAB mutant and
PR:PROJECT_SUMMARY               	overexpression of clpC suppressed the puromycin sensitivity of the ΔsrrAB
PR:PROJECT_SUMMARY               	strain. Genetic studies suggested that phosphorylated SrrA is required to
PR:PROJECT_SUMMARY               	influence puromycin resistance. ClpC positively influenced respiration in
PR:PROJECT_SUMMARY               	association with ClpP. ClpP was also required for optimal fermentative growth,
PR:PROJECT_SUMMARY               	whereas ClpC was dispensable. Metabolomics studies demonstrated that
PR:PROJECT_SUMMARY               	intracellular metabolic profiles of the ΔclpC and ΔsrrAB mutants are distinct
PR:PROJECT_SUMMARY               	from the wild type strain, supporting the notion that both ClpC and SrrAB affect
PR:PROJECT_SUMMARY               	central metabolism. We propose a model wherein SrrAB regulates energy
PR:PROJECT_SUMMARY               	homeostasis, in part, via modulation of regulated proteolysis.
PR:INSTITUTE                     	Montana State University
PR:DEPARTMENT                    	Chemistry and Biochemistry
PR:LABORATORY                    	Copie Lab
PR:LAST_NAME                     	Eilers
PR:FIRST_NAME                    	Brian
PR:ADDRESS                       	103 Chemistry and Biochemistry Bldg, RM 144, Valerie Copie Lab, Bozeman,
PR:ADDRESS                       	Montana, 59717, USA
PR:EMAIL                         	brian.eilers@montana.edu
PR:PHONE                         	4069945116
PR:PUBLICATIONS                  	Journal of Bacteriology (accepted with revisions)
#STUDY
ST:STUDY_TITLE                   	Role of ClpCP in respiratory and fermentative growth
ST:STUDY_SUMMARY                 	To determine metabolite concentrations and differences at the 48 hour time point
ST:STUDY_SUMMARY                 	for WT, ClpC mutant, srrAB mutant, and ClpC:srrAB double mutant
ST:INSTITUTE                     	Montana State University
ST:DEPARTMENT                    	Chemistry and Biochemistry
ST:LABORATORY                    	Copie Lab
ST:LAST_NAME                     	Eilers
ST:FIRST_NAME                    	Brian
ST:ADDRESS                       	103 Chemistry and Biochemistry Bldg, RM 144, Valerie Copie Lab, Bozeman,
ST:ADDRESS                       	Montana, 59717, USA
ST:EMAIL                         	brian.eilers@montana.edu
ST:PHONE                         	4069945116
ST:NUM_GROUPS                    	4 (WT, ClpC, srrAB, and ClpC:srrAB)
ST:TOTAL_SUBJECTS                	4
#SUBJECT
SU:SUBJECT_TYPE                  	Bacteria
SU:SUBJECT_SPECIES               	Staphylococcus aureus
SU:TAXONOMY_ID                   	1280
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	WT_1	Genotype:wild type	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	WT_2	Genotype:wild type	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	WT_3	Genotype:wild type	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	WT_4	Genotype:wild type	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	ClpC_1	Genotype:ClpC deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	ClpC_2	Genotype:ClpC deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	ClpC_3	Genotype:ClpC deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	ClpC_4	Genotype:ClpC deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	SrrAB_1	Genotype:SrrAB deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	SrrAB_2	Genotype:SrrAB deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	SrrAB_3	Genotype:SrrAB deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	SrrAB_4	Genotype:SrrAB deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	ClpC-SrrAB_1	Genotype:ClpC-SrrAB double deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	ClpC-SrrAB_2	Genotype:ClpC-SrrAB double deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	ClpC-SrrAB_3	Genotype:ClpC-SrrAB double deletion mutant	Treatment=48 hour growth
SUBJECT_SAMPLE_FACTORS           	-	ClpC-SrrAB_4	Genotype:ClpC-SrrAB double deletion mutant	Treatment=48 hour growth
#COLLECTION
CO:COLLECTION_SUMMARY            	Aerobic growth for all strains was performed on four biological replicates for
CO:COLLECTION_SUMMARY            	each cell group. Overnight cultures diluted 1:1000 were used to inoculate 25 mL
CO:COLLECTION_SUMMARY            	of fresh TSB in a 250 mL flask with 220 rpm agitation at 37° C. Aliquots of 10
CO:COLLECTION_SUMMARY            	mL were collected at 48 hrs , centrifuged at 5000 rpm for 5 minutes, then rinsed
CO:COLLECTION_SUMMARY            	once with 1 mL of 1X PBS, and centrifuged at 5000 rpm for 5 minutes. The
CO:COLLECTION_SUMMARY            	supernatant was discarded, and cell pellets were frozen at -80 °C until further
CO:COLLECTION_SUMMARY            	use.
CO:SAMPLE_TYPE                   	Bacterial cells
#TREATMENT
TR:TREATMENT_SUMMARY             	deletion mutants were compared to the wild-type after 48 hours of growth
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Frozen cell pellets were resuspended in 1 mL of a 2:1 methanol/chloroform
SP:SAMPLEPREP_SUMMARY            	mixture and transferred to FastPrep lysis B matrix tubes (MP Biomedicals). Cells
SP:SAMPLEPREP_SUMMARY            	were lysed using the FastPrep-24 5G instrument and designated S. aureus settings
SP:SAMPLEPREP_SUMMARY            	(2 cycles at a speed of 6.0 m/s for 40 s)]. 300 μL of each layer of a 1:1
SP:SAMPLEPREP_SUMMARY            	aqueous chloroform solution was added to each cell lysate. The tubes were
SP:SAMPLEPREP_SUMMARY            	vortexed, placed at -20 °C for 20 min, and centrifuged at 14,000 g for 10
SP:SAMPLEPREP_SUMMARY            	minutes. 800 μL of the aqueous phase was transferred to microfuge tubes and
SP:SAMPLEPREP_SUMMARY            	placed in a Speedvac (no heat, manual run, volatile solvent) to dry overnight.
SP:SAMPLEPREP_SUMMARY            	Samples were resuspended in 600 μL of NMR buffer [0.25 mM
SP:SAMPLEPREP_SUMMARY            	4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), 0.4 mM imidazole, 25 mM
SP:SAMPLEPREP_SUMMARY            	phosphate buffer, 90% H2O/10% D2O] and transferred to 5 mm Bruker NMR tubes.
#ANALYSIS
AN:OPERATOR_NAME                 	Brian Tripet
#NMR
NM:INSTRUMENT_NAME               	Bruker 600-MHz AVANCE III solution NMR spectrometer
NM:INSTRUMENT_TYPE               	FT-NMR
NM:NMR_EXPERIMENT_TYPE           	1D-1H
NM:NMR_COMMENTS                  	equipped with an automatic SampleJet sample loading system, as well as a 5 mm
NM:NMR_COMMENTS                  	triple resonance (1H, 15N, 13C) liquid-helium-cooled TCI probe (cryoprobe) and
NM:NMR_COMMENTS                  	Topspin software (Bruker version 3.2).
NM:SPECTROMETER_FREQUENCY        	1H Larmor frequency
NM:NMR_PROBE                     	cryoprobe
NM:NMR_SOLVENT                   	10%D20
NM:NMR_TUBE_SIZE                 	5mm
NM:SHIMMING_METHOD               	topshim
NM:PULSE_SEQUENCE                	zgesgp
NM:WATER_SUPPRESSION             	qfil
#NMR_METABOLITE_DATA
NMR_METABOLITE_DATA:UNITS	Attomoles/CFU
NMR_METABOLITE_DATA_START
Samples	WT_1	WT_2	WT_3	WT_4	ClpC_1	ClpC_2	ClpC_3	ClpC_4	SrrAB_1	SrrAB_2	SrrAB_3	SrrAB_4	ClpC-SrrAB_1	ClpC-SrrAB_2	ClpC-SrrAB_3	ClpC-SrrAB_4
Factors	Genotype:wild type	Genotype:wild type	Genotype:wild type	Genotype:wild type	Genotype:ClpC deletion mutant	Genotype:ClpC deletion mutant	Genotype:ClpC deletion mutant	Genotype:ClpC deletion mutant	Genotype:SrrAB deletion mutant	Genotype:SrrAB deletion mutant	Genotype:SrrAB deletion mutant	Genotype:SrrAB deletion mutant	Genotype:ClpC-SrrAB double deletion mutant	Genotype:ClpC-SrrAB double deletion mutant	Genotype:ClpC-SrrAB double deletion mutant	Genotype:ClpC-SrrAB double deletion mutant
ADP	67.3	87.5	65.4	69.2	139.5	139.7	129.4	139.9	127.4	132	134.8	109.5	117.6	105.3	137.1	104.7
AMP	498.8	464	444.5	424	503.7	462.5	456.3	467.2	504.3	543.1	640	552.9	294	317.7	456.6	388.8
Acetate	1244.9	1317.9	1774.4	1605.9	2306.8	2406.2	2211.4	2455.2	2033.5	2135.1	2376.3	1614.2	2563.2	2707.2	2366.7	2269.8
Asparagine	10666.1	10999.3	10826.1	10963.5	13310.6	13739.8	12333.8	13152.9	18197.8	19128.3	16820.9	13053.5	12881	14657.4	17763.3	12881.1
Aspartate	5642.4	5511.8	3270.4	3160.2	4210	4880.7	3271.2	3117.2	3785.7	3785.9	6748.9	5683.7	4114.8	4666.2	7078.2	6468.6
Betaine	7077.9	7164.5	8023.8	7880.2	9240.6	9691.3	10984.5	10976.4	5446.2	4980.9	10506.2	9073.8	6321	7438.2	9760.5	9024.6
Citrulline	179.5	193.6	176.2	131.5	186.1	199.5	299.8	187.9	208.6	267.7	230.8	244.9	109.8	112.5	109.2	114.9
Dimethylamine	1598.1	1584	3024.2	2827.3	1285.1	1301.6	1598	1722.1	4015.4	4053.5	3585.8	2985.5	1598.7	1820.7	1169.1	1080.6
Formate	140.7	151.8	134.8	144.7	196.4	191.7	198.9	210.7	152.6	164.1	198.8	164	155.7	180.9	179.4	177.9
GTP	63.3	70.6	65.6	68.2	56.1	45.5	63.3	55.2	98.1	92.9	98.2	90.8	31.5	35.4	30.6	29.4
Glutamate	1635.3	1691.1	1713.9	1647.8	1847.4	1736.4	2182.8	1607.1	823.3	823.4	1580.9	1442.5	802.8	760.2	945	885
Glutarate	713.2	703.8	1621.9	1603.5	569.8	626.2	591	586	3412.3	3412.2	2360.3	1961.8	1092.6	1226.7	855	794.7
Glycine	69.9	78.4	49.9	43.3	149.8	149.9	150	162.1	55.6	55.7	55.8	68.9	105.2	105.3	133.8	132.6
Histamine	109.2	109.2	115.3	119.1	167.1	207.3	216.9	202.3	95.6	75.4	95.7	81.2	152.1	160.5	218.7	203.4
Histidine	90.4	93.4	113.6	108.2	63.3	86.6	141.5	128.1	100.6	113.2	140.6	120.6	124.2	144.6	143.1	136.2
Isoleucine	283.1	226.8	250.4	243.8	269.6	230.3	297.7	305.1	357.8	320	330.5	284.6	346.5	238.5	246.9	235.5
Lactate	187.2	187	169.4	156.2	247.8	231.6	229.4	231.9	242.2	256.3	320.3	249.5	153.6	208.2	246.3	219.6
Leucine	631.1	674.6	810.6	761.2	635.4	682.6	635.6	838.8	1041.5	989.2	1100.6	927.7	913.5	703.5	743.4	694.2
Lysine	10400.7	8755.8	10425.9	10263.3	13124.2	11183.4	13124.4	11431.8	14144.9	12574.5	14728.6	11981.2	11912.4	12479.1	12071.1	11647.8
Methionine	306.8	341	340.9	342.6	645.5	694.4	590.6	611.5	402.2	396	561.5	511.7	469.2	569.1	814.8	777.3
NAD+	316.9	350.1	373.4	347.5	610	702.5	741.2	640.5	489.8	480	432.9	424.3	521.1	618	744.9	722.4
NADP+	21.2	26.4	36.5	36.9	30.5	33.7	48	30.9	30.8	33.2	32.6	33.8	25.2	30.3	33.6	26.1
Niacinamide	25.9	25.8	23.8	26	39.6	55.5	35.8	44.9	28.3	43.1	26.8	28.3	27.6	37.8	52.5	40.8
Nicotinamide N-oxide	68.9	74.6	100	97.2	77.6	110.6	127.8	109.4	169.5	153.8	101.8	111.1	100.5	113.4	99	84.6
N-alpha-Acetyllysine	1437.9	1349.9	3033.9	2894.8	3469.1	3416.1	3899.2	4030.1	3424	3421.2	1859.1	1561.8	3491.7	3954	2452.8	2278.2
O-Phosphocholine	63.3	58.4	100	86.6	81	94.8	93.5	106.9	125.8	126.5	133.2	89.5	118.5	130.8	117.9	115.8
Phenylalanine	269.4	288.5	319.3	313.6	356.8	357	442.9	405.5	404.1	393.2	404	332.3	442.5	390	399.6	343.8
Succinate	238.1	246.4	222.8	214.8	225.7	238.1	199.8	199.5	320.9	318.2	684	610.5	184.8	239.1	368.7	346.8
Tartrate	244.2	244.3	244.6	244.7	280	281.1	281.4	281.5	277.2	277.3	277.6	277.5	239.3	239.4	239.5	239.8
Theophylline	15.8	9.2	14.6	12.7	98.5	98.6	98.8	98.9	12	15.4	30.8	12.9	91.1	91.2	91.3	91.4
Threonine	247.5	218.4	201.4	192	314.8	280.8	283.9	269.6	300.7	300.8	382.2	300.9	199.8	249.6	296.1	260.7
Trigonelline	137.6	147.3	71.3	71.5	196.1	235.9	203.2	179.5	50.1	50.2	190.8	169.8	117	136.2	206.1	195.6
Trimethylamine N-oxide	3502.1	3584.9	3949.2	3582.6	4225.9	4013.3	4353.7	4562.5	1662.2	1986.5	5197.5	4246.8	2391	2692.5	4089.3	3806.7
Tryptophan	191.3	198.4	236.5	235.5	265.6	274.6	335.7	277.4	265.8	240.2	299.7	240	288	283.5	282.3	284.7
Tyrosine	468.1	468.2	468.3	472.2	649.9	612.2	675.1	668.3	514.5	499.7	684.6	612	487.5	570.6	693	659.1
UMP	106.1	100	94.8	90.8	108.8	108.2	109.7	97.6	130.8	133.8	178.5	139.1	67.5	72.9	102.3	92.4
Valine	557.6	544	603.5	608.2	540.2	467.2	546.1	587.5	759.4	689.8	747.7	648.3	666	511.8	530.1	503.1
sn-Glycero-3-phosphocholine	140.9	135.1	160.9	163.8	212.9	217.6	278	234.7	201.5	194.5	172.9	187.1	157.5	185.1	168.9	163.2
NMR_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
ADP
AMP
Acetate
Asparagine
Aspartate
Betaine
Citrulline
Dimethylamine
Formate
GTP
Glutamate
Glutarate
Glycine
Histamine
Histidine
Isoleucine
Lactate
Leucine
Lysine
Methionine
NAD+
NADP+
Niacinamide
Nicotinamide N-oxide
N-alpha-Acetyllysine
O-Phosphocholine
Phenylalanine
Succinate
Tartrate
Theophylline
Threonine
Trigonelline
Trimethylamine N-oxide
Tryptophan
Tyrosine
UMP
Valine
sn-Glycero-3-phosphocholine
METABOLITES_END
#END