Summary of Study ST003711

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 PR002304. The data can be accessed directly via it's Project DOI: 10.21228/M8T82V This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST003711
Study Title	(p)ppGpp and DksA play crucial role in reducing the efficacy of ꞵ-lactam antibiotics by modulating bacterial membrane permeability
Study Type	Metabolomics
Study Summary	The key signaling molecules in the bacterial stress sensing pathway, the alarmone (p)ppGpp and transcription factor DksA, help in survival during nutritional deprivation and exposure to xenobiotics by modulating cellular metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked with the functions of three proteins: RelA, SpoT, and RelV. At threshold or elevated concentrations of (p)ppGpp, the level of cellular metabolites and proteins in the presence and absence of DksA in V. cholerae and other bacteria has not yet been comprehensively studied. We engineered the genome of V. cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp biosynthetic enzymes. We observed a higher sensitivity of the (p)ppGpp0ΔdksA V. cholerae mutant to different ꞵ-lactam antibiotics compared to the wild-type (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the cell membrane and peptidoglycan biosynthesis pathways are significantly altered in the (p)ppGpp0, ΔdksA, and (p)ppGpp0ΔdksA V. cholerae strains. Further, the mutant strains displayed enhanced inner and outer membrane permeability in comparison to the WT strains. These results directly correlate with the tolerance and survival of V. cholerae to ꞵ-lactam antibiotics. These findings may help in the development of adjuvants for ꞵ-lactam antibiotics by inhibiting the functions of stringent response modulators.
Institute	Translational Health Science And Technology Institute (THSTI)
Department	Biology
Laboratory	Biomarker lab
Last Name	Kumar
First Name	Yashwant
Address	NCR Biotech Science Cluster, Faridabad, Haryana, 121001, India
Email	y.kumar@thsti.res.in
Phone	01292876796
Submit Date	2025-02-05
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2025-03-04
Release Version	1

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Project:

Project ID:	PR002304
Project DOI:	doi: 10.21228/M8T82V
Project Title:	(p)ppGpp and DksA play crucial role in reducing the efficacy of ꞵ-lactam antibiotics by modulating bacterial membrane permeability
Project Type:	Metabolomics
Project Summary:	The key signaling molecules in the bacterial stress sensing pathway, the alarmone (p)ppGpp and transcription factor DksA, help in survival during nutritional deprivation and exposure to xenobiotics by modulating cellular metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked with the functions of three proteins: RelA, SpoT, and RelV. At threshold or elevated concentrations of (p)ppGpp, the level of cellular metabolites and proteins in the presence and absence of DksA in V. cholerae and other bacteria has not yet been comprehensively studied. We engineered the genome of V. cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp biosynthetic enzymes. We observed a higher sensitivity of the (p)ppGpp0ΔdksA V. cholerae mutant to different ꞵ-lactam antibiotics compared to the wild-type (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the cell membrane and peptidoglycan biosynthesis pathways are significantly altered in the (p)ppGpp0, ΔdksA, and (p)ppGpp0ΔdksA V. cholerae strains. Further, the mutant strains displayed enhanced inner and outer membrane permeability in comparison to the WT strains. These results directly correlate with the tolerance and survival of V. cholerae to ꞵ-lactam antibiotics. These findings may help in the development of adjuvants for ꞵ-lactam antibiotics by inhibiting the functions of stringent response modulators.
Institute:	Translational health science and technology institute
Department:	Biology
Laboratory:	Biomarker lab
Last Name:	Kumar
First Name:	Yashwant
Address:	NCR Biotech Science Cluster, Faridabad, Haryana, 121001, India
Email:	y.kumar@thsti.res.in
Phone:	01292876496
Funding Source:	THSTI

Subject:

Subject ID:	SU003843
Subject Type:	Bacteria
Subject Species:	Vibrio cholerae
Taxonomy ID:	666

Factors:

Subject type: Bacteria; Subject species: Vibrio cholerae (Factor headings shown in green)

mb_sample_id	local_sample_id	factor	Sample source
SA406536	JV8_5	mutanat	Bacterial origin
SA406537	JV9_5	mutanat	Bacterial origin
SA406538	JV9_4	mutanat	Bacterial origin
SA406539	JV9_3	mutanat	Bacterial origin
SA406540	JV9_2	mutanat	Bacterial origin
SA406541	JV9_1	mutanat	Bacterial origin
SA406542	JV8_6	mutanat	Bacterial origin
SA406543	JV8_4	mutanat	Bacterial origin
SA406544	MC3_1	mutanat	Bacterial origin
SA406545	JV8_3	mutanat	Bacterial origin
SA406546	JV8_2	mutanat	Bacterial origin
SA406547	JV8_1	mutanat	Bacterial origin
SA406548	JV7_6	mutanat	Bacterial origin
SA406549	JV7_5	mutanat	Bacterial origin
SA406550	JV7_4	mutanat	Bacterial origin
SA406551	JV9_6	mutanat	Bacterial origin
SA406552	MC3_2	mutanat	Bacterial origin
SA406553	JV7_2	mutanat	Bacterial origin
SA406554	MC4_6	mutanat	Bacterial origin
SA406555	MCI_6	mutanat	Bacterial origin
SA406556	MCI_5	mutanat	Bacterial origin
SA406557	MCI_4	mutanat	Bacterial origin
SA406558	MCI_3	mutanat	Bacterial origin
SA406559	MCI_2	mutanat	Bacterial origin
SA406560	MCI_1	mutanat	Bacterial origin
SA406561	MC4_5	mutanat	Bacterial origin
SA406562	MC3_3	mutanat	Bacterial origin
SA406563	MC4_4	mutanat	Bacterial origin
SA406564	MC4_3	mutanat	Bacterial origin
SA406565	MC4_2	mutanat	Bacterial origin
SA406566	MC4_1	mutanat	Bacterial origin
SA406567	MC3_6	mutanat	Bacterial origin
SA406568	MC3_5	mutanat	Bacterial origin
SA406569	MC3_4	mutanat	Bacterial origin
SA406570	JV7_3	mutanat	Bacterial origin
SA406571	JV7_1	mutanat	Bacterial origin
SA406572	RRVI_1	mutanat	Bacterial origin
SA406573	RRVI_2	mutanat	Bacterial origin
SA406574	NR13_1	mutanat	Bacterial origin
SA406575	NR13_2	mutanat	Bacterial origin
SA406576	NR13_3	mutanat	Bacterial origin
SA406577	NR13_4	mutanat	Bacterial origin
SA406578	NR13_5	mutanat	Bacterial origin
SA406579	NR13_6	mutanat	Bacterial origin
SA406580	NRVI_1	mutanat	Bacterial origin
SA406581	NRVI_2	mutanat	Bacterial origin
SA406582	NRVI_3	mutanat	Bacterial origin
SA406583	NRVI_4	mutanat	Bacterial origin
SA406584	NRVI_5	mutanat	Bacterial origin
SA406585	NRVI_6	mutanat	Bacterial origin
SA406586	BS1_1_6	mutanat	Bacterial origin
SA406587	RRVI_3	mutanat	Bacterial origin
SA406588	RRVI_4	mutanat	Bacterial origin
SA406589	RRVI_5	mutanat	Bacterial origin
SA406590	RRVI_6	mutanat	Bacterial origin
SA406591	BRVI_1	mutanat	Bacterial origin
SA406592	BRVI_2	mutanat	Bacterial origin
SA406593	BRVI_3	mutanat	Bacterial origin
SA406594	BRVI_4	mutanat	Bacterial origin
SA406595	BRVI_5	mutanat	Bacterial origin
SA406596	BRVI_6	mutanat	Bacterial origin
SA406597	BS1_1_1	mutanat	Bacterial origin
SA406598	BS1_1_2	mutanat	Bacterial origin
SA406599	BS1_1_3	mutanat	Bacterial origin
SA406600	BS1_1_4	mutanat	Bacterial origin
SA406601	BS1_1_5	mutanat	Bacterial origin
SA406602	N16_4	wild type	Bacterial origin
SA406603	N16_3	wild type	Bacterial origin
SA406604	N16_2	wild type	Bacterial origin
SA406605	N16_5	wild type	Bacterial origin
SA406606	N16_6	wild type	Bacterial origin
SA406607	N16_1	wild type	Bacterial origin

Showing results 1 to 72 of 72

Showing results 1 to 72 of 72

Collection:

Collection ID:	CO003836
Collection Summary:	The WT V. cholerae strain N16961 and previously constructed (p)ppGpp variant strains of V. cholerae and other strains constructed and used in this study are mentioned in Table 1. (p)ppGpp variant strains used were N16961-R13(N16961::∆relA), N16961-RV1(N16961::∆relV), BS1.1 (N16961::∆relA, ∆spoT) and BRV1(N16961::∆relA, ∆spoT, ∆relV) (11, 12). We have constructed DksA mutant strains of N16961 and of (p)ppGpp variant strains. All the plasmids used in this study are mentioned in Table S2. For liquid culture, the strains were grown in Luria Broth (LB) at 37°C in a shaker with 180 rpm while LB agar plates were used for solid culture. The following antibiotic concentrations were used: streptomycin (100 μg/mL), spectinomycin (50 μg/mL), kanamycin (40 μg/mL), zeocin (25 μg/mL), ampicillin (100μg/mL) and chloramphenicol (30 μg/mL for E. coli and 2 μg/mL for V. cholerae). The bacteria were tested for sucrose sensitivity by plating them onto LA supplemented with 15% sucrose and incubating them at 24°C. For long term storage at –80ºC, we used LB supplemented with 20% glycerol.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR003852
Treatment Summary:	Antibiotic susceptibility testing Antibiotic susceptibility test by disc diffusion method was done to measure the zone of inhibition by different antibiotics in all (p)ppGpp and DksA mutant strains. For the disc diffusion method, all the strains were grown overnight aerobically at 37°C in MHB medium and the primary cultures were diluted 1:100 in fresh MHB medium and incubated aerobically at 37°C, when OD600 reached 0.5. The 1 mL of this culture was plated onto Mueller-Hinton agar (MHA, Difco, USA) plate (23” x 23” cm) using sterile cotton swabs and commercially available discs (Liofilchem) containing defined amounts of interested antibiotics were placed on it. Plates were incubated overnight at 37°C in a static incubator and the zone of clearance was measured with the help of antibiotic zone scale.

Treatment ID:

TR003852

Treatment Summary:

Antibiotic susceptibility testing Antibiotic susceptibility test by disc diffusion method was done to measure the zone of inhibition by different antibiotics in all (p)ppGpp and DksA mutant strains. For the disc diffusion method, all the strains were grown overnight aerobically at 37°C in MHB medium and the primary cultures were diluted 1:100 in fresh MHB medium and incubated aerobically at 37°C, when OD600 reached 0.5. The 1 mL of this culture was plated onto Mueller-Hinton agar (MHA, Difco, USA) plate (23” x 23” cm) using sterile cotton swabs and commercially available discs (Liofilchem) containing defined amounts of interested antibiotics were placed on it. Plates were incubated overnight at 37°C in a static incubator and the zone of clearance was measured with the help of antibiotic zone scale.

Sample Preparation:

Sampleprep ID:	SP003849
Sampleprep Summary:	The cells were pelleted down again by centrifugation (10,000 rpm at 4°C for 10 min), washed with 0.9% normal saline and stored at -80°C. To extract the intracellular metabolites cold 100% methanol was added (Sigma Aldrich; Cat no. 34860) followed by vortexing and bath sonication for 10 min (Bransonic® Ultrasonic M Cleaning Bath 1510). The cell debris was pelleted down by centrifugation (10,000 rpm at 4°C for 10 min) and supernatant was collected in two separate microcentrifuge tubes (120 µL each tube), vacuum dried (Thermo Scientific™ Savant™ SPD1010) and stored at -80°C. For the analysis of metabolites, the dried supernatant was dissolved in 60 µL of 15% methanol or 50% acetonitrile (Cat no. 271004) followed by vortexing for 5 min and centrifuged (10,000 rpm for 10 min). The supernatant was collected in a separate sample vial (Supelco™ Analytical).

Sampleprep ID:

SP003849

Sampleprep Summary:

The cells were pelleted down again by centrifugation (10,000 rpm at 4°C for 10 min), washed with 0.9% normal saline and stored at -80°C. To extract the intracellular metabolites cold 100% methanol was added (Sigma Aldrich; Cat no. 34860) followed by vortexing and bath sonication for 10 min (Bransonic® Ultrasonic M Cleaning Bath 1510). The cell debris was pelleted down by centrifugation (10,000 rpm at 4°C for 10 min) and supernatant was collected in two separate microcentrifuge tubes (120 µL each tube), vacuum dried (Thermo Scientific™ Savant™ SPD1010) and stored at -80°C. For the analysis of metabolites, the dried supernatant was dissolved in 60 µL of 15% methanol or 50% acetonitrile (Cat no. 271004) followed by vortexing for 5 min and centrifuged (10,000 rpm for 10 min). The supernatant was collected in a separate sample vial (Supelco™ Analytical).

Combined analysis:

Analysis ID	AN006088	AN006089	AN006090	AN006091
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS
Column	Waters ACQUITY UPLC HSS T3 (100 x 2.1 mm, 1.8 µm)	Waters ACQUITY UPLC HSS T3 (100 x 2.1 mm, 1.8 µm)	Waters XBridge BEH Amide (100 x 2.1 mm, 2.5 µm)	Waters XBridge BEH Amide (100 x 2.1 mm, 2.5 µm)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Fusion Orbitrap	Thermo Fusion Orbitrap	Thermo Fusion Orbitrap	Thermo Fusion Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	relative intensity	relative intensity	relative intensity	relative intensity

Chromatography:

Chromatography ID:	CH004622
Chromatography Summary:	Solvent A for the RP was water, and Solvent B was methanol, with 0.1% formic acid in each. At a flow rate of 0.3 mL/min, the elution gradient proceeds from 1% B to 95% B in 10 minutes.
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Waters ACQUITY UPLC HSS T3 (100 x 2.1 mm, 1.8 µm)
Column Temperature:	40°C
Flow Gradient:	1% B to 95% B in 10 minutes
Flow Rate:	300 µL/min
Solvent A:	100% Water; 0.1% Formic acid
Solvent B:	100% Methanol; 0.1% Formic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH004623
Chromatography Summary:	Solvent A consisted of 20 mM ammonium acetate (pH-9.0) water for polar compound separation, while mobile phase B consisted of 100% acetonitrile. At a flow rate of 0.35 mL/min, the elution gradient commences at 85% B and proceeds to 10% B over 14 minutes.
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Waters XBridge BEH Amide (100 x 2.1 mm, 2.5 µm)
Column Temperature:	40°C
Flow Gradient:	85% B and proceeds to 10% B over 14 minutes
Flow Rate:	350 µL/min
Solvent A:	100% Water; 20 mM ammonium acetate (pH-9.0)
Solvent B:	100% Acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS005795
Analysis ID:	AN006088
Instrument Name:	Thermo Fusion Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	mass resolution was retained at 120,000 for MS1 mode and 30,000 for MS2 acquisition. The data acquisition mass range was 60-900Da Feature list has M/z_retention time (m/z is first then retention time and separated by underscore)
Ion Mode:	POSITIVE

MS ID:	MS005796
Analysis ID:	AN006089
Instrument Name:	Thermo Fusion Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	mass resolution was retained at 120,000 for MS1 mode and 30,000 for MS2 acquisition. The data acquisition mass range was 60-900Da Feature list has M/z_retention time (m/z is first then retention time and separated by underscore)
Ion Mode:	NEGATIVE

MS ID:	MS005797
Analysis ID:	AN006090
Instrument Name:	Thermo Fusion Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	mass resolution was retained at 120,000 for MS1 mode and 30,000 for MS2 acquisition. The data acquisition mass range was 60-900Da Feature list has M/z_retention time (m/z is first then retention time and separated by underscore)
Ion Mode:	POSITIVE

MS ID:	MS005798
Analysis ID:	AN006091
Instrument Name:	Thermo Fusion Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	mass resolution was retained at 120,000 for MS1 mode and 30,000 for MS2 acquisition. The data acquisition mass range was 60-900Da Feature list has M/z_retention time (m/z is first then retention time and separated by underscore)
Ion Mode:	NEGATIVE