#METABOLOMICS WORKBENCH yash_thsti_20250205_223714 DATATRACK_ID:5597 STUDY_ID:ST003711 ANALYSIS_ID:AN006090 PROJECT_ID:PR002304 VERSION 1 CREATED_ON February 10, 2025, 11:34 pm #PROJECT PR:PROJECT_TITLE (p)ppGpp and DksA play crucial role in reducing the efficacy of -lactam PR:PROJECT_TITLE antibiotics by modulating bacterial membrane permeability PR:PROJECT_TYPE Metabolomics PR:PROJECT_SUMMARY The key signaling molecules in the bacterial stress sensing pathway, the PR:PROJECT_SUMMARY alarmone (p)ppGpp and transcription factor DksA, help in survival during PR:PROJECT_SUMMARY nutritional deprivation and exposure to xenobiotics by modulating cellular PR:PROJECT_SUMMARY metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked PR:PROJECT_SUMMARY with the functions of three proteins: RelA, SpoT, and RelV. At threshold or PR:PROJECT_SUMMARY elevated concentrations of (p)ppGpp, the level of cellular metabolites and PR:PROJECT_SUMMARY proteins in the presence and absence of DksA in V. cholerae and other bacteria PR:PROJECT_SUMMARY has not yet been comprehensively studied. We engineered the genome of V. PR:PROJECT_SUMMARY cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp PR:PROJECT_SUMMARY biosynthetic enzymes. We observed a higher sensitivity of the (p)ppGpp0ΔdksA V. PR:PROJECT_SUMMARY cholerae mutant to different -lactam antibiotics compared to the wild-type PR:PROJECT_SUMMARY (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the PR:PROJECT_SUMMARY cell membrane and peptidoglycan biosynthesis pathways are significantly altered PR:PROJECT_SUMMARY in the (p)ppGpp0, ΔdksA, and (p)ppGpp0ΔdksA V. cholerae strains. Further, the PR:PROJECT_SUMMARY mutant strains displayed enhanced inner and outer membrane permeability in PR:PROJECT_SUMMARY comparison to the WT strains. These results directly correlate with the PR:PROJECT_SUMMARY tolerance and survival of V. cholerae to -lactam antibiotics. These findings PR:PROJECT_SUMMARY may help in the development of adjuvants for -lactam antibiotics by PR:PROJECT_SUMMARY inhibiting the functions of stringent response modulators. PR:INSTITUTE Translational health science and technology institute PR:DEPARTMENT Biology PR:LABORATORY Biomarker lab PR:LAST_NAME Kumar PR:FIRST_NAME Yashwant PR:ADDRESS NCR Biotech Science Cluster,, Faridabad, Haryana, 121001, India PR:EMAIL y.kumar@thsti.res.in PR:PHONE 01292876496 PR:FUNDING_SOURCE THSTI #STUDY ST:STUDY_TITLE (p)ppGpp and DksA play crucial role in reducing the efficacy of -lactam ST:STUDY_TITLE antibiotics by modulating bacterial membrane permeability ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY The key signaling molecules in the bacterial stress sensing pathway, the ST:STUDY_SUMMARY alarmone (p)ppGpp and transcription factor DksA, help in survival during ST:STUDY_SUMMARY nutritional deprivation and exposure to xenobiotics by modulating cellular ST:STUDY_SUMMARY metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked ST:STUDY_SUMMARY with the functions of three proteins: RelA, SpoT, and RelV. At threshold or ST:STUDY_SUMMARY elevated concentrations of (p)ppGpp, the level of cellular metabolites and ST:STUDY_SUMMARY proteins in the presence and absence of DksA in V. cholerae and other bacteria ST:STUDY_SUMMARY has not yet been comprehensively studied. We engineered the genome of V. ST:STUDY_SUMMARY cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp ST:STUDY_SUMMARY biosynthetic enzymes. We observed a higher sensitivity of the (p)ppGpp0ΔdksA V. ST:STUDY_SUMMARY cholerae mutant to different -lactam antibiotics compared to the wild-type ST:STUDY_SUMMARY (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the ST:STUDY_SUMMARY cell membrane and peptidoglycan biosynthesis pathways are significantly altered ST:STUDY_SUMMARY in the (p)ppGpp0, ΔdksA, and (p)ppGpp0ΔdksA V. cholerae strains. Further, the ST:STUDY_SUMMARY mutant strains displayed enhanced inner and outer membrane permeability in ST:STUDY_SUMMARY comparison to the WT strains. These results directly correlate with the ST:STUDY_SUMMARY tolerance and survival of V. cholerae to -lactam antibiotics. These findings ST:STUDY_SUMMARY may help in the development of adjuvants for -lactam antibiotics by ST:STUDY_SUMMARY inhibiting the functions of stringent response modulators. ST:INSTITUTE Translational Health Science And Technology Institute (THSTI) ST:DEPARTMENT Biology ST:LABORATORY Biomarker lab ST:LAST_NAME Kumar ST:FIRST_NAME Yashwant ST:ADDRESS NCR Biotech Science Cluster, ST:EMAIL y.kumar@thsti.res.in ST:PHONE 01292876796 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Vibrio cholerae SU:TAXONOMY_ID 666 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS 1 N16_1 factor:wild type | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=N16_1 SUBJECT_SAMPLE_FACTORS 2 N16_2 factor:wild type | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=N16_2 SUBJECT_SAMPLE_FACTORS 3 N16_3 factor:wild type | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=N16_3 SUBJECT_SAMPLE_FACTORS 4 N16_4 factor:wild type | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=N16_4 SUBJECT_SAMPLE_FACTORS 5 N16_5 factor:wild type | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=N16_5 SUBJECT_SAMPLE_FACTORS 6 N16_6 factor:wild type | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=N16_6 SUBJECT_SAMPLE_FACTORS 7 NR13_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NR13_1 SUBJECT_SAMPLE_FACTORS 8 NR13_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NR13_2 SUBJECT_SAMPLE_FACTORS 9 NR13_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NR13_3 SUBJECT_SAMPLE_FACTORS 10 NR13_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NR13_4 SUBJECT_SAMPLE_FACTORS 11 NR13_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NR13_5 SUBJECT_SAMPLE_FACTORS 12 NR13_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NR13_6 SUBJECT_SAMPLE_FACTORS 13 NRVI_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NRVI_1 SUBJECT_SAMPLE_FACTORS 14 NRVI_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NRVI_2 SUBJECT_SAMPLE_FACTORS 15 NRVI_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NRVI_3 SUBJECT_SAMPLE_FACTORS 16 NRVI_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NRVI_4 SUBJECT_SAMPLE_FACTORS 17 NRVI_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NRVI_5 SUBJECT_SAMPLE_FACTORS 18 NRVI_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=NRVI_6 SUBJECT_SAMPLE_FACTORS 19 RRVI_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=RRVI_1 SUBJECT_SAMPLE_FACTORS 20 RRVI_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=RRVI_2 SUBJECT_SAMPLE_FACTORS 21 RRVI_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=RRVI_3 SUBJECT_SAMPLE_FACTORS 22 RRVI_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=RRVI_4 SUBJECT_SAMPLE_FACTORS 23 RRVI_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=RRVI_5 SUBJECT_SAMPLE_FACTORS 24 RRVI_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=RRVI_6 SUBJECT_SAMPLE_FACTORS 25 BRVI_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BRVI_1 SUBJECT_SAMPLE_FACTORS 26 BRVI_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BRVI_2 SUBJECT_SAMPLE_FACTORS 27 BRVI_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BRVI_3 SUBJECT_SAMPLE_FACTORS 28 BRVI_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BRVI_4 SUBJECT_SAMPLE_FACTORS 29 BRVI_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BRVI_5 SUBJECT_SAMPLE_FACTORS 30 BRVI_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BRVI_6 SUBJECT_SAMPLE_FACTORS 31 BS1_1_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BS1_1_1 SUBJECT_SAMPLE_FACTORS 32 BS1_1_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BS1_1_2 SUBJECT_SAMPLE_FACTORS 33 BS1_1_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BS1_1_3 SUBJECT_SAMPLE_FACTORS 34 BS1_1_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BS1_1_4 SUBJECT_SAMPLE_FACTORS 35 BS1_1_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BS1_1_5 SUBJECT_SAMPLE_FACTORS 36 BS1_1_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=BS1_1_6 SUBJECT_SAMPLE_FACTORS 37 JV7_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV7_1 SUBJECT_SAMPLE_FACTORS 38 JV7_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV7_2 SUBJECT_SAMPLE_FACTORS 39 JV7_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV7_3 SUBJECT_SAMPLE_FACTORS 40 JV7_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV7_4 SUBJECT_SAMPLE_FACTORS 41 JV7_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV7_5 SUBJECT_SAMPLE_FACTORS 42 JV7_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV7_6 SUBJECT_SAMPLE_FACTORS 43 JV8_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV8_1 SUBJECT_SAMPLE_FACTORS 44 JV8_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV8_2 SUBJECT_SAMPLE_FACTORS 45 JV8_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV8_3 SUBJECT_SAMPLE_FACTORS 46 JV8_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV8_4 SUBJECT_SAMPLE_FACTORS 47 JV8_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV8_5 SUBJECT_SAMPLE_FACTORS 48 JV8_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV8_6 SUBJECT_SAMPLE_FACTORS 49 JV9_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV9_1 SUBJECT_SAMPLE_FACTORS 50 JV9_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV9_2 SUBJECT_SAMPLE_FACTORS 51 JV9_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV9_3 SUBJECT_SAMPLE_FACTORS 52 JV9_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV9_4 SUBJECT_SAMPLE_FACTORS 53 JV9_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV9_5 SUBJECT_SAMPLE_FACTORS 54 JV9_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=JV9_6 SUBJECT_SAMPLE_FACTORS 55 MC3_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC3_1 SUBJECT_SAMPLE_FACTORS 56 MC3_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC3_2 SUBJECT_SAMPLE_FACTORS 57 MC3_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC3_3 SUBJECT_SAMPLE_FACTORS 58 MC3_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC3_4 SUBJECT_SAMPLE_FACTORS 59 MC3_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC3_5 SUBJECT_SAMPLE_FACTORS 60 MC3_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC3_6 SUBJECT_SAMPLE_FACTORS 61 MC4_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC4_1 SUBJECT_SAMPLE_FACTORS 62 MC4_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC4_2 SUBJECT_SAMPLE_FACTORS 63 MC4_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC4_3 SUBJECT_SAMPLE_FACTORS 64 MC4_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC4_4 SUBJECT_SAMPLE_FACTORS 65 MC4_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC4_5 SUBJECT_SAMPLE_FACTORS 66 MC4_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MC4_6 SUBJECT_SAMPLE_FACTORS 67 MCI_1 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MCI_1 SUBJECT_SAMPLE_FACTORS 68 MCI_2 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MCI_2 SUBJECT_SAMPLE_FACTORS 69 MCI_3 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MCI_3 SUBJECT_SAMPLE_FACTORS 70 MCI_4 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MCI_4 SUBJECT_SAMPLE_FACTORS 71 MCI_5 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MCI_5 SUBJECT_SAMPLE_FACTORS 72 MCI_6 factor:mutanat | Sample source:Bacterial origin RAW_FILE_NAME(raw file name)=MCI_6 #COLLECTION CO:COLLECTION_SUMMARY The WT V. cholerae strain N16961 and previously constructed (p)ppGpp variant CO:COLLECTION_SUMMARY strains of V. cholerae and other strains constructed and used in this study are CO:COLLECTION_SUMMARY mentioned in Table 1. (p)ppGpp variant strains used were CO:COLLECTION_SUMMARY N16961-R13(N16961::∆relA), N16961-RV1(N16961::∆relV), BS1.1 CO:COLLECTION_SUMMARY (N16961::∆relA, ∆spoT) and BRV1(N16961::∆relA, ∆spoT, ∆relV) (11, 12). CO:COLLECTION_SUMMARY We have constructed DksA mutant strains of N16961 and of (p)ppGpp variant CO:COLLECTION_SUMMARY strains. All the plasmids used in this study are mentioned in Table S2. For CO:COLLECTION_SUMMARY liquid culture, the strains were grown in Luria Broth (LB) at 37°C in a shaker CO:COLLECTION_SUMMARY with 180 rpm while LB agar plates were used for solid culture. The following CO:COLLECTION_SUMMARY antibiotic concentrations were used: streptomycin (100 μg/mL), spectinomycin CO:COLLECTION_SUMMARY (50 μg/mL), kanamycin (40 μg/mL), zeocin (25 μg/mL), ampicillin (100μg/mL) CO:COLLECTION_SUMMARY and chloramphenicol (30 μg/mL for E. coli and 2 μg/mL for V. cholerae). The CO:COLLECTION_SUMMARY bacteria were tested for sucrose sensitivity by plating them onto LA CO:COLLECTION_SUMMARY supplemented with 15% sucrose and incubating them at 24°C. For long term CO:COLLECTION_SUMMARY storage at –80ºC, we used LB supplemented with 20% glycerol. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY Antibiotic susceptibility testing Antibiotic susceptibility test by disc TR:TREATMENT_SUMMARY diffusion method was done to measure the zone of inhibition by different TR:TREATMENT_SUMMARY antibiotics in all (p)ppGpp and DksA mutant strains. For the disc diffusion TR:TREATMENT_SUMMARY method, all the strains were grown overnight aerobically at 37°C in MHB medium TR:TREATMENT_SUMMARY and the primary cultures were diluted 1:100 in fresh MHB medium and incubated TR:TREATMENT_SUMMARY aerobically at 37°C, when OD600 reached 0.5. The 1 mL of this culture was TR:TREATMENT_SUMMARY plated onto Mueller-Hinton agar (MHA, Difco, USA) plate (23” x 23” cm) using TR:TREATMENT_SUMMARY sterile cotton swabs and commercially available discs (Liofilchem) containing TR:TREATMENT_SUMMARY defined amounts of interested antibiotics were placed on it. Plates were TR:TREATMENT_SUMMARY incubated overnight at 37°C in a static incubator and the zone of clearance was TR:TREATMENT_SUMMARY measured with the help of antibiotic zone scale. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cells were pelleted down again by centrifugation (10,000 rpm at 4°C for 10 SP:SAMPLEPREP_SUMMARY min), washed with 0.9% normal saline and stored at -80°C. To extract the SP:SAMPLEPREP_SUMMARY intracellular metabolites cold 100% methanol was added (Sigma Aldrich; Cat no. SP:SAMPLEPREP_SUMMARY 34860) followed by vortexing and bath sonication for 10 min (Bransonic® SP:SAMPLEPREP_SUMMARY Ultrasonic M Cleaning Bath 1510). The cell debris was pelleted down by SP:SAMPLEPREP_SUMMARY centrifugation (10,000 rpm at 4°C for 10 min) and supernatant was collected in SP:SAMPLEPREP_SUMMARY two separate microcentrifuge tubes (120 µL each tube), vacuum dried (Thermo SP:SAMPLEPREP_SUMMARY Scientific™ Savant™ SPD1010) and stored at -80°C. For the analysis of SP:SAMPLEPREP_SUMMARY metabolites, the dried supernatant was dissolved in 60 µL of 15% methanol or SP:SAMPLEPREP_SUMMARY 50% acetonitrile (Cat no. 271004) followed by vortexing for 5 min and SP:SAMPLEPREP_SUMMARY centrifuged (10,000 rpm for 10 min). The supernatant was collected in a separate SP:SAMPLEPREP_SUMMARY sample vial (Supelco™ Analytical). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Solvent A consisted of 20 mM ammonium acetate (pH-9.0) water for polar compound CH:CHROMATOGRAPHY_SUMMARY separation, while mobile phase B consisted of 100% acetonitrile. At a flow rate CH:CHROMATOGRAPHY_SUMMARY of 0.35 mL/min, the elution gradient commences at 85% B and proceeds to 10% B CH:CHROMATOGRAPHY_SUMMARY over 14 minutes. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Waters XBridge BEH Amide (100 x 2.1 mm, 2.5 µm) CH:SOLVENT_A 100% Water; 20 mM ammonium acetate (pH-9.0) CH:SOLVENT_B 100% Acetonitrile CH:FLOW_GRADIENT 85% B and proceeds to 10% B over 14 minutes CH:FLOW_RATE 350 µL/min CH:COLUMN_TEMPERATURE 40°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fusion Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS mass resolution was retained at 120,000 for MS1 mode and 30,000 for MS2 MS:MS_COMMENTS acquisition. The data acquisition mass range was 60-900Da Feature list has MS:MS_COMMENTS M/z_retention time (m/z is first then retention time and separated by MS:MS_COMMENTS underscore) MS:MS_RESULTS_FILE ST003711_AN006090_Results.txt UNITS:relative intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END