Summary of study ST000884

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 PR000613. The data can be accessed directly via it's Project DOI: 10.21228/M8F681 This work is supported by NIH grant, U2C- DK119886.

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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 IDST000884
Study TitleEvidence that COG0325 proteins are involved in PLP homeostasis
Study SummaryPyridoxal 5'-phosphate (PLP) is an essential cofactor for nearly 60 Escherichia coli enzymes but is a highly reactive molecule that is toxic in its free form. How PLP levels are regulated and how PLP is delivered to target enzymes are still open questions. The COG0325 protein family belongs to the fold-type III class of PLP enzymes and binds PLP but has no known biochemical activity although it occurs in all kingdoms of life. Various pleiotropic phenotypes of the E. coli COG0325 (yggS) mutant have been reported, some of which were reproduced and extended in this study. Comparative genomic, genetic and metabolic analyses suggest that these phenotypes reflect an imbalance in PLP homeostasis. The E. coli yggS mutant accumulates the PLP precursor pyridoxine 5'-phosphate (PNP) and is sensitive to an excess of pyridoxine but not of pyridoxal. The pyridoxine toxicity phenotype is complemented by the expression of eukaryotic yggS orthologs. It is also suppressed by the presence of amino acids, specifically isoleucine, threonine and leucine, suggesting the PLP-dependent enzyme transaminase B (IlvE) is affected. These genetic results lay a foundation for future biochemical studies of the role of COG0325 proteins in PLP homeostasis.
Institute
University of California, Davis
DepartmentGenome and Biomedical Sciences Facility
LaboratoryWCMC Metabolomics Core
Last NameFiehn
First NameOliver
Address1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616
Emailofiehn@ucdavis.edu
Phone(530) 754-8258
Submit Date2017-09-14
Study CommentsLB stands for Luria Bertani medium. MG stands for minimal growth For strains grown on MG OD 0.5 is the mid log growth phase and OD 1.0 is the late log growth phase For strains grown on LB OD 1 is the mid log growth phase and OD 2.0 is the late log growth phase
Raw Data AvailableYes
Raw Data File Type(s).cdf
Analysis Type DetailGC-MS
Release Date2017-10-14
Release Version1
Oliver Fiehn Oliver Fiehn
https://dx.doi.org/10.21228/M8F681
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000613
Project DOI:doi: 10.21228/M8F681
Project Title:Evidence that COG0325 proteins are involved in Pyridoxal 5'-phosphate (PLP) homeostasis
Project Summary:Pyridoxal 5'-phosphate (PLP) is an essential cofactor for nearly 60 Escherichia coli enzymes but is a highly reactive molecule that is toxic in its free form. How PLP levels are regulated and how PLP is delivered to target enzymes are still open questions. The COG0325 protein family belongs to the fold-type III class of PLP enzymes and binds PLP but has no known biochemical activity although it occurs in all kingdoms of life. Various pleiotropic phenotypes of the E. coli COG0325 (yggS) mutant have been reported, some of which were reproduced and extended in this study. Comparative genomic, genetic and metabolic analyses suggest that these phenotypes reflect an imbalance in PLP homeostasis. The E. coli yggS mutant accumulates the PLP precursor pyridoxine 5'-phosphate (PNP) and is sensitive to an excess of pyridoxine but not of pyridoxal. The pyridoxine toxicity phenotype is complemented by the expression of eukaryotic yggS orthologs. It is also suppressed by the presence of amino acids, specifically isoleucine, threonine and leucine, suggesting the PLP-dependent enzyme transaminase B (IlvE) is affected. These genetic results lay a foundation for future biochemical studies of the role of COG0325 proteins in PLP homeostasis.
Institute:University of Florida
Department:Microbiology and Cell Science
Last Name:de Crecy-Lagard
First Name:Valerie
Address:Gainesville, FL 32611
Email:vcrecy@ufl.edu
Phone:352-392-9416

Subject:

Subject ID:SU000918
Subject Type:Cells
Subject Species:Escherichia coli
Taxonomy ID:562
Species Group:Microorganism

Factors:

Subject type: Cells; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id local_sample_id Treatment SampleType Strain
SA051220150707bctsa38_1LB OD 1.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051221150707bctsa42_2LB OD 1.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051222150706bctsa05_3LB OD 1.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051223150706bctsa48_3LB OD 1.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051224150708bctsa12_1LB OD 1.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051214150707bctsa16_1LB OD 1.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051215150707bctsa30_1LB OD 1.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051216150708bctsa47_1LB OD 1.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051217150709bctsa06_1LB OD 1.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051218150708bctsa27_1LB OD 1.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051219150706bctsa24_1LB OD 1.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051203150706bctsa06_3LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051204150707bctsa37_1LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051205150706bctsa44_1LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051206150706bctsa15_3LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051207150706bctsa01_2LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051208150708bctsa15_1LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051209150706bctsa28_2LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051210150706bctsa37_1LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051211150706bctsa40_1LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051212150708bctsa24_1LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051213150708bctsa35_1LB OD 1.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051231150707bctsa36_1LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051232150707bctsa48_2LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051233150707bctsa01_1LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051234150708bctsa48_1LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051235150708bctsa06_1LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051236150706bctsa29_1LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051225150707bctsa25_1LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051226150708bctsa01_1LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051227150706bctsa03_2LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051228150706bctsa19_3LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051229150707bctsa41_2LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051230150706bctsa42_2LB OD 1.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051256150706bctsa12_2LB OD 2.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051257150706bctsa13_2LB OD 2.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051258150706bctsa23_2LB OD 2.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051259150707bctsa28_1LB OD 2.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051260150707bctsa03_1LB OD 2.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051261150707bctsa14_1LB OD 2.0 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051250150708bctsa20_1LB OD 2.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051251150708bctsa50_1LB OD 2.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051252150708bctsa04_1LB OD 2.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051253150706bctsa02_2LB OD 2.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051254150708bctsa45_1LB OD 2.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051255150706bctsa18_2LB OD 2.0 Cell Pellets E.coli Delta glyA (strain​ VDC6665)
SA051237150706bctsa08_2LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051238150706bctsa10_2LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051239150707bctsa33_1LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051240150708bctsa44_1LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051241150706bctsa49_2LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051242150708bctsa19_1LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6594)
SA051243150706bctsa47_1LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051244150707bctsa13_1LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051245150706bctsa45_2LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051246150707bctsa24_1LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051247150708bctsa07_1LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051248150707bctsa23_1LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051249150707bctsa34_1LB OD 2.0 Cell Pellets E.coli Delta yggS (strain VDC6595)
SA051268150706bctsa41_1LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051269150707bctsa43_2LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051270150707bctsa27_1LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051271150706bctsa22_2LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051272150709bctsa10_1LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051273150707bctsa06_3LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone1)
SA051262150707bctsa10_3LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051263150706bctsa09_3LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051264150707bctsa49_2LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051265150706bctsa07_2LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051266150707bctsa26_1LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051267150708bctsa46_1LB OD 2.0 Cell Pellets E.coli WT BW25113 (clone 2)
SA051281150623bctsa15_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli Delta glyA (strain VDC6664)
SA051282150624bctsa09_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli Delta glyA (strain VDC6664)
SA051279150623bctsa10_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli Delta glyA (strain​ VDC6665)
SA051280150624bctsa10_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli Delta glyA (strain​ VDC6665)
SA051274150624bctsa06_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli Delta yggS (strain VDC6594)
SA051275150623bctsa05_4LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli Delta yggS (strain VDC6594)
SA051276150624bctsa08_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli Delta yggS (strain VDC6595)
SA051277150623bctsa28_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli Delta yggS (strain VDC6595)
SA051278150624bctsa02_4LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli Delta yggS (strain VDC6595)
SA051285150624bctsa03_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli WT BW25113 (clone1)
SA051286150623bctsa18_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli WT BW25113 (clone1)
SA051283150624bctsa11_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli WT BW25113 (clone 2)
SA051284150623bctsa17_2LB Supernatant (SN) OD 1.0 extracellular supernatant E.coli WT BW25113 (clone 2)
SA051293150624bctsa05_2LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli Delta glyA (strain VDC6664)
SA051291150623bctsa03_3LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli Delta glyA (strain​ VDC6665)
SA051292150624bctsa12_2LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli Delta glyA (strain​ VDC6665)
SA051287150624bctsa07_2LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli Delta yggS (strain VDC6595)
SA051288150624bctsa01_2LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli Delta yggS (strain VDC6595)
SA051289150623bctsa08_2LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli Delta yggS (strain VDC6595)
SA051290150623bctsa26_1LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli Delta yggS (strain VDC6595)
SA051296150624bctsa13_2LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli WT BW25113 (clone1)
SA051297150623bctsa06_4LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli WT BW25113 (clone1)
SA051294150624bctsa04_2LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli WT BW25113 (clone 2)
SA051295150623bctsa16_2LB Supernatant (SN) OD 2.0 extracellular supernatant E.coli WT BW25113 (clone 2)
SA051303150706bctsa46_1MG OD 0.5 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051304150706bctsa43_1MG OD 0.5 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051305150707bctsa50_2MG OD 0.5 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051306150706bctsa39_1MG OD 0.5 Cell Pellets E.coli Delta glyA (strain VDC6664)
SA051307150706bctsa50_1MG OD 0.5 Cell Pellets E.coli Delta glyA (strain VDC6664)
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Collection:

Collection ID:CO000912
Collection Summary:Cells were grown overnight in M9 glucose 0.2% (w/v) supplemented with 1 mM glycine at 37 °C, and then were diluted to an OD of 0.03 in M9 glucose 0.2% (w/v) supplemented with 1 mM glycine. Mid-log (OD600=0.5) or late-log (OD600=1) samples were centrifuged for 20 s at 13,500 g and stored at -80 °C.
Sample Type:Cell

Treatment:

Treatment ID:TR000932
Treatment Summary:Bacteria were grown on Luria Bertani (LB) medium (BD Diagnostics Systems) at 37°C or on M9 minimal medium supplemented with 0.2 % (w/v) glucose and 1 mM glycine

Sample Preparation:

Sampleprep ID:SP000925
Sampleprep Summary:B6 species [PLP, PNP, pyridoxamine 5'-phosphate (PMP), PL, PN and PM] were extracted from WT and ΔyggS pellets (1.0 ml culture of OD60051.0) in 0.9 ml 5 % (w/v) metaphosphoric acid and 0.1 ml internal standard 4-deoxypyridoxine (73 nmol/ml in 5 % metaphosphoric acid). The suspension was vortexed and sonicated. After centrifugation at 10 000 g for 15 min, the supernatant was filtered and a 50 ml sample was taken for HPLC analysis using fluorescence detection (excitation 328 nm, emission 393 nm).

Combined analysis:

Analysis ID AN001441
Analysis type MS
Chromatography type GC
Chromatography system Agilent 6890N
Column Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um)
MS Type EI
MS instrument type GC-TOF
MS instrument name Leco Pegasus III GC TOF
Ion Mode POSITIVE
Units Counts

Chromatography:

Chromatography ID:CH001012
Instrument Name:Agilent 6890N
Column Name:Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um)
Column Pressure:7.7 PSI (initial condition)
Column Temperature:50 - 330°C
Flow Rate:1 ml/min
Injection Temperature:50°C ramped to 250°C by 12°C/s
Sample Injection:0.5 uL
Oven Temperature:50°C for 1 min, then ramped at 20°C/min to 330°C, held constant for 5 min
Transferline Temperature:230°C
Washing Buffer:Ethyl Acetate
Sample Loop Size:30 m length x 0.25 mm internal diameter
Randomization Order:Excel generated
Chromatography Type:GC

MS:

MS ID:MS001331
Analysis ID:AN001441
Instrument Name:Leco Pegasus III GC TOF
Instrument Type:GC-TOF
MS Type:EI
Ion Mode:POSITIVE
Ion Source Temperature:250°C
Ionization Energy:70eV
Mass Accuracy:Nominal
Scan Range Moverz:85-500
Scanning Cycle:17 Hz
Scanning Range:80-500 Da
Skimmer Voltage:1850
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