Dataset Information

E.coli Proteome Carbamate Search

ABSTRACT: Carbon dioxide regulates cellular processes as diverse as metabolism, cellular homeostasis, chemosensing and pathogenesis. CO2 is generally unreactive but can interact with neutral amines on protein under normal introcellular conditions to form a carbamate post-translational modification. This data set contains the results of a screening of E.coli to search for CO2-binding proteins and the identification of several such PTM sites.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Escherichia Coli

SUBMITTER: Victoria Linthwaite

LAB HEAD: Martin Cann

PROVIDER: PXD019606 | Pride | 2022-05-19

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	180515_03_MartinCann_122_A.mgf	Mgf
	180515_03_MartinCann_122_A.wiff	Wiff
	180515_03_MartinCann_122_A.wiff.scan	Wiff
	180515_04_MartinCann_122_A_E1.mgf	Mgf
	180515_04_MartinCann_122_A_E1.wiff	Wiff

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Publications

A methodology for carbamate post-translational modification discovery and its application in Escherichia coli.

Linthwaite Victoria L VL Cann Martin J MJ

Interface focus 20210212 2

Carbon dioxide can influence cell phenotypes through the modulation of signalling pathways. CO2 regulates cellular processes as diverse as metabolism, cellular homeostasis, chemosensing and pathogenesis. This diversity of regulated processes suggests a broadly conserved mechanism for CO2 interactions with diverse cellular targets. CO2 is generally unreactive but can interact with neutral amines on protein under normal intracellular conditions to form a carbamate ...[more]

PMID: 33633830

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Project description:To understand cell responses against high succinate stress, a continuous culture was performed for 9 months using a wild Escherichia coli under gradually increasing succinate stress. The final adapted strain against high succinate stress, named DST160, was able to grow with no lag phase in medium containing 1.35 M of succinate stress while wild W3110 showed more than 38 h lag phase. The maximum growth rate and growth yield of DST160 under the stress condition were 0.20 h-1 and 0.192 g-cell/g-glucose, which were 53.8 % and 12.3 % higher than those of W3110 (0.13 h-1 and 0.171 g-cell/g-glucose, respectively). A single colony of E. coli W3110 was inoculated into a 15-mL tube containing 4 mL of LB medium. After 12 h cultivation in a shaking incubator at 37℃ and 220 rpm, pre-culture (1 mL) was transferred to a 250 mL-fermentor (Mini-chemostat fermentor; Biotron Inc., Bucheon, Korea) containing 100 mL medium consisted of M9-glucose minimal medium (3 g glucose , 0.8 g NH4Cl, 0.5 g NaCl, 7.5 g Na2HPO4•2H2O, 3 g KH2PO4 with separately added trace elements of 0.2 g MgSO4•7H2O, 0.1 g CaCl2, 1 mg thiamine) per liter supplemented with 254 mmole of succinate (30 g succinic acid) per liter. The initial pH of the medium was set to be pH 7.5 by adding 6N NaOH. The fermentor was operated at 37℃, 350 rpm with air flushing (100 mL/min.). After 12 h batch mode growth, feeding and outlet pumps were turned on at flow rate of 0.167 mL/min (Dilution rate=0.1 h-1). The fresh medium reservoir (10 L) contained the same contents and pH with the initial medium. Every 10 turnover time (100 h), the reservoir was changed with the same medium composition except succinate concentration. The succinate concentration of the reservoir was gradually increased by 25.4 ~ 84.7 mM (3 ~ 10 g/L of succinic acid) at a time than previous states depending on the cell concentrations at the outlet. When the cell concentration was reduced below O.D.=0.2, succinate feeding pump was temporarily stopped to go back to the slightly lower succinate concentration. The continuous culture was performed until the steady state when succinate in the reservoir reached 1.35 M (160 g/L in succinic acid form). The final adapted cells in 100 mL culture were harvested by centrifuge and frozen rapidly in a liquid nitrogen tank for further analyses including DNA microarray. To understand the change of up/down regulations in the high-succinate adapted E. coli, total mRNAs of control cells and the adapted E. coli cells were extracted using a RNA extraction kit (RNeasy mini kit; Quiagen, Hilden, Germany), and the transcriptomes were compared using DNA microarray. Hybridization and washing were accomplished by a facility of Digital Genomics Co (Seoul, Korea). DNA microarray were scanned using an Axon GenePix 4000B Scanner (Axon Instruments, Union City, CA, USA), and the scanned images were analyzed with GenePix Pro 3.0 software to obtain gene expression ratios.

Dataset Information

E.coli Proteome Carbamate Search

Dataset's files

Publications

A methodology for carbamate post-translational modification discovery and its application in <i>Escherichia coli</i>.

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