Dataset Information

Fumarate effect on the Escherichia coli proteome at aerobic and anaerobic growth conditions

ABSTRACT: The changes in protein composition of E. coli were studied in a global proteomic approach for aerobic growth without or with fumarate as carbon source (glycerol + O2, fumarate + O2, respectively) and anaerobic growth without or with fumarate as carbon source (glycerol + DMSO, glycerol + DMSO + fumarate, respectively). The experiments should unravel the changes in response to fumarate under aerobic (i) and anaerobic (ii) conditions, and more generally (iii) the fumarate proteome under aerobic versus anaerobic conditions. Fumarate was used as the C4DC due to its capability to support aerobic and anaerobic growth, and glycerol was used as the alternative carbon source that exerts no or only weak carbon catabolite repression.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Escherichia Coli

SUBMITTER: Elke Hammer

LAB HEAD: Elke Hammer

PROVIDER: PXD011760 | Pride | 2019-11-20

REPOSITORIES: Pride

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Publications

Fumarate dependent protein composition under aerobic and anaerobic growth conditions in Escherichia coli.

Surmann Kristin K Stopp Marius M Wörner Sebastian S Dhople Vishnu M VM Völker Uwe U Unden Gottfried G Hammer Elke E

Journal of proteomics 20191114

In the absence of sugars, C4-dicarboxylates (C4DC) like fumarate represent important substrates for growth of Escherichia coli. Aerobically, C4DCs are oxidized to CO<sub>2</sub> whereas anaerobically, C4DCs are used for fumarate respiration. In order to determine the impact of fumarate under aerobic and anaerobic conditions, proteomes of E. coli W3110 grown aerobically or anaerobically with fumarate and/or the non-C4DC substrate glycerol were comparatively profiled by nanoLC-MS/MS. Membrane enri ...[more]

PMID: 31734389

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Project description:Anaerobic respiration in the metal reducer Shewanella oneidensis MR-1, unlike other bacteria, is regulated by the cAMP receptor protein, CRP. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways that include DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP. To explore the roles of CyaC and CRP in the regulation of anaerobic respiration in Shewanella oneidensis MR-1, the genome-wide transcriptome profiles of ÎcyaC and Îcrp mutants were compared to those of the wild type. Given the growth deficiencies displayed by Îcrp and ÎcyaC under anaerobic conditions, all strains were cultured in chemostats under O2 limitation. Since both Îcrp and ÎcyaC strains are not impaired in aerobic respiration, oxygen-limited conditions provided the means to maintain equal growth rates (0.06 h-1) among the mutant and wild-type strains thus eliminating the effect of growth on the overall transcriptome profiles. At the same time, while growing aerobically, the cells decreased dissolved O2 concentration in the medium below the detection threshold (DOTâ¤1%) allowing for the expression of anaerobic respiratory genes. The latter was confirmed by the reduction of fumarate by the wild-type culture which produced stoichiometric amounts of succinate under O2-limited conditions. The optical density (OD600) of the wild type, ÎcyaC, and Îcrp chemostat cultures used for transcriptome profiling was 0.50, 0.45 and 0.40, respectively. All the experiments were carried out in duplicate.

Project description:The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box. The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators. We performed three comparisons to identify genes induced under anaerobic denitrifying conditions in the P. aeruginosa wild type strain and genes which are under control of the Anr or Dnr regulators under these anaerobic conditions. Since the anr and dnr mutant strains do not grow under anaerobic denitrifying conditions, we applied anaerobic shift experiments. Pseudomonas aeruginosa was grown in a modified AB minimal medium, containing 25 ÂµM FeSO4, 20 mM glucose and 50 mM NaNO3. The 200 ml aerobic cultures were grown in 1 l Erlenmeyer flasks at 37 oC and 300 rpm. The aerobic culture was grown to an OD578 of 0.3. For the aerobic culture, cells were harvested at this point. For the anaerobic shift experiments 130 ml of the respective aerobic culture were transferred to a 135 ml sealed serum flask. Control experiments verified that oxygen tension decreased within 3 - 5 min below the detection limit of an oxygen electrode. The cells were harvested after incubation for additional 2h under anaerobic conditions. Within these 2h incubation period no growth of the wild type, the anr mutant or the dnr mutant strain was observed. First comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown under aerobic conditions up to an OD578 of 0.3 with the transcriptome profile of the PAO1 strain, which was first grown under aerobic conditions up to an OD578 of 0.3 and than shifted to anaerobic conditions by transfer to a sealed serum flask and further incubated for two hours under anaerobic conditions. Second comparison: Identification of genes regulated differently in the anr mutant strain PAO6261. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa anr mutant strain PAO6261. Both strains were harvested after 2h incubation under anaerobic conditions. Third comparison: Identification of genes regulated differently in the dnr mutant strain RM536. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa dnr mutant strain RM536. Both strains were harvested after 2h incubation under anaerobic conditions.

Dataset Information

Fumarate effect on the Escherichia coli proteome at aerobic and anaerobic growth conditions

Publications

Fumarate dependent protein composition under aerobic and anaerobic growth conditions in Escherichia coli.

Similar Datasets

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