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EttA alleviates early ribosome stalling events induced by acidic residues

ABSTRACT: In this study we have used an unbiased approach to determine the impact of the ettA deletion (delta ettA) on the physiology of E. coli. The gene deletion creates a hypersensitivity to salt when the bacteria are grown on carbon sources metabolized by the TCA cycle. This phenotype is due to the reduced translation of several genes of the TCA cycle and of genes involved in stress responses. In this work we have systematically compared three strains MG1655 WT, delta ettA and the delta ettA complemented with an exogenous chromosomal copy of ettA (CettA). Label-Free mass spectrometry studies performed on light (S15) and heavy (S150) fractions of proteins extracts from cultures grown in MMAA-NaCl medium, identified several protein expression changes in the delta ettA strain compared to the WT or the CettA strains.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: Gregory Boel

PROVIDER: MSV000093143 | MassIVE | Wed Oct 18 08:04:00 BST 2023

SECONDARY ACCESSION(S): PXD046209

REPOSITORIES: MassIVE

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Project description:Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. The bacterium Sinorhizobium meliloti has a NAD(P)-malic enzyme (DME) and a NADP-malic enzyme (TME) and DME is required for symbiotic N2-fixation. To help understand the role of these enzymes, we examined growth, metabolic and transcriptional consequences resulting from the deletion of these enzymes. Few effects were observed upon growth with glucose, whereas growth with the gluconeogenic substrate, succinate, resulted in transcriptional and metabolic effects particularly in the dme mutant strains. When grown with succinate, DME mutant cells accumulated hexose sugar phosphates and trehalose, while TME mutants accumulated putrescine. Succinate-grown DME mutant cells also showed increased transcription of genes for gluconeogenesis and for pathways such as amino acid and fatty acid synthesis that divert metabolites away from the TCA cycle. These data suggested that, DME is required to regulate the levels of TCA cycle intermediates and that the activity of TME is insufficient to prevent the accumulation of TCA cycle intermediates in cells utilizing succinate as carbon source. Consistent with this, in short-1-3 hour incubations with succinate, dme mutant cells excreted large amounts of malate whereas little malate was excreted from tme or wild-type cells. These results support the suggestion that DME is required for N2-fixation in alfalfa because it is required for synthesis of pyruvate and acetyl-CoA and the rapid metabolism of C4-dicarboxylates supplied by the plant. RNA expression was measured for S. meliloti in exponential phase grown in MOPS (morpholinpropanesulfonic acid) buffered minimal media under 2 growth conditions: 1) 15 mM succinate, 2) 15 mM glucose; using wild type cells as well as dme and tme mutant strains (6 experiments, 2 replicates: total 12 samples)

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EttA alleviates early ribosome stalling events induced by acidic residues

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