Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Christensen, David G.; Orr, James S.; Rao, Christopher V.; Wolfe, Alan J.

doi:10.1128/aem.03034-16

Cited by 54 publications

(53 citation statements)

References 27 publications

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“…And the acetylation level of eMDH increased with the incubation time, which is consistent with the previous study on protein acetylation dynamics [47]. Such acetylation accumulation may also result from a carbon-to-nitrogen or a carbon-to-magnesium imbalance in vivo [39, 86]. …”

Section: Resultssupporting

confidence: 89%

Studying the Lysine Acetylation of Malate Dehydrogenase

Venkat

Gregory

Sturges

et al. 2017

Journal of Molecular Biology

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Protein acetylation plays important roles in many biological processes. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid (TCA) cycle, has been identified to be acetylated in bacteria by proteomic studies, but no further characterization has been reported. One challenge for studying protein acetylation is to get purely acetylated proteins at specific positions. Here we applied the genetic code expansion strategy to site-specifically incorporate Nε-acetyllysine into MDH. The acetylation of lysine residues in MDH could enhance its enzyme activity. The Escherichia coli deacetylase CobB could deacetylate acetylated MDH, while the E. coli acetyltransferase YfiQ cannot acetylate MDH efficiently. Our results also demonstrated that acetyl-CoA or acetyl-phosphate could acetylate MDH chemically in vitro. Furthermore, the acetylation level of MDH was shown to be affected by carbon sources in the growth medium.

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Section: Resultssupporting

confidence: 89%

Studying the Lysine Acetylation of Malate Dehydrogenase

Venkat

Gregory

Sturges

et al. 2017

Journal of Molecular Biology

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“…Note that the band in RimJ was not reproducible. The positive control contains one additional YfiQ-dependent band around 72 kDa, which corresponds to Acs (82). YncA and AstA each produce an acetylated band that can be observed in the Coomassie stained gel at the expected molecular weight of these proteins.…”

Section: Resultsmentioning

confidence: 99%

Identification of novel protein lysine acetyltransferases inEscherichia coli

Christensen

Meyer

Baumgartner

et al. 2018

Preprint

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Post-translational modifications, such as Nε-lysine acetylation, regulate protein function. Nε-lysine acetylation can occur either non-enzymatically or enzymatically. The non-enzymatic mechanism uses acetyl phosphate (AcP) or acetyl coenzyme A (AcCoA) as acetyl donors to modify an Nε-lysine residue of a protein. The enzymatic mechanism uses Nε-lysine acetyltransferases (KATs) to specifically transfer an acetyl group from AcCoA to Nε-lysine residues on proteins. To date, only one KAT (YfiQ, also known as Pka and PatZ) has been identified in E. coli. Here, we demonstrate the existence of 4 additional E. coli KATs: RimI, YiaC, YjaB, and PhnO. In a genetic background devoid of all known acetylation mechanisms (most notably AcP and YfiQ) and one deacetylase (CobB), overexpression of these putative KATs elicited unique patterns of protein acetylation. We mutated key active site residues and found that most of them eliminated enzymatic acetylation activity. We used mass spectrometry to identify and quantify the specificity of YfiQ and the four novel KATs. Surprisingly, our analysis revealed a high degree of substrate specificity. The overlap between KAT-dependent and AcP-dependent acetylation was extremely limited, supporting the hypothesis that these two acetylation mechanisms play distinct roles in the post-translational modification of bacterial proteins. We further showed that these novel KATs are conserved across broad swaths of bacterial phylogeny. Finally, we determined that one of the novel KATs (YiaC) and the known KAT (YfiQ) can negatively regulate bacterial migration. Together, these results emphasize distinct and specific non-enzymatic and enzymatic protein acetylation mechanisms present in bacteria.ImportanceNε-lysine acetylation is one of the most abundant and important post-translational modifications across all domains of life. One of the best-studied effects of acetylation occurs in eukaryotes, where acetylation of histone tails activates gene transcription. Although bacteria do not have true histones, Nε-lysine acetylation is prevalent; however, the role of these modifications is mostly unknown. We constructed an E. coli strain that lacked both known acetylation mechanisms to identify four new Nε-lysine acetyltransferases (RimI, YiaC, YjaB, and PhnO). We used mass spectrometry to determine the substrate specificity of these acetyltransferases. Structural analysis of selected substrate proteins revealed site-specific preferences for enzymatic acetylation that had little overlap with the preferences of the previously reported acetyl-phosphate non-enzymatic acetylation mechanism. Finally, YiaC and YfiQ appear to regulate flagellar-based motility, a phenotype critical for pathogenesis of many organisms. These acetyltransferases are highly conserved and reveal deeper and more complex roles for bacterial post-translational modification.

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“…Magnesium supplementation is necessary for robust glucose consumption in buffered tryptone broth containing glucose (8). In the absence of magnesium, the rate of glucose consumption is much lower, with most occurring during stationary phase.…”

Section: Resultsmentioning

confidence: 99%

“…However, this inhibition was only observed when the environment also included significant (Ͼ10 mM) acetate, which occurs when the glucose-containing growth medium also contains magnesium and amino acids. The magnesium and amino acids help support sufficiently high growth rates such that overflow metabolism, and thus acetate excretion, becomes significant (8).…”

Section: Discussionmentioning

confidence: 99%

Extracellular Acidic pH Inhibits Acetate Consumption by Decreasing Gene Transcription of the Tricarboxylic Acid Cycle and the Glyoxylate Shunt

et al. 2019

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Escherichia coli produces acetate during aerobic growth on various carbon sources. After consuming the carbon substrate, E. coli can further grow on the acetate. This phenomenon is known as the acetate switch, where cells transition from producing acetate to consuming it. In this study, we investigated how pH governs the acetate switch. When E. coli was grown on a glucose-supplemented medium initially buffered to pH 7, the cells produced and then consumed the acetate. However, when the initial pH was dropped to 6, the cells still produced acetate but were only able to consume it when little (<10 mM) acetate was produced. When significant acetate was produced in acidic medium, which occurs when the growth medium contains magnesium, amino acids, and sugar, the cells were unable to consume the acetate. To determine the mechanism, we characterized a set of metabolic mutants and found that those defective in the tricarboxylic acid (TCA) cycle or glyoxylate shunt exhibited reduced rates of acetate consumption. We further found that the expression of the genes in these pathways was reduced during growth in acidic medium. The expression of the genes involved in the AckA-Pta pathway, which provides the principal route for both acetate production and consumption, was also inhibited in acidic medium but only after glucose was depleted, which correlates with the acetate consumption phase. On the basis of these results, we conclude that growth in acidic environments inhibits the expression of the acetate catabolism genes, which in turn prevents acetate consumption. IMPORTANCE Many microorganisms produce fermentation products during aerobic growth on sugars. One of the best-known examples is the production of acetate by Escherichia coli during aerobic growth on sugars. In E. coli, acetate production is reversible: once the cells consume the available sugar, they can consume the acetate previously produced during aerobic fermentation. We found that pH affects the reversibility of acetate production. When the cells produce significant acetate during growth in acidic environments, they are unable to consume it. Unconsumed acetate may accumulate in the cell and inhibit the expression of pathways required for acetate catabolism. These findings demonstrate how acetate alters cell metabolism; they also may be useful for the design of aerobic fermentation processes.

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Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Cited by 54 publications

References 27 publications

Studying the Lysine Acetylation of Malate Dehydrogenase

Studying the Lysine Acetylation of Malate Dehydrogenase

Identification of novel protein lysine acetyltransferases inEscherichia coli

Extracellular Acidic pH Inhibits Acetate Consumption by Decreasing Gene Transcription of the Tricarboxylic Acid Cycle and the Glyoxylate Shunt

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