Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli

Abstract

Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation.

Keywords: flagella biosynthesis; isocitrate lyase; metabolic regulation; sirtuin.

Figure 1. Frequency histogram of the acetylated peptide ratios (log₂) of Escherichia coli ΔpatZ and ΔcobB mutants referred to the wild-type strain

1. A–D Bacteria were grown in minimal media: glucose-limited chemostat cultures (A, B) and batch acetate cultures (C, D). Acetylation data are expressed as the ratios for ΔpatZ/wt (A, C) and ΔcobB/wt (B, D) strains. Frequency histograms represent the median of the log₂ ratios from four biological replicates. Insert figures represent the correlation of acetylated peptide ratios in two biological replicates (Pearson's correlation of each condition/mutant comparison is shown on the plot).

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Figure 2. Differential protein acetylation in patZ and cobB mutants in acetate cultures

1. Representation of the log₂ acetylation ratio for ΔpatZ/wt (y-axis) and ΔcobB/wt (x-axis) of peptides in acetate cultures. Values used were obtained from four biological replicates (n = 1,875 different acetylated peptides detected). Peptides with significantly different ratios in the patZ and cobB mutants are colored in blue and yellow, respectively (two-sample t-test, adjusted for multiple testing using permutation-based FDR < 0.05).

2. Venn diagram showing the overlap of significant acetylated peptides with an acetylation ratio compared with the wild-type strain higher than 2 (FDR < 0.05). In blue are represented the number of significant acetylated peptides of ΔpatZ and in yellow those for ΔcobB mutant. The overlapping grey region represents those peptides which are significantly acetylated in both mutants.

3. Representative examples of proteins acetylated in the ΔcobB mutant in acetate cultures, but not altered in the patZ mutant, whose function is probably affected. Further information is detailed in the main text and in the Supplementary Materials and Methods.

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Figure 3. Analysis of acetylated proteins: acetylation motif and effect of protein abundance on acetylation

1. Sequence motif surrounding acetylated lysines. Logo was created using the Icelogo software package. All acetylated peptides found with an acetyl (K) probability higher than 0.9 and a cutoff P-value < 0.01 were used.

2. Frequency of protein acetylation detection as a function of protein abundance in the cell. Proteins in whole-cell extract protein digests were analyzed by LC-MS. Quantified proteins were sorted as a function of their relative abundance into five quantiles Q1–Q5. The less abundant proteins belong to Q1, and the more abundant ones to Q5. This analysis was performed for each experimental replicate and in all conditions assayed in this study: A, acetate cultures; B, chemostat cultures; C, glucose batch cultures exponential phase; D, glucose batch cultures stationary phase (n = 4 per condition). Upper bar plot: Orange bars represent the percentage of total proteins detected belonging to each quantile at each condition (n = 4). Lower bar plot: Purple bars represent the percentage of acetylated proteins belonging to each protein quantile. Further information is detailed in the Supplementary Materials and Methods.

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Figure 4. Regulation of acetate metabolism enzymes by lysine acetylation

1. A, B In vitro deacetylation assays of acetyl-CoA synthetase (Acs). Affinity-purified enzymes were deacetylated with purified CobB. Negative controls were performed in the absence of CobB and in the presence of the CobB inhibitor nicotinamide (NAM). The effect of deacetylation was assessed by specific enzyme activity assays, Western blotting using an anti-acetyl-lysine antibody (A) and mass spectrometry (B). Relative intensities of the acetylated peptides found in each deacetylation reaction are shown for Acs in (B). Peptide intensities were normalized.

2. C Metabolic fluxes in ¹³C-labeled glucose-limited chemostat cultures run at D = 0.2 h⁻¹. Fluxes are normalized to the glucose uptake rate (100%). Glucose uptake flux (mmol/g/h) was 8.66 ± 0.21 in the wild-type strain, 9.08 ± 0.06 in the ΔcobB mutant and 8.17 ± 0.01 in ΔpatZ mutant. Chemostat cultures were carried out in triplicate.

3. D, E In vitro deacetylation assays of isocitrate lyase (AceA). Affinity-purified enzymes were deacetylated with purified CobB. Negative controls were performed in the absence of CobB and in the presence of the CobB inhibitor nicotinamide (NAM). The effect of deacetylation was assessed by specific enzyme activity assays, Western blotting using an anti-acetyl-lysine antibody (D) and mass spectrometry (E). Relative intensities of the acetylated peptides found in each deacetylation reaction are shown for AceA in (E). Peptide intensities were normalized.

Source data are available online for this figure.

Figure 5. Microarray analysis of transcriptional response to cobB and patZ deletions in E. coli in batch and chemostat glucose cultures

1. A Annotation matrix obtained from the gene expression microarray data where the main functions of the up- and down-regulated genes are grouped by their expression with a P-value threshold of 0.005; for further information see Geiger et al (2013). Gene expression data were referred to the wild-type strain and expressed as fold-change.

2. B, C Transcriptional regulation of the flagellar regulon (B) and acid stress response genes (C) by RcsB (Keseler et al, 2013).

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Figure 6. Physiological effects of the inactivation of RcsB due to the acetylation of lysine 154 in E. coli

1. A, B Presence of flagella (A) and mobility assays in semisolid agar (B) of the E. coli wild-type strain (1) and mutants ΔcobB (2), ΔrcsB (3), ΔrcsB+prcsB-K154R (4), ΔrcsB+prcsB-K154Q (5) and ΔrcsB+prcsB-K154E (6) was assessed. For both assays, E. coli wild-type and its mutants were harvested at the exponential phase of cultures.

Figure 7. Effect of RcsB inactivation on acid stress response

1. Glutamate decarboxylase (Gad) enzyme activity. Colorimetric enzyme activity assay was followed by the consumption of H⁺ increasing the pH of the reaction in vitro. The increase of pH was detected by the color turn from yellow to blue of the pH indicator bromocresol green.

2. Acid stress survival of the different E. coli mutants. The effect of cobB and rcsB deletion on acid stress survival of E. coli was analyzed. The rcsB knockout mutant was complemented with the rcsB wild-type gene (K154) and its different mutants mimicking different acylation states of lysine 154 (K154R, K154Q and K154E). Bacteria were grown overnight in minimal medium with pH 5.5 to pre-adapt them to growth at low pH. These bacteria were subjected to acid stress for 2 h (pH 2.5), and cell survival was measured afterward. Mean values ± SD are shown (n = 3).

Source data are available online for this figure.