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. 2010 Jun 1;76(5):1162-74.
doi: 10.1111/j.1365-2958.2010.07125.x. Epub 2010 Mar 16.

CobB regulates Escherichia coli chemotaxis by deacetylating the response regulator CheY

Ru Li  1 , Jing Gu, Yuan-Yuan Chen, Chuan-Le Xiao, Li-Wei Wang, Zhi-Ping Zhang, Li-Jun Bi, Hong-Ping Wei, Xu-De Wang, Jiao-Yu Deng, Xian-En Zhang
Affiliations
Free PMC article

CobB regulates Escherichia coli chemotaxis by deacetylating the response regulator CheY

Ru Li et al. Mol Microbiol. .
Free PMC article

Abstract

The silent information regulator (Sir2) family proteins are NAD+-dependent deacetylases. Although a few substrates have been identified, functions of the bacteria Sir2-like protein (CobB) still remain unclear. Here the role of CobB on Escherichia coli chemotaxis was investigated. We used Western blotting and mass spectrometry to show that the response regulator CheY is a substrate of CobB. Surface plasmon resonance (SPR) indicated that acetylation affects the interaction between CheY and the flagellar switch protein FliM. The presence of intact flagella in knockout strains DeltacobB, Deltaacs, Delta(cobB) Delta(acs), Delta(cheA) Delta(cheZ), Delta(cheA) Delta(cheZ) Delta(cobB) and Delta(cheA) Delta(cheZ) Delta(acs) was confirmed by electron microscopy. Genetic analysis of these knockout strains showed that: (i) the DeltacobB mutant exhibited reduced responses to chemotactic stimuli in chemotactic assays, whereas the Deltaacs mutant was indistinguishable from the parental strain, (ii) CheY from the DeltacobB mutant showed a higher level of acetylation, indicating that CobB can mediate the deacetylation of CheY in vivo, and (iii) deletion of cobB reversed the phenotype of Delta(cheA) Delta(cheZ). Our findings suggest that CobB regulates E. coli chemotaxis by deacetylating CheY. Thus a new function of bacterial cobB was identified and also new insights of regulation of bacterial chemotaxis were provided.

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Figures

Fig. 1
Fig. 1
CobB deacetylates AcCheY in vitro. The acetylation levels of all proteins were determined by Western blotting using a specific anti-acetyl-lysine antibody. Experiments were replicated three times, and representative results are shown. A and B. (A) SDS-PAGE of samples. (B) Western blot of the gel in (A). Lane 1, AcCheY + NAD+; lane 2, AcCheY + CobB; lane 3, AcCheY + CobB + NAD+; lane 4, AcCheY + CobB + NAD+ + NAM; lane 5, marker. The concentration of AcCheY and CobB was 15 µM and 3 µM respectively. The reaction was carried out for 6 h at 25°C. C. Average levels of CheY acetylation, quantified from the Western blots of three experiments (mean ± SD) using AlphaView image analysis software and normalized relative to the value obtained in absence of CobB (lane 1). An asterisk indicates a statistically significant difference from lane 1 (P < 0.01; anova analysis).
Fig. 2
Fig. 2
LC-MS/MS analysis confirms that lysine 91 and 109 are acetylated in AcCheY, and can be deacetylated by CobB. A. Tryptic digestion of AcCheY with Lys91 acetylated. The boxed b(3) and b(5) ions have a mass of 42 Da greater than the corresponding ions in the CobB-treated sample. B. Tryptic digestion of AcCheY with Lys109 acetylated. The boxed b(18)++ and y(11)++ ions have a mass of 21 Da greater than the corresponding ions in the CobB-treated sample. C. Tryptic digestion of D-CheY.
Fig. 3
Fig. 3
SPR analysis of CheY and AcCheY binding to FliM. Approximately 1120 RU FliM was immobilized on CM5 sensor surface using amine coupling method. This experiment was replicated at least three times. A. Responses of CheY at the concentrations 1–16 µM (from lower to upper). B. Responses of AcCheY at the concentrations 1–32 µM (from lower to upper). The red lines represent protein injections at the indicated concentration. The black lines represent the global fit of the entire data set to 1:1 Langmuir binding model. The inserted table lists the kinetic constants derived from the sensorgrams.
Fig. 4
Fig. 4
Swarm assays on semi-solid TB plates. Cells were inoculated near the centre of tryptone swarm plates containing 0.2% agar and incubated at 35°C. Experiments were replicated four times, and representative results are shown. A. Swarm ring formation by: (1) wild-type strain W3110, (2) W3110 ΔcobB mutant and (3) W3110 Δacs mutant. Photographs were taken after 3 h. B. Displacement (diameter) of the outermost edge of swarms of strains used in (A). Data are means ± SD from four independent experiments. An asterisk indicates a statistically significant difference from W3110 (P < 0.05; anova analysis). C. Swarm ring formation by: (1) wild-type strain W3110, (2) W3110 Δ(cheA) Δ(cheZ) mutant, (3) W3110 Δ(cheA) Δ(cheZ) Δ(cobB) mutant and (4) W3110 Δ(cheA) Δ(cheZ) Δ(acs) mutant. Photographs were taken after 3.5 h. D. Displacement (diameter) of the outermost edge of swarms of strains used in (C). Data are means ± SD from four independent experiments.
Fig. 5
Fig. 5
Chemotactic responses of W3110, ΔcobB mutant, Δacs mutant and Δ(cobB) Δ(acs) mutant. A. Plug assays and drop assays. The concentrations of galactose plug, NiSO4 plug and leucine drop were 50 mM, 25 mM and 100 mM respectively. The medium was supplemented with 30 mM acetate. The results were quantified with a ruler. Data are means ± SD from four independent experiments. An asterisk indicates a statistically significant difference from W3110 (P < 0.05; anova analysis). B. Capillary assays. Assays were carried out at 35°C for 1 h. Only concentrations indicated by the symbols were tested. Data are means ± SD from three independent experiments. (▾) W3110; (○) W3110 ΔcobB mutant; (▾) W3110 Δacs mutant; (▿) W3110 Δ(cobB) Δ(acs) mutant.
Fig. 6
Fig. 6
Western blotting analysis of CheY acetylation in vivo. The 30 µg protein lysate was resolved in 12–15% SDS-PAGE and analysed by Western blotting. Experiments were replicated three times, and representative results are shown. A–C. (A) SDS-PAGE of the extracts. (B) Western blot with a CheY antibody. (C) Western blot with an anti-acetyl-lysine antibody. Lane 1, marker; lane 2, W3110; lane 3, W3110 ΔcobB mutant; lane 4, W3110 Δacs mutant. D. Average levels of CheY acetylation, quantified from the Western blots of three experiments (mean ± SD) using AlphaView image analysis software and normalized relative to the value obtained in W3110 (lane 2). An asterisk indicates a statistically significant difference from lane 2 (P < 0.01; anova analysis).
Fig. 7
Fig. 7
Chemotactic responses of W3110, Δ(cheA) Δ(cheZ), Δ(cheA) Δ(cheZ) Δ(cobB) and Δ(cheA) Δ(cheZ) Δ(acs). A. Plug assays and drop assays. The concentrations of NiSO4 plug, leucine drop and galactose plug were 25 mM, 100 mM and 50 mM respectively. The results were quantified with a ruler. Data are means ± SD from four independent experiments. An asterisk indicates a statistically significant difference from W3110 (P < 0.05; anova analysis). B. Capillary assays. Only the concentrations indicated by the symbols were tested. Data are means ± SD from three independent experiments. (▪) W3110; (formula image) W3110 Δ(cheA) Δ(cheZ) mutant; (▿) W3110 Δ(cheA) Δ(cheZ) Δ(cobB) mutant; (◊) W3110 Δ(cheA) Δ(cheZ) Δ(acs) mutant.
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