Rina Barak

Acetylation of the response regulator, CheY, is involved in bacterial chemotaxis.

It is well established that the response regulator of the chemotaxis system of Escherichia coli, CheY, can undergo acetylation at lysine residues 92 and 109 via a reaction mediated by acetyl‐CoA synthetase (Acs). The outcome is activation of CheY, which results in increased clockwise rotation. Nevertheless, it has not been known whether CheY acetylation is involved in chemotaxis. To address this question, we examined the chemotactic behaviour of two mutants, one lacking the acetylating enzyme Acs, and the other having an arginine‐for‐lysine substitution at residue 92 of CheY – one of the acetylation sites. The Δacs mutant exhibited much reduced sensitivity to chemotactic stimuli (both attractants and repellents) in tethering assays and greatly reduced responses in ring‐forming, plug and capillary assays. Likewise, the cheY(92KR) mutant had reduced sensitivity to repellents in tethering assays and a reduced response in capillary assays. However, its response to the addition or removal of attractants was normal. These observations suggest that Acs‐mediated acetylation of CheY is involved in chemotaxis and that the acetylation site Lys‐92 is only involved in the response to repellents. The observation that, in the cheY(92KR) mutant, the addition of a repellent was not chemotactically equivalent to the removal of an attractant also suggests that there are different signalling pathways for attractants and repellents in E. coli.

Acetylation represses the binding of CheY to its target proteins

The ability of CheY, the response regulator of bacterial chemotaxis, to generate clockwise rotation is regulated by two covalent modifications – phosphorylation and acetylation. While the function and signal propagation of the former are widely understood, the mechanism and role of the latter are still obscure. To obtain information on the function of this acetylation, we non‐enzymatically acetylated CheY to a level similar to that found in vivo, and examined its binding to its kinase CheA, its phosphatase CheZ and the switch protein FliM – its target at the flagellar switch complex. Acetylation repressed the binding to all three proteins. These results suggest that both phosphorylation and acetylation determine CheYs ability to bind to its target proteins, thus providing two levels of regulation, fast and slow respectively. The fast level is modulated by environmental signals (e.g. chemotactic and thermotactic stimuli). The slow one is regulated by the metabolic state of the cell and it determines, at each metabolic state, the fraction of CheY molecules that can participate in signalling.

In Vivo Acetylation of CheY, a Response Regulator in Chemotaxis of Escherichia coli

CheY, the excitatory response regulator in the chemotaxis system of Escherichia coli, can be modulated by two covalent modifications: phosphorylation and acetylation. Both modifications have been detected in vitro only. The role of CheY acetylation is still obscure, although it is known to be involved in chemotaxis and to occur in vitro by two mechanisms--acetyl-CoA synthetase-catalyzed transfer of acetyl groups from acetate to CheY and autocatalyzed transfer from AcCoA. Here, we succeeded in detecting CheY acetylation in vivo by three means--Western blotting with a specific anti-acetyl-lysine antibody, mass spectrometry, and radiolabeling with [(14)C]acetate in the presence of protein-synthesis inhibitor. Unexpectedly, the level and rate of CheY acetylation in vivo were much higher than that in vitro. Thus, before any treatment, 9-13% of the lysine residues were found acetylated, depending on the growth phase, meaning that, on average, essentially every CheY molecule was acetylated in vivo. This high level was mainly the outcome of autoacetylation. Addition of acetate caused an incremental increase in the acetylation level, in which acetyl-CoA synthetase was involved too. These findings may have far-reaching implications for the structure-function relationship of CheY.

The specificity of fumarate as a switching factor of the bacterial flagellar motor

Fumarate restores to flagella of cytoplasm‐free, CheY‐ containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild‐type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching‐promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo‐taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.

Regulation of interaction between signaling protein CheY and flagellar motor during bacterial chemotaxis.

Publisher Summary This chapter describes the regulation of interaction between the signaling protein CheY and flagellar motor during bacterial chemotaxis. This chapter discusses the regulation of the interaction between two key players in the signal transduction pathway in bacterial chemotaxis: the protein CheY and the “switch” at the base of the flagellar motor. One of the major modes of communication between a cell and its environment is by chemotaxis— namely, by attraction to some chemicals and repulsion from others. The interaction of CheY with the switch, a key step in signal transduction during chemotaxis, may be regulated in a number of ways. CheY phosphorylation is an established mechanism of regulation, but the experimental results indicate that this alone is not sufficient. AcAMP, Ac∼P, Ca2+, fumarate, and perhaps additional compounds may be involved in the regulation. AcAMP causes an additional chemical modification of CheY— that is, acetylation; the mechanism of the function of fumarate is not known. It is reasonable to assume that while the phosphorylation activates CheY to bind to the switch, CheY causes it to detach from the switch; the acetylation and the fumarate affect switching step(s) subsequent to the binding.

Chemotactic-like response of Escherichia coli cells lacking the known chemotaxis machinery but containing overexpressed CheY

We describe a chemotactic‐like response of Escherichia coli strains lacking most of the known chemotaxis machinery but containing high levels of the response regulator CheY. The bacteria accumulated in aspartate‐containing capillaries, they formed rings on tryptone‐containing semisolid agar, and the probability of counterclockwise flagellar rotation transiently increased in response to stimulation with aspartate (10−10–10−5 M; the response was inverted at > 10−4 M). The temporal response was partial and delayed, as was the response of a control wild‐type strain having a high CheY level. α‐Methyl‐DL‐aspartate, a non‐metabolizable analogue of aspartate as well as other known attractants of E. Coli, glucose and, to a lesser extent, galactose, maltose and serine caused a similar response. So did low concentrations of acetate and benzoate (which, at higher concentrations, act as repellents for wild‐type E. coli ). Other tested repellents such as indole, Ni2+ and Co2+ increased the clockwise bias. These observations raise the possibility that, at least when the conventional signal transduction components are missing, a non‐conventional chemotactic signal transduction pathway might be functional in E. coli. Potential molecular mechanisms are discussed.

Acetylation represses the binding of CheY to its target proteins: Corrigendum

Accepted 14 August, 2010. *For correspondence. E-mail m.eisenbach@weizmann.ac.il; Tel. (+972) 8 9343923; Fax (+972) 8 9472722. †Department of Neurobiology and Ethology, Faculty of Science and Science Education, University of Haifa, Mt. Carmel 31905, Israel; Department of Medicine, Division of Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA. Molecular Microbiology (2010) 77(6), 1606 doi:10.1111/j.1365-2958.2010.07354.x