Phosphorylation of the Response Regulator CheV Is Required for Adaptation to Attractants during Bacillus subtilisChemotaxis

Abstract

In the Gram-positive soil bacterium Bacillus subtilis, the chemoreceptors are coupled to the central two-component kinase CheA via two proteins, CheW and CheV. CheV is a two-domain protein with an N-terminal CheWlike domain and a C-terminal two-component receiver domain. In this study, we show that CheV is phosphorylated in vitro on a conserved aspartate in the presence of phosphorylated CheA (CheA-P). This reaction is slower compared with the phospho-transfer reaction between CheA-P and one other response regulator of the system, CheB. CheV-P is also highly stable in comparison with CheB-P. Both of these properties are more pronounced in the full-length protein compared with a truncated form composed only of the receiver domain, that is, deletion of the CheW-like domain results in increase in the rate of the phospho-transfer reaction and decrease in stability of the phosphorylated protein. Phosphorylation of CheV is required for adaptation to the addition of the chemoattractant asparagine. In tethered-cell assays, strains expressing an unphosphorylatable point mutant of cheV or a truncated mutant lacking the entire receiver domain are severely impaired in adaptation to the addition of asparagine. Both of these strains, however, show near normal counterclockwise biases, suggesting that in the absence of the attractant the chemoreceptors are efficiently coupled to CheA kinase by the mutant CheV proteins. Inability of the CheW-like domain of CheV to support complete adaptation to the addition of asparagine also suggests that unlike CheW, this domain by itself may lead to the formation of signaling complexes that stay overactive in the presence of the attractant. A possible structural basis for this feature is discussed.

Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis

“…Interestingly, deletion of either gene encoding the coupling proteins cheW or cheV (Karatan et al, 2001;Rosario et al, 1994) to make a cheRBCDW or cheRBCDV mutant made the bacteria again responsive to stimuli, and in the former strain also significantly increased the mean prestimulus CCW bias (Table 5). In this sense cheW had a moderating effect on cheD, the only allele that we observed to have this effect.…”

“…Besides the CheC mechanism, there is one that involves methylation/demethylation of the receptors (Hanlon & Ordal, 1994;Kirby et al, 1997;Zimmer et al, 2000Zimmer et al, , 2002 and another that involves phosphorylation of the response regulator CheV (Karatan et al, 2001). We sought to understand how deletion of these adaptational systems affected the unstimulated behaviour of bacteria and their response to attractant.…”

“…The latter two strains each lack one of the two proteins that couple the CheA autokinase to the MCP receptor complex. A cheWV strain has a very tumbly bias and is unresponsive to all stimuli (Karatan et al, 2001;Rosario et al, 1994). The cheRBCD strain had a very low prestimulus CCW bias and was unresponsive to the addition and removal of asparagine (Fig.…”

“…Furthermore, the ability of this strain to partially adapt to the addition of asparagine is significant because it suggests that there still exists some measure of post-additional adaptation, despite the lack of all three known adaptation mechanisms: CheR/CheB methylation/demethylation (Hanlon et al, 1993;Kirby et al, 1997;Zimmer et al, 2000Zimmer et al, , 2002, the CheC mechanism (Kirby et al, 2001;Rosario et al, 1995), and CheV phosphorylation (Karatan et al, 2001). It is possible that a fourth (as-yet-unidentified) adaptation mechanism is also present in the chemotaxis system of B. subtilis.…”

“…The other two proteins -CheC and CheV -are involved in mechanisms of adaptation. CheV has two domains, a CheW-type coupling domain and a CheY-type responseregulator domain , which undergoes phosphorylation to help bring about adaptation (Karatan et al, 2001). CheC works by an unknown mechanism.…”