The above results suggest there are two mechanisms that protect against cross-talk from CpxA to OmpR. Both mechanisms depend on the cognate partners of the cross-talking pair, CpxR and EnvZ.
Cross-talk to a response regulator in the absence of its histidine kinase has been reported previously (Amemura et al., 1990
; Silva et al., 1998
; Stock et al., 1989
; Verhamme et al., 2002
; Wanner et al., 1988
) (reviewed in (Laub and Goulian, 2007
)). Many histidine kinases not only phosphorylate but also dephosphorylate their cognate response regulators. The dephosphorylation activity may result from a phosphatase catalytic activity of the histidine kinase or may be due to the ability of the histidine kinase to stimulate an auto-phosphatase activity intrinsic to the response regulator (Stock et al., 2000
). For simplicity, we will simply use the term phosphatase activity to refer to this histidine kinase-dependent dephosphorylation. It has been suggested that in the absence of stimulus, the phosphatase activity is important for maintaining a two-component system in an off state by preventing inappropriate phosphorylation of the response regulator by other phosphodonors (Alves and Savageau, 2003
; Laub and Goulian, 2007
; Stock et al., 1989
; Wanner, 1992
). In light of the results described here, it seems likely that this is the case for EnvZ. However, the EnvZ-OmpR system does not simply switch between off and on states. Instead, transcription is regulated in a continuous or graded fashion, which presumably reflects a continuous or graded variation in OmpR-P levels (Batchelor et al., 2004
). It is possible that under conditions in which EnvZ produces intermediate levels of OmpR-P, another phosphodonor could have a significant effect on OmpR phosphorylation. This is not what we observed for the action of CpxA on OmpR, however. Based on measurements of ompC
expression (as judged by CFP fluorescence), we did not see any evidence of an increase in OmpR-P levels due to the combined action of EnvZ and CpxA when compared with the levels in an envZ+ cpxA−
strain ( lanes 5, 4, and 8). In addition, experiments comparing wild-type and ackA− pta−
strains suggest that EnvZ has a similar ability to mask the effects of OmpR phosphorylation by the phosphodonor acetyl phosphate (Fig. S6, Supplementary Materials
A simple model can account for these observations (summarized in ): For conditions in which EnvZ activity leads to intermediate levels of OmpR-P, the model assumes the rates of OmpR phosphorylation and OmpR-P dephosphorylation by EnvZ are both high. At steady state, the balance between the reactions leads to intermediate levels of OmpR-P (). In the absence of EnvZ, if OmpR is phosphorylated by other phospho donors such as CpxA, there is presumably a relatively low rate of OmpR-P dephosphorylation A low rate of OmpR phosphorylation by CpxA, balanced by this low rate of OmpR-P dephosphorylation, can lead to intermediate levels of OmpR-P at steady state (). (We have not observed any evidence of CpxA phosphatase activity towards OmpR-P - this point is discussed further below.) When both CpxA and EnvZ are present (but CpxR is still absent), the low OmpR phosphorylation rate by CpxA is negligible compared with that of EnvZ (). For this reason, cross-talk from CpxA is unobservable when EnvZ is present. Note that this is consistent with in vitro studies showing that a cytoplasmic fragment of CpxA phosphorylates OmpR with much slower kinetics compared with a similar fragment of EnvZ (Skerker et al., 2005
). The same argument should apply for cross-talk from other phosphodonors provided they have relatively low rates of OmpR phosphorylation. Thus, in this model the balance between strong phosphorylation and dephosphorylation activities of EnvZ provides insulation from inappropriate cross-talk, even under conditions that lead to intermediate levels of OmpR-P at steady state.
Proposed mechanisms for cross-talk suppression by EnvZ and CpxR
We find that cross-talk from CpxA to OmpR is blind to the level of CpxA stimulation. Indeed, we observed cross-talk to OmpR without taking steps to stimulate CpxA. In addition, stimulation of CpxA by NlpE over-expression had no effect on cross-talk to OmpR. It is possible that in the absence of CpxR, CpxA is not stimulated by NlpE overexpression. However we observed similar results for cross-talk from EnvZ to CpxR when we examined the effect of EnvZ stimulation with procaine. It is possible that these observations are related to the fact that we do not see any evidence of cross-talk in the phosphatase activity of CpxA or EnvZ (data not shown). In particular, these results taken together may indicate that the input stimulus affects the phosphatase activity but not the kinase activity for CpxA and EnvZ. An alternative explanation is that phosphotransfer from CpxA-P to OmpR is rate-limiting and that virtually all of the CpxA in the cell is phosphorylated under steady state conditions. In this case one would expect crosstalk to be blind to stimulus irrespective of whether stimulus increases the rate of CpxA autophosphorylation or decreases the CpxA phosphatase activity. (This latter point assumes only the unphosphorylated form of CpxA can function as a phosphatase). Note that this explanation would also account for the lack of any observable phosphatase cross-talk, assuming CpxA-P.
We did not detect cross-talk from CpxA to OmpR in envZ−
strains unless the cells also lacked CpxR. We observed similar results for cross-talk from EnvZ to CpxR. Similar behavior has been reported previously for cross-talk between PhoR/PhoB and the ectopically expressed VanS/VanR system in E. coli
(Silva et al., 1998
). Our results suggest a model in which CpxR out-competes OmpR for interaction with CpxA (). If the phosphorylation of CpxR and OmpR by CpxA can be described by standard competitive inhibition, then the ratio of the phosphorylation rates for the two response regulators is the ratio of catalytic efficiencies for the two reactions, which is independent of the total CpxA concentration: VOmpR
). The steady-state concentration of phosphorylated response regulator is determined by the balance of the phosphorylation and dephosphorylation rates. Therefore, the corresponding ratio of the concentrations of phosphorylated response regulators at steady-state, [OmpR-P]/[CpxR-P], depends on the details of the dephosphorylation mechanism. If the dephosphorylation reactions were independent of CpxA and followed first order kinetics with rate constants kCpxR-P
, then [OmpR-P]/[CpxR-P] = (kCpxR-P/kOmpR-P
), which is again independent of CpxA concentration. However, the situation is different when the histidine kinase plays a role in response regulator dephosphorylation. If CpxA mediates dephosphorylation of CpxR-P but not of OmpR-P, then a a simple model of the cycle of phosphorylation and dephosphorylation predicts that [CpxR-P] is independent of CpxA concentration ((Batchelor and Goulian, 2003
) and results not shown). This implies the ratio [OmpR-P]/[CpxR-P] decreases with decreasing [CpxA]. Thus, cross-talk is kept to a minimum for low histidine kinase concentrations. Interestingly, histidine kinase concentrations appear to be relatively low compared with the concentrations of the cognate response regulators in two-component systems for which the ratio has been measured ((Cai and Inouye, 2002
) and unpublished observations).
Studies of several other pairs of homologous two-component systems suggest that similar insulation mechanisms depending on phosphatase activity and response regulator competition suppress cross-talk between these pairs as well. We observed cross-talk from EnvZ to CpxR (Fig. S4
), PhoR to OmpR, and CusS to OmpR and CpxR (data not shown) only when the reciprocal response regulator and histidine kinase were absent. Similar results have also been observed for cross-talk from several different two-component systems to the PhoR/PhoB system (Kim et al., 1996
; Silva et al., 1998
; Zhou et al., 2005
). Thus, the mechanisms described here may quite generally provide insulation between pairs of homologous two-component systems comprised of bifunctional histidine kinases. In some cases we did not observe cross-talk even when these protective layers were removed. For example, we did not observe cross-talk from RstB to OmpR and from BaeS and PhoR to CpxR under our growth conditions. In these cases, this may indicate that the relevant domains in the histidine kinases and response regulators that make contact during phospho-transfer do not share sufficient similarity. Indeed, molecular specificity is undoubtedly the dominant mechanism for minimizing cross-talk between two-component systems (Skerker et al., 2008
). However, the molecular determinants of this specificity are not sufficiently well-understood to rank two-component system pairs based on their potential for cross-talk. Nevertheless, for those pairs that are sufficiently similar that cross-phosphorylation can occur, the protective mechanisms described here can provide a significant level of insulation. In effect, these mechanisms amplify or enhance the effects of molecular specificity. Without them, the level of specificity is not sufficient to completely block the effects of cross-talk in vivo (e.g. cross-talk from CpxA to OmpR).
These mechanisms for suppressing cross-talk may also have important implications for the evolution of two-component systems. Whether new circuits with new inputs and outputs evolve through co-evolution of histidine kinase-response regulator pairs, or by establishment of new interacting partners, the evolutionary path is likely to be constrained by selection against inappropriate cross-talk with other two-component systems. Mechanisms that suppress crosstalk should partially relax some of these constraints and therefore make it possible for well-insulated two-component systems to emerge in fewer mutational steps. The same argument implies that these mechanisms should make it more difficult to isolate mutants with increased cross-talk. Consistent with this prediction, we observed that in an envZ− cpxR− background, ompC expression was highly susceptible to mutations in cpxA (). In contrast, the presence of CpxR masked or buffered the effects of this variation so that the same pool of mutated cpxA showed no significant variation in ompC expression.
The suppression mechanisms may also make it more difficult to evolve circuits that use cross-regulation as part of their design. However, this does not preclude the possibility of crosstalk among two-component systems. Sufficiently strong interactions between a bifunctional histidine kinase and non-cognate response regulator should result in robust cross-phosphorylation even in the presence of their cognate partners. Indeed, there are at least a few reports of true cross-regulation between two-component systems (Laub and Goulian, 2007
). Nevertheless, even in such cases, the cross-talking pair presumably must be protected from cross-phosphorylation with other two-component systems; the mechanisms described here may provide part of this insulation.