two - component systems , which are found in large numbers within bacteria , offer a rich setting for exploring the evolution of cell signaling ( wuichet et al , 2010 ) . these systems are composed of a sensor kinase ( sk ) that receives input signals and controls the phosphorylation state of a partner response regulator ( rr ) . two - component systems generally show a high level of specificity , with sks controlling only their partner rrs ( laub and goulian , 2007 ) . weak phosphorylation between non - partner proteins can be detected in vitro ( skerker et al , 2005 ; yamamoto et al , 2005 ) but , in at least several examples , has been shown to be suppressed in vivo in wild - type strains ( silva et al , 1998 ; siryaporn and goulian , 2008 ; groban et al , 2009 ) . the weak interactions underlying this phosphorylation cross - talk may provide an efficient starting point for the evolution of new signaling circuits . however , signal transduction in many two - component systems depends on the sk mediating two distinct and antagonistic reactions : rr phosphorylation and dephosphorylation . the critical role played by these opposing reactions in controlling the level of rr phosphorylation could significantly increase the barrier to evolving a functional signaling system . in several systems , weak cross - phosphorylation between a non - partner sk and rr can be detected in vivo in strains for which specific partner regulators have been deleted ( silva et al , 1998 ; siryaporn and goulian , 2008 ; groban et al , 2009 ) . however , the output is blind to input stimulus to the sk ( silva et al , 1998 ; siryaporn and goulian , 2008 ) . thus , cross - talk in these cases appears as a fixed offset in rr activity rather than weak signal transduction activity . analysis of interacting partners have identified residues in sks and rrs that are important for controlling the specificity of interaction ( white et al , 2007 ; burger and van nimwegen , 2008 ; skerker et al , 2008 ; weigt et al , 2009 ; bell et al , 2010 ; szurmant and hoch , 2010 ) . by modifying specificity - determining residues , sk and rr variants that specifically interact with non - partner proteins have been engineered , effectively rewiring the flow of phosphorylation ( skerker et al , 2008 ; bell et al , 2010 ) . however , for bifunctional sks , re - engineering the specificity for phosphotransfer alone ( as in skerker et al , 2008 ) , without a comparable switch in phosphatase specificity , may not necessarily produce a new system that is signal responsive . indeed , while engineered sk variants tested in vivo showed strong and specific phosphorylation of new partners ( skerker et al , 2008 ) , we have found that , at least for the cases that we tested , the variants were unresponsive to changes in input stimulus ( supplementary figure s1 ) . taken together , the above observations suggest that to produce a signal - responsive system , it may be necessary to change both phosphotransfer and phosphatase specificity of a bifunctional sk . here , we use a directed evolution approach to explore interactions between the non - partner regulators cpxa and ompr . we show that by combining mutations in cpxa that individually increase ompr phosphorylation , phosphatase activity emerges and results in a circuit that is responsive to changes in cpxa stimulus . to explore the connections between specificity and signal response , we screened for increased cross - talk between a pair of non - partner regulators in escherichia coli : cpxa and ompr ( figure 1a ) . cross - talk from cpxa to ompr is detectable in vitro ( skerker et al , 2005 ) and in vivo in strains that lack the partners for these regulators , envz and cpxr ( siryaporn and goulian , 2008 ) . to identify amino - acid substitutions in cpxa that produce increased cross - talk , individual sites along the dhp domain of cpxa , the portion of the protein associated with sk rr interaction ( zapf et al , 2000 ; marina et al , 2005 ; laub and goulian , 2007 ; casino et al , 2009 ; szurmant and hoch , 2010 ) , were mutated by saturation mutagenesis . for each site , a library with the corresponding codon randomized was screened for increased expression of cfp from an ompr - regulated promoter and was characterized for its propensity to produce increased cross - talk ( supplementary figure s2 ) . individual mutants that showed significantly increased cross - talk were selected from the libraries for five sites ( i  v  supplementary figure s2b  d ) and analyzed in greater detail ( figure 1 ) . for all of these single - site mutants , the level of ompr - regulated transcription was comparable to or surpassed the level set by ompr 's partner kinase envz ( figure 1b ) . in addition , the effect of these mutants on transcriptional regulation by cpxr , the partner rr to cpxa , suggests the increased cross - talk is not due to a general hyperactivity of the mutants ( supplementary figure s4 ) . to provide further evidence that these mutants produce increased cross - talk to ompr , we used a previously developed assay for co - localization of an ompr - yfp protein fusion with plasmids containing ompr - binding sites in live cells ( batchelor and goulian , 2006 ; siryaporn and goulian , 2008 ) . we tested two mutants ( substitutions at sites i and iii ) using this assay . both showed dramatically increased localization of ompr - yfp when compared with wild - type cpxa ( figure 1c ) . an in vitro phosphorylation assay also demonstrated that these mutants phosphorylate ompr with significantly faster kinetics relative to wild - type cpxa ( figure 1d ; supplementary figure s5 ) , again consistent with increased cross - talk to ompr . in an attempt to further increase cross - talk from cpxa to ompr , individual substitutions at sites i  v were combined , resulting in cpxamut1 ( figure 2a ) . this could indicate a defect in cpxa resulting from combining mutations or that the protein functions as a phosphatase . to test for evidence of phosphatase activity , we used a strain with an envz allele that encodes a kinase+ phosphatase envz mutant . expression of wild - type cpxa ( in the absence of cpxr ) resulted in an increase in reporter gene expression , suggesting a further increase in ompr phosphorylation due to cross - talk from cpxa ( figure 2b ) . we note that the kinase+ phosphatase envz mutant does not show the cross - talk suppression observed for wild - type envz ( siryaporn and goulian , 2008 ; groban et al , 2009 ) . this is consistent with a mechanism of cross - talk suppression that depends on the cycle of phosphorylation and dephosphorylation of the wild - type protein ( siryaporn and goulian , 2008 ; groban et al , 2009 ) . in contrast with the behavior of wild - type cpxa , expression of cpxamut1 resulted in a considerable decrease in ompr - regulated transcription ( figure 2b ) . similar results were observed in an envz strain in growth conditions that produced high levels of the phosphodonor acetyl phosphate ( supplementary figure s6 ) . cpxa responds to envelope stress ( raivio and silhavy , 2000 ) and is relatively unstimulated under ordinary growth conditions . over - expression of the lipoprotein nlpe , a condition that stimulates cpxa ( snyder et al , 1995 ) , produced a small increase in ompr - regulated transcription in the cpxamut1 strain ( figure 2c ) , suggesting cross - talk from cpxamut1 to ompr is weakly signal responsive . to improve the signal response , we performed a genetic screen using cpxamut1 as a starting point for mutagenesis . by screening for colonies that showed increased reporter gene expression upon induction of nlpe , we identified cpxamut2 , which differs from cpxamut1 by a single substitution at site iv ( figure 2a ) . in contrast to cpxamut1 , cross - talk from cpxamut2 to ompr shows a strong dependence on activating and repressing stimuli ( figure 2c ; supplementary figure s7 ) . cpxamut2 also shows behavior in vivo consistent with phosphatase activity toward ompr - p ( figure 2b ; supplementary figure s6 ) and shows significant phosphatase activity toward ompr - p in an in vitro assay ( figure 2d ) . it is also possible that cpxamut2 decreases ompc - cfp transcription by sequestering ompr - p , although we note that we have not detected localization of ompr - gfp to the periphery in cells expressing cpxamut2 at wild - type levels ( data not shown ) . the above results suggest that cpxamut2 responds to input signals and functions as a bifunctional sk , phosphorylating and dephosphorylating ompr . for a bifunctional sk , rr phosphorylation is expected to be robust or insensitive to changes in sk expression level ( batchelor and goulian , 2003 ; shinar et al , 2007 ; shinar and feinberg , 2010 ) . this robust behavior is evident for cpxamut2 , in marked contrast with the behavior of wild - type cpxa ( figure 2e ) , providing further support that cpxamut2 functions as a bifunctional sk . combining five amino - acid substitutions in cpxa , each of which individually increases ompr phosphorylation , produces a signal - responsive mutant that phosphorylates and dephosphorylates ompr . remarkably , in the context of a simple model of the cycle of phosphorylation and dephosphorylation mediated by a bifunctional sk ( figure 3a ; see supplementary information for details ) , this complex change in phenotype can be accounted for by the change in a single parameter : the strength of the interaction or affinity between cpxa and ompr . we used this model to ask how rr phosphorylation level depends on the sk  rr interaction strength . the model predicts qualitatively different behaviors for the limits of weak and strong interaction , corresponding to low and high affinity between sk and rr , respectively . in the weak - interaction limit , . with increasing interaction strength , rr phosphorylation first increases and then decreases ( figure 3a , middle and right panels ) , indicating the emergence of phosphatase activity . this counterintuitive result reflects the asymmetry between sk - mediated phosphorylation and dephosphorylation of the rr : the phosphorylation reaction requires that the sk first autophosphorylates , whereas the dephosphorylation reaction does not require any modification of the sk . the model predicts that bifunctionality and signal responsiveness emerge in the strong - interaction limit . rr interaction provides a simple interpretation of the cpxa mutants described above ( figure 3a , right panel ) . in particular , the model suggests the emergence of phosphatase activity , signal responsiveness , and robustness in cpxamut2 can result simply from a greatly increased interaction with ompr . the model suggests that ompr interacts significantly more strongly with cpxamut2 than with wild - type cpxa . to test this , we over - expressed the sks in a strain expressing a fusion of gfp to the c - terminus of the ompr receiver domain ( ompr'-gfp ) , the region that interacts with envz . over - expression of envz produces increased fluorescence at the cell periphery ( figure 3b ) , consistent with a strong interaction of the ompr receiver domain with the membrane protein envz . over - expression of wild - type cpxa or a single - site mutant , on the other hand , showed a uniform distribution of fluorescence , which is consistent with a much weaker interaction of the ompr receiver domain with these membrane proteins . in particular , although the cpxa single - site mutant shows cross - talk to ompr that is comparable to that of envz ( figure 1b ) , it nevertheless interacts relatively weakly with ompr . in contrast , over - expression of cpxamut2 produced strong fluorescence at the cell periphery , similar to the fluorescence distribution observed for over - expression of envz , suggesting a strong interaction with ompr and consistent with the predictions of the model ( figure 3a ) . over - expression of cpxamut1 produced fluorescence localization that was similar to that of cpxamut2 ( data not shown ) . in the context of our model , the weaker signal response of cpxamut1 compared with cpxamut2 could be due to a stronger sk rr interaction for cpxamut1 , although this effect is not distinguishable through our measurements of ompr'-gfp localization . the subtle synergistic and antagonistic interactions between amino acids in proteins present challenges for interpreting the relations between sequence , structure , and function in protein evolution and design ( depristo et al , 2005 ; camps et al , 2007 ; bloom and arnold , 2009 ) . indeed , it is not uncommon to find that the outcome of combining multiple mutations in a gene can not be easily predicted from the effects of each mutation alone . however , if a protein carries out more than one function , such as the phosphorylation and dephosphorylation reactions controlled by bifunctional sks , its activity may have a complex dependence on relatively simple physicochemical properties . this can give the appearance of complex intragenic interactions between mutations and produce dramatic changes in activity with relatively few amino - acid substitutions . considering that phosphorylation cross - talk from cpxa to ompr is not detectable in wild - type cells ( siryaporn and goulian , 2008 ) , a remarkably small number of amino - acid substitutions were required to produce a robust signaling pathway connecting these two proteins . this is consistent with the above analysis , which suggests that the complex behavior resulting from combining mutations can be explained by a simple increase in the strength of interaction between sk and rr . interestingly , weak cross - phosphorylation has been identified in vitro between numerous pairs of two - component systems ( skerker et al , 2005 ; yamamoto et al , 2005 ) . the suppression of cross - talk in vivo ( silva et al , 1998 ; siryaporn and goulian , 2008 ; groban et al , 2009 ) presumably prevents these interactions from having a deleterious effect on fitness , enabling them to persist . based on the above results , we suggest that this latent cross - talk may endow networks of bifunctional sks and rrs with a considerable level of evolutionary plasticity . when additional bifunctional sks or rrs appear by gene duplication or lateral gene transfer , new pathways could initially be established from basal cross - talk as signal - blind connections with fixed levels of phosphorylation . strengthening the interactions leads to phosphatase activity , signal responsiveness , and insulation from competing phosphorylation sources ( alves and savageau , 2003 ; siryaporn and goulian , 2008 ; groban et al , 2009 ) . by analogy with our observations for cpxa and from our modeling work , this complex behavior may emerge by simply combining mutations that increase cross - phosphorylation , which would be a relatively simple path in protein sequence space for the evolution of new signaling systems . this also simplifies the process of screening for new signaling pathways , which may be useful for synthetic applications involving the engineering of new microbial signaling circuits . to explore the connections between specificity and signal response , we screened for increased cross - talk between a pair of non - partner regulators in escherichia coli : cpxa and ompr ( figure 1a ) . cross - talk from cpxa to ompr is detectable in vitro ( skerker et al , 2005 ) and in vivo in strains that lack the partners for these regulators , envz and cpxr ( siryaporn and goulian , 2008 ) . to identify amino - acid substitutions in cpxa that produce increased cross - talk , individual sites along the dhp domain of cpxa , the portion of the protein associated with sk rr interaction ( zapf et al , 2000 ; marina et al , 2005 ; laub and goulian , 2007 ; casino et al , 2009 ; szurmant and hoch , 2010 ) , were mutated by saturation mutagenesis . for each site , a library with the corresponding codon randomized was screened for increased expression of cfp from an ompr - regulated promoter and was characterized for its propensity to produce increased cross - talk ( supplementary figure s2 ) . individual mutants that showed significantly increased cross - talk were selected from the libraries for five sites ( i  v  supplementary figure s2b  d ) and analyzed in greater detail ( figure 1 ) . for all of these single - site mutants , the level of ompr - regulated transcription was comparable to or surpassed the level set by ompr 's partner kinase envz ( figure 1b ) . in addition , the effect of these mutants on transcriptional regulation by cpxr , the partner rr to cpxa , suggests the increased cross - talk is not due to a general hyperactivity of the mutants ( supplementary figure s4 ) . to provide further evidence that these mutants produce increased cross - talk to ompr , we used a previously developed assay for co - localization of an ompr - yfp protein fusion with plasmids containing ompr - binding sites in live cells ( batchelor and goulian , 2006 ; siryaporn and goulian , 2008 ) . we tested two mutants ( substitutions at sites i and iii ) using this assay . both showed dramatically increased localization of ompr - yfp when compared with wild - type cpxa ( figure 1c ) . an in vitro phosphorylation assay also demonstrated that these mutants phosphorylate ompr with significantly faster kinetics relative to wild - type cpxa ( figure 1d ; supplementary figure s5 ) , again consistent with increased cross - talk to ompr . in an attempt to further increase cross - talk from cpxa to ompr , individual substitutions at sites i  v were combined , resulting in cpxamut1 ( figure 2a ) . this could indicate a defect in cpxa resulting from combining mutations or that the protein functions as a phosphatase . to test for evidence of phosphatase activity , we used a strain with an envz allele that encodes a kinase+ phosphatase envz mutant . expression of wild - type cpxa ( in the absence of cpxr ) resulted in an increase in reporter gene expression , suggesting a further increase in ompr phosphorylation due to cross - talk from cpxa ( figure 2b ) . we note that the kinase+ phosphatase envz mutant does not show the cross - talk suppression observed for wild - type envz ( siryaporn and goulian , 2008 ; groban et al , 2009 ) . this is consistent with a mechanism of cross - talk suppression that depends on the cycle of phosphorylation and dephosphorylation of the wild - type protein ( siryaporn and goulian , 2008 ; groban et al , 2009 ) . in contrast with the behavior of wild - type cpxa , expression of cpxamut1 resulted in a considerable decrease in ompr - regulated transcription ( figure 2b ) . similar results were observed in an envz strain in growth conditions that produced high levels of the phosphodonor acetyl phosphate ( supplementary figure s6 ) . cpxa responds to envelope stress ( raivio and silhavy , 2000 ) and is relatively unstimulated under ordinary growth conditions . over - expression of the lipoprotein nlpe , a condition that stimulates cpxa ( snyder et al , 1995 ) , produced a small increase in ompr - regulated transcription in the cpxamut1 strain ( figure 2c ) , suggesting cross - talk from cpxamut1 to ompr is weakly signal responsive . to improve the signal response , we performed a genetic screen using cpxamut1 as a starting point for mutagenesis . by screening for colonies that showed increased reporter gene expression upon induction of nlpe , we identified cpxamut2 , which differs from cpxamut1 by a single substitution at site iv ( figure 2a ) . in contrast to cpxamut1 , cross - talk from cpxamut2 to ompr shows a strong dependence on activating and repressing stimuli ( figure 2c ; supplementary figure s7 ) . cpxamut2 also shows behavior in vivo consistent with phosphatase activity toward ompr - p ( figure 2b ; supplementary figure s6 ) and shows significant phosphatase activity toward ompr - p in an in vitro assay ( figure 2d ) . it is also possible that cpxamut2 decreases ompc - cfp transcription by sequestering ompr - p , although we note that we have not detected localization of ompr - gfp to the periphery in cells expressing cpxamut2 at wild - type levels ( data not shown ) . the above results suggest that cpxamut2 responds to input signals and functions as a bifunctional sk , phosphorylating and dephosphorylating ompr . for a bifunctional sk , rr phosphorylation is expected to be robust or insensitive to changes in sk expression level ( batchelor and goulian , 2003 ; shinar et al , 2007 ; shinar and feinberg , 2010 ) . this robust behavior is evident for cpxamut2 , in marked contrast with the behavior of wild - type cpxa ( figure 2e ) , providing further support that cpxamut2 functions as a bifunctional sk . combining five amino - acid substitutions in cpxa , each of which individually increases ompr phosphorylation , produces a signal - responsive mutant that phosphorylates and dephosphorylates ompr . remarkably , in the context of a simple model of the cycle of phosphorylation and dephosphorylation mediated by a bifunctional sk ( figure 3a ; see supplementary information for details ) , this complex change in phenotype can be accounted for by the change in a single parameter : the strength of the interaction or affinity between cpxa and ompr . we used this model to ask how rr phosphorylation level depends on the sk  rr interaction strength . the model predicts qualitatively different behaviors for the limits of weak and strong interaction , corresponding to low and high affinity between sk and rr , respectively . in the weak - interaction limit , . with increasing interaction strength , rr phosphorylation first increases and then decreases ( figure 3a , middle and right panels ) , indicating the emergence of phosphatase activity . this counterintuitive result reflects the asymmetry between sk - mediated phosphorylation and dephosphorylation of the rr : the phosphorylation reaction requires that the sk first autophosphorylates , whereas the dephosphorylation reaction does not require any modification of the sk . the model predicts that bifunctionality and signal responsiveness emerge in the strong - interaction limit . rr interaction provides a simple interpretation of the cpxa mutants described above ( figure 3a , right panel ) . in particular , the model suggests the emergence of phosphatase activity , signal responsiveness , and robustness in cpxamut2 can result simply from a greatly increased interaction with ompr . the model suggests that ompr interacts significantly more strongly with cpxamut2 than with wild - type cpxa . to test this , we over - expressed the sks in a strain expressing a fusion of gfp to the c - terminus of the ompr receiver domain ( ompr'-gfp ) , the region that interacts with envz . over - expression of envz produces increased fluorescence at the cell periphery ( figure 3b ) , consistent with a strong interaction of the ompr receiver domain with the membrane protein envz . over - expression of wild - type cpxa or a single - site mutant , on the other hand , showed a uniform distribution of fluorescence , which is consistent with a much weaker interaction of the ompr receiver domain with these membrane proteins . in particular , although the cpxa single - site mutant shows cross - talk to ompr that is comparable to that of envz ( figure 1b ) , it nevertheless interacts relatively weakly with ompr . in contrast , over - expression of cpxamut2 produced strong fluorescence at the cell periphery , similar to the fluorescence distribution observed for over - expression of envz , suggesting a strong interaction with ompr and consistent with the predictions of the model ( figure 3a ) . over - expression of cpxamut1 produced fluorescence localization that was similar to that of cpxamut2 ( data not shown ) . in the context of our model , the weaker signal response of cpxamut1 compared with cpxamut2 could be due to a stronger sk rr interaction for cpxamut1 , although this effect is not distinguishable through our measurements of ompr'-gfp localization . the subtle synergistic and antagonistic interactions between amino acids in proteins present challenges for interpreting the relations between sequence , structure , and function in protein evolution and design ( depristo et al , 2005 ; camps et al , 2007 ; bloom and arnold , 2009 ) . indeed , it is not uncommon to find that the outcome of combining multiple mutations in a gene can not be easily predicted from the effects of each mutation alone . however , if a protein carries out more than one function , such as the phosphorylation and dephosphorylation reactions controlled by bifunctional sks , its activity may have a complex dependence on relatively simple physicochemical properties . this can give the appearance of complex intragenic interactions between mutations and produce dramatic changes in activity with relatively few amino - acid substitutions . considering that phosphorylation cross - talk from cpxa to ompr is not detectable in wild - type cells ( siryaporn and goulian , 2008 ) , a remarkably small number of amino - acid substitutions were required to produce a robust signaling pathway connecting these two proteins . this is consistent with the above analysis , which suggests that the complex behavior resulting from combining mutations can be explained by a simple increase in the strength of interaction between sk and rr . interestingly , weak cross - phosphorylation has been identified in vitro between numerous pairs of two - component systems ( skerker et al , 2005 ; yamamoto et al , 2005 ) . the suppression of cross - talk in vivo ( silva et al , 1998 ; siryaporn and goulian , 2008 ; groban et al , 2009 ) presumably prevents these interactions from having a deleterious effect on fitness , enabling them to persist . based on the above results , we suggest that this latent cross - talk may endow networks of bifunctional sks and rrs with a considerable level of evolutionary plasticity . when additional bifunctional sks or rrs appear by gene duplication or lateral gene transfer , new pathways could initially be established from basal cross - talk as signal - blind connections with fixed levels of phosphorylation . strengthening the interactions leads to phosphatase activity , signal responsiveness , and insulation from competing phosphorylation sources ( alves and savageau , 2003 ; siryaporn and goulian , 2008 ; groban et al , 2009 ) . by analogy with our observations for cpxa and from our modeling work , this complex behavior may emerge by simply combining mutations that increase cross - phosphorylation , which would be a relatively simple path in protein sequence space for the evolution of new signaling systems . this also simplifies the process of screening for new signaling pathways , which may be useful for synthetic applications involving the engineering of new microbial signaling circuits . a detailed description of the experimental procedures , with specific references to each figure , is given in supplementary information .
we have evolved a robust two - component signal transduction pathway from a sensor kinase ( sk ) and non - partner response regulator ( rr ) that show weak cross - talk in vitro and no detectable cross - talk in vivo in wild - type strains . 
 the sk , cpxa , is bifunctional , with both kinase and phosphatase activities for its partner rr . 
 we show that by combining a small number of mutations in cpxa that individually increase phosphorylation of the non - partner rr ompr , phosphatase activity against phospho - ompr emerges . 
 the resulting circuit also becomes responsive to input signal to cpxa . 
 the effects of combining these mutations in cpxa appear to reflect complex intragenic interactions between multiple sites in the protein . 
 however , by analyzing a simple model of two - component signaling , we show that the behavior can be explained by a monotonic change in a single parameter controlling protein  protein interaction strength . 
 the results suggest one possible mode of evolution for two - component systems with bifunctional sks whereby the remarkable properties and competing reactions that characterize these systems can emerge by combining mutations of the same effect .