Vertical inheritance of PhoR and PhoB
To examine the divergent evolution of two-component signaling pathways, we focused on the PhoR-PhoB signaling pathway (Wanner and Chang, 1987
), which is found throughout the bacterial kingdom and helps a wide range of organisms respond to phosphate starvation. To systematically identify orthologs of E. coli phoR
, we used a modified version of reciprocal best hits in BLAST analysis that allows for the identification of putative duplications. Most proteobacteria, except a small number of δ-proteobacteria, were found to encode a single ortholog of each phoR
, suggesting that these genes are rarely duplicated, particularly in the α-, β-, and γ-proteobacteria (). Additionally, gene trees for phoR
closely matched a species tree (, S1A–B
), indicating that this signaling system has likely been vertically inherited in these clades.
Phosphotransfer specificity of PhoR is different in α– and γ-proteobacteria
Given that phoR
genes were rarely duplicated during the evolution of proteobacteria, it might be expected that the residues dictating phosphotransfer specificity would be relatively constant in order to preserve the interaction between PhoR and PhoB. We thus examined the six residues in PhoR and seven residues in PhoB previously identified as critical determinants of specificity in two-component signaling proteins (Capra et al., 2010
). We extracted these residues, hereafter referred to simply as specificity residues, from 149 PhoR orthologs and 92 PhoB orthologs, and built sequence logos representing the relative frequency of amino acids at each specificity position (, Table S1
). The difference in number of PhoR and PhoB orthologs results from the independent identification of kinase and regulator orthologs; most organisms encode both PhoR and PhoB.
The specificity residues of both PhoR and PhoB are generally well-conserved (, S1C
), although several positions showed substantial variability. We then split the PhoR and PhoB sequences into groups corresponding to the three major proteobacterial subdivisions, α, β, γ. Sequence logos built for each phylogenetic group revealed that differences between subdivisions can account for nearly all of the variability in the combined sequence logos (). For instance, in γ- and β-proteobacteria the first two positions are almost always threonine and valine, whereas in α-proteobacteria these positions are usually alanine and serine or two alanines. Similar observations were made for the specificity residues of PhoB orthologs grouped according to phylogenetic subdivision. Importantly, each PhoR and PhoB sequence logo was built using species that are highly diverged. The strong conservation within each clade thus suggests that specificity residues are usually subject to strong purifying selection. Why, though, have specificity residues diverged between clades?
Identification of adaptive mutations that prevent cross-talk in vitro
The clade-specific differences in PhoR and PhoB specificity residues may simply reflect degeneracy in the residues that enable PhoR and PhoB to interact. Alternatively, the differences may have produced functional changes such that a PhoR from one clade is less efficient at interacting with a PhoB from a different clade. To distinguish between these possibilities, we purified PhoR kinases from representative γ– and α–proteobacteria, E. coli
and C. crescentus
, and examined their ability to phosphorylate a panel of 11 PhoB orthologs from α, β, and γ-proteobacteria (, S1D
). For each PhoB from a γ-proteobacterium, phosphotransfer from the E. coli
(γ) PhoR was significantly faster than from C. crescentus
(α) PhoR. Similarly, each PhoB from an α-proteobacterium was preferentially phosphorylated by the α–PhoR. For the two chosen β–PhoB orthologs, we observed more rapid phosphorylation by the γ–PhoR than the α–PhoR, consistent with the specificity residues of the β–PhoR and β–PhoB orthologs being more similar to those found in γ–proteobacteria than those in α–proteobacteria. We conclude that within each proteobacterial subdivision, the phosphotransfer specificity of PhoR and PhoB orthologs is relatively static. However, substitutions in the specificity residues of α-PhoR and α-PhoB orthologs have led to significant differences in phosphotransfer specificity between clades.
The changes in PhoR-PhoB specificity residues, and consequent alteration of interaction specificity, could have resulted from neutral drift. However, the strong conservation of specificity residues within each clade, which includes species that are widely divergent, suggests that such drift is extremely rare. Instead, the alternative PhoR-PhoB specificity residues in α-proteobacteria may be adaptive and provide an important selective advantage. We hypothesized that the substitutions in α-PhoR and α-PhoB specificity residues prevent unwanted cross-talk with another pathway that is specific to the α-proteobacteria, i.e. negative selection led to changes in α-PhoR and α-PhoB. This model predicts that PhoR orthologs from γ-proteobacteria may phosphorylate response regulators found exclusively in α-proteobacteria, which the α-PhoR orthologs have adapted to avoid phosphorylating.
To test this possibility, we performed comprehensive phosphotransfer profiling of E. coli
(γ) and C. crescentus
(α) PhoR. Both PhoR constructs were autophosphorylated in vitro
and then examined, in parallel, for phosphotransfer to the 44 response regulators encoded by C. crescentus
(). Both PhoR constructs phosphorylated the C. crescentus
PhoB, consistent with their orthologous relationship; although as noted above, phosphotransfer from the α-PhoR is more robust. Interestingly, the γ-PhoR showed significant phosphotransfer to NtrX, whereas the α-PhoR construct did not. Notably, most α-proteobacteria encode two paralogous Ntr systems, NtrB-NtrC and NtrX-NtrY, while the γ–proteobacteria typically encode only one, NtrB-NtrC (, Table S1
). The two α–Ntr systems, which likely arose through duplication and divergence, do not cross-talk with each other in vitro
() and, consistently, have different specificity residues (). Collectively, our observations suggest that the different PhoR specificity residues seen in α–proteobacteria may have evolved to accommodate the presence of a second, lineage-specific pathway, NtrX-NtrY. Such a change in PhoR was presumably accompanied by changes in the PhoB specificity residues (see ) to maintain phosphotransfer from PhoR.
Substituting γ-like specificity residues into α-PhoR increases phosphorylation of NtrX
The divergent evolution of NtrX after duplication led initially to cross-talk with PhoR in α-proteobacteria
Thus, we hypothesized that the alanine, serine, and phenylalanine found at specificity positions 1, 2, and 4 of α–PhoR proteins represent adaptive mutations that prevent crosstalk to NtrX. To test this hypothesis, we created a series of C. crescentus PhoR mutants in which specificity residues were replaced with the corresponding residues from γ–proteobacterial PhoR. We made each single mutant, three double mutants, and the triple mutant. Each mutant kinase was then profiled against the complete set of C. crescentus response regulators to examine what effect, if any, these residues have on phosphotransfer specificity. Strikingly, each mutant led to a significant increase in NtrX phosphorylation ().
We also examined detailed time courses of phosphotransfer from each mutant PhoR, as well as the wild-type kinases, to the C. crescentus
regulators PhoB and NtrX. Each mutant kinase exhibited an increase in cross-talk with NtrX compared to the wild-type C. crescentus
(α) PhoR, but retained the ability to phosphorylate C. crescentus
PhoB at rates comparable to the wild-type PhoR (, S2A–C
). Although some mutant PhoR kinases phosphorylated several substrates (see ), we focused on PhoB and NtrX as time-courses of phosphotransfer indicated these as the two preferred targets of mutant PhoR constructs (Figure S2D–E
The most significant cross-talk to NtrX occurred for PhoR mutants with a valine substituted for serine at specificity position 2. Importantly, substantial cross-talk was not observed when this serine was substituted with other residues including leucine, aspartate, glutamate, and threonine. Only valine, corresponding to that found in γ-proteobacterial PhoR orthologs, produced significant cross-talk (Figure S2F–G
Taken togther, our in vitro studies support the notion that alanine, serine, and phenylalanine at specificity positions 1, 2, and 4 represent adaptive mutations that prevent cross-talk to NtrX in α-proteobacteria.
Avoidance of cross-talk is a significant selective pressure
To test whether these mutations also prevent cross-talk in vivo, we engineered the chromosomal copy of phoR in the α-proteobacterium C. crescentus to produce a mutant PhoR in which specificity positions 1 and 2 are threonine and valine, respectively, as they are in γ-proteobacteria; hereafter this mutant strain is referred to as PhoR(TV). Based on our in vitro experiments, we expected that cross-talk from PhoR(TV) to NtrX would be induced during growth in phosphate-limited media (). During growth in such conditions, wild-type PhoR is stimulated to autophosphorylate and phosphotransfer to PhoB, which then activates genes involved in responding to phosphate limitation. Thus, any effects of increased cross-talk to NtrX by the PhoR(TV) kinase should be manifest specifically during growth in phosphate-limited media.
Cross-talk between PhoR(TV) and NtrX leads to a growth defect and fitness disadvantage in phosphate-limited media
We grew cells to mid-logarithmic phase in phosphate-limited media and measured the rate of growth by monitoring the accumulation of optical density at 600 nm. In minimal media containing either 50 μM phosphate or 5 μM phosphate, the PhoR(TV) mutant grew significantly more slowly than wild type, with a doubling time ~30% longer than wild type in each case (, S3A–B
). This growth defect was almost as severe as that observed for a ΔphoR
strain which cannot mount a proper transcriptional response to phosphate-limitation. To assess whether cross-talk from PhoR(TV) to NtrX contributed to the slow growth phenotype observed, we deleted ntrX
in the PhoR(TV) strain. Indeed, the deletion of ntrX
significantly reduced the growth deficiency of the PhoR(TV) mutant (, S3A–B
) suggesting that cross-talk with NtrX contributes significantly to the slow growth phenotype of a PhoR(TV) strain. The suppression observed was not a non-specific acceleration of growth as the ntrX
deletion alone had no effect on growth in phosphate-limited medium.
In phosphate-replete medium, the PhoR(TV) mutant strains grew at a rate nearly identical to the wild type (), indicating that, as expected, cross-talk to NtrX requires PhoR to be activated as a kinase. The ntrX deletion and PhoR(TV)/ΔntrX strains grew more slowly in phosphate-replete medium, as the NtrY-NtrX pathway is likely necessary for responding to a signal or metabolite produced in M2G medium.
To corroborate our growth rate measurements, we performed competitive fitness assays in which each mutant strain was mixed with the wild type at a ratio of 1:1 and grown in the same flask for 104 hours, or approximately 40 wild-type generations. The mutant and wild-type strains were engineered to constitutively produce CFP or YFP, allowing for a rapid assessment of relative strain abundance using fluorescence microscopy. In phosphate-limited conditions, the PhoR(TV) strain showed a significant growth disadvantage, being almost completely eliminated from the population after 104 hours (, S3C
). The fitness disadvantage of the PhoR(TV) mutant was comparable to that of ΔphoR
competed against wild type in the same phosphate-limited medium. Consistent with our growth measurements, deleting ntrX
in the PhoR(TV) background improved competitive fitness (, S3C–D
). In phosphate-replete conditions, the PhoR(TV) and ΔphoR
mutants retained a ratio with wild type close to 1:1, demonstrating that the selective disadvantage of introducing ancestral specificity residues into PhoR likely occurs only in conditions in which PhoR is a kinase. Collectively, these data further support a model in which the α-specific substitutions in PhoR specificity residues (T→A and V→S at specificity positions 1 and 2) are selectively advantageous because they help prevent phosphotransfer cross-talk to NtrX, and perhaps other response regulators.
The growth and competitive fitness defects of PhoR(TV) in phosphate-limited media were comparable to that seen for ΔphoR
. This similarity suggested that the detrimental effect of cross-talk in the PhoR(TV) strain stems from an inability to phosphorylate PhoB and activate PhoB-dependent genes in phosphate-limited conditions. To test this hypothesis directly, we examined global gene expression patterns in the PhoR(TV) and ΔphoR
strains during growth in phosphate-limited conditions. These expression profiles exhibited strong similarity with a Pearson correlation coefficient of ~0.9 (Table S2
), supporting a model in which phosphorylation cross-talk from PhoR(TV) to NtrX comes at the expense of phosphorylating PhoB. The inappropriate phosphorylation of NtrX could also contribute to the growth defect of the PhoR(TV) mutant. However, NtrX-dependent genes (see Experimental Procedures) were not significantly affected in the PhoR(TV) strain during growth in phosphate-limited conditions; NtrX-dependent genes behaved similarly in the PhoR(TV) and ΔphoR
strains in phosphate-limited conditions (Table S2
). This may result from NtrY, the cognate kinase for NtrX, functioning as a phosphatase to prevent the accumulation of phosphorylated NtrX in phosphate-limited media. Consistent with this notion, ntrX
are not required for growth in phosphate-limited media, suggesting that in this condition NtrY is likely in a phosphatase state.
Importantly, and in contrast to NtrY, PhoR functions as a kinase, not a phosphatase, in phosphate-limited media. Thus, our results indicate that the α-specific substitutions in PhoR specificity residues (T→A and V→S) impact fitness by affecting cross-talk at the level of phosphotransfer. Consistently, in phosphate-replete media, when PhoR is primarily active as a phosphatase, these substitutions had little to no effect on competitive fitness (, S3C–D
). To further confirm that these substitutions do not significantly impact the phosphatase activity of PhoR, we examined global patterns of gene expression in the PhoR(TV) mutant grown in a phosphate-replete medium. Under these conditions, PhoR likely acts as a phosphatase to eliminate any errant phosphorylation of PhoB. Accordingly, the expression levels of known PhoB-dependent genes, such as pstC, pstA,
, were modestly elevated in a ΔphoR
strain grown in phosphate-replete medium (). By contrast, these genes were not affected, or were slightly downregulated, in the PhoR(TV) strain grown in the same phosphate-replete conditions, indicating that PhoR(TV) retains phosphatase activity in vivo
. Collectively, our data demonstrate that the growth and fitness defect of the PhoR(TV) mutant stems from inappropriate phosphotransfer to NtrX, and perhaps other non-cognate substrates.
Different adaptive mutations prevent cross-talk in other proteobacterial clades
Our results suggest that α-proteobacteria have accumulated substitutions in PhoR that prevent unwanted cross-talk with the non-cognate substrate NtrX. There could, however, be other ways to avoid cross-talk between these systems in other clades. Like the α-proteobacteria, most β-proteobacteria encode NtrY-NtrX orthologs (). However, the β-PhoR orthologs have specificity residues at positions 1 and 2, similar to those found in γ-PhoR orthologs. This observation suggests that either the β-proteobacteria can tolerate cross-talk between PhoR and NtrX, or other mutations have emerged to prevent PhoR from phosphorylating NtrX. We favored the latter possibility as a comparison of sequence logos for the NtrX orthologs from α- and β-proteobacteria revealed differences at two critical positions (). Whereas most α-NtrX orthologs have aspartate, glycine, and lysine at specificity positions 2, 5, and 7, respectively, the β-NtrX orthologs typically have glycine, glutamate, and alanine at these same three respective positions. We speculated that the different specificity residues in a given β-NtrX may eliminate cross-talk from a β-PhoR; that is, β-proteobacteria may have evolved to avoid cross-talk by accumulating substitutions in NtrX rather than PhoR and PhoB.
To test this hypothesis, we asked whether introducing the β-NtrX specificity residues into an α-NtrX would eliminate cross-talk from α-PhoR(TV) which, as shown above, phosphotransfers to α-NtrX in vitro and in vivo. Indeed, whereas C. crescentus NtrX was robustly phosphorylated by PhoR(TV), a mutant NtrX harboring the β-like substitutions D13G, G20E, F107I, and K108A was not detectably phosphorylated (). This mutant NtrX was not simply unfolded or unphosphorylatable as it was still robustly phosphorylated by α-NtrY. Hence, the substitutions introduced specifically eliminated cross-talk from PhoR(TV), while still allowing for interaction with the cognate kinase NtrY. Taken together, these results suggest that in β-proteobacteria, substitutions in NtrX alleviated cross-talk with PhoR while in α-proteobacteria substitutions in PhoR prevented cross-talk with NtrX. Although the substitutions are different, the net result in both cases was an insulation of the Ntr and Pho systems.
Global optimization of signaling fidelity
Our results with the Pho and Ntr signaling pathways indicate that the avoidance of crosstalk following gene duplication is a major selective pressure that drives the accumulation of adapative substitutions in the specificity-determining residues of two-component signaling proteins. More generally, this model predicts that the specificity residues of two-component signaling proteins in extant organisms should be sufficiently different from, or orthogonal to, one another to prevent cross-talk. To test this prediction, we extracted the six major specificity residues from each of the 22 canonical histidine kinases encoded in the E. coli
K12 genome (). Pairwise comparisons indicated that kinases typically had no more than three identities with every other kinase at these six specificity sites, often with non-conservative differences at the remaining sites. One notable exception is NarX and NarQ, which contain two identities and four conservative differences. However, these kinases, which likely arose through gene duplication, each phosphorylate the response regulators NarL and NarP in vitro
and likely in vivo
, and hence represent a case of physiologically beneficial cross-regulation (Noriega et al., 2010
). Aside from these two kinases, there is a general pattern of orthogonality between specificity residues in the system-wide set of E. coli
histidine kinases. This orthogonality is further reflected by a lack of information in a sequence logo built from the specificity residues of the 22 E. coli
histidine kinases (), particularly in comparison to the sequence logos built from orthologous histidine kinases (, ). A similar pattern of orthogonality was evident in the specificity residues of the 20 canonical histidine kinases in C. crescentus
, as well as the specificity residues of the response regulators from both E. coli
and C. crescentus
). These observations, in combination with our detailed investigation of the Ntr and Pho proteins across phylogenies, suggest that the avoidance of cross-talk is a pervasive and significant selective pressure driving the system-wide insulation of two-component signaling pathways, and consequently, that in extant organisms, two-component systems are largely insulated from one another ().
Extant two-component signaling pathways are insulated from each other at the level of phosphotransfer