Evolutionary history expands the range of signaling interactions in hybrid multikinase networks

Two-component systems (TCSs) are ubiquitous signaling pathways, typically comprising a sensory histidine kinase (HK) and a response regulator, which communicate via intermolecular kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute non-canonical TCS signaling pathways, with transmitter and receiver domains within a single protein communicating via intramolecular phosphotransfer. Here, we report how evolutionary relationships between hybrid HKs can be used as predictors of potential intermolecular and intramolecular interactions (‘phylogenetic promiscuity’). We used domain-swap genes chimeras to investigate the specificity of phosphotransfer within hybrid HKs of the GacS–GacA multikinase network of Pseudomonas brassicacearum. The receiver domain of GacS was replaced with those from nine donor hybrid HKs. Three chimeras with receivers from other hybrid HKs demonstrated correct functioning through complementation of a gacS mutant, which was dependent on strains having a functional gacA. Formation of functional chimeras was predictable on the basis of evolutionary heritage, and raises the possibility that HKs sharing a common ancestor with GacS might remain components of the contemporary GacS network. The results also demonstrate that understanding the evolutionary heritage of signaling domains in sophisticated networks allows their rational rewiring by simple domain transplantation, with implications for the creation of designer networks and inference of functional interactions.


Scientific Reports
| (2021) 11:11763 | https://doi.org/10.1038/s41598-021-91260-w www.nature.com/scientificreports/ In Table 1, we list the results obtained for all the receiver domains considered in our experimental study. They are sorted from smallest to largest interaction score, and we provide their rank among all the receiver domains involved in P. brassicacearum hybrid HKs, as well as the interaction score values, and the score gap with the next ranked one. We also indicated the standard deviation on the interaction scores, and the reliability of our ranking, both obtained by bootstrapping. We found that the top predicted partner for the GacS HK domain is the GacS receiver domain, with a gap substantially larger than the standard deviation, and a reliability equal to 1 (which is the maximum possible). The next predicted potential partners of the GacS HK domain are the receiver domains from 4122, 3082 and 1633. Gaps between their interaction scores are smaller than standard deviations, and accordingly, ranking reliabilities are smaller than 1. In contrast, the gap between the last of these three domains (1633) and the next ranked one (625_2, second receiver of RetS) is substantially larger than standard deviations. In summary, this analysis predicts that the three top potential cross-talk partners for the GacS HK domain among all the receiver domains involved in P. brassicacearum hybrid HKs, are the receiver domains of 4122, 3082 and 1633. While this approach cannot order them reliably, it predicts that they are consistently better candidates than all other ones. These results are consistent with the phylogeny-based analysis. Sequence analysis of the receiver and kinase domains shows amino acid residues common to all hybrid HKs of the GacS cluster, including phosphorylation sites: aspartic acid and histidine residues. Only one residue (asparagine) is common and exclusive to the group of receivers consisting of GacS and the three phylogenetically closest HKs ( Supplementary Fig. S2). Coevolution analysis has shown that hybrid HKs do not exhibit the same extended amino acid coevolution between kinase and receiver domains as canonical kinase-receiver pairs 6 .
Collectively, these results suggest the seven hybrid HKs of the GacS kinase cluster share a common evolutionary history (recently duplicated from a common ancestor), and are therefore potentially able to engage in cross-talk/cross-communication with each other. To explore potential phosphotransfer interactions between the different hybrid HKs of the GacS kinase cluster, we focused experimentally on the receiver domains of the hybrid HKs, creating chimeric proteins by replacing the GacS native receiver domain with receiver domains from each of the GacS receiver cluster hybrid HKs.   swapped with other receiver domains from the GacS receiver cluster. The other two chimeras were engineered to contain receiver domains from outside the GacS receiver cluster-the receiver of 2650 (a hybrid HK from outside the GacS cluster) and the first receiver domain of RetS (625_1), only the second receiver domain of which (625_2) belongs to the GacS receiver cluster (Fig. 1). In all chimeras the C-terminal GacS HPt domain was retained, potentially allowing phosphotransfer to GacA when the transplanted receiver domain was able to substitute functionally for the original GacS receiver domain. Expressing the chimeras in a gacS mutant and monitoring GacA-dependent phenotypes (protease activity and colony size) thus provided a read-out of whether the chimeric HK was functional. Table 2 shows the phenotypes of wild-type P. brassicacearum, the gacS mutant and the gacS mutant strain complemented with gacS. The wild-type and complemented gacS strains showed protease activity and small non-fluorescent colonies, while the gacS mutant exhibited no protease activity and large fluorescent colonies due to enhanced pyoverdine production. Three chimeras (containing receiver domains from the three hybrid HKs closest to GacS in the receiver cluster-1633, 3082_3 and 4122) successfully restored wild-type phenotypes, with the exception of the colony size for chimera 1633. In contrast, chimeras harboring receiver domains from Table 1. Direct coupling analysis. The results show the interaction scores (effective interaction energies) with the kinase domain of GacS. All the receiver domains involved in P. brassicacearum hybrid HKs were considered, and ranked from smallest to largest interaction scores. The gap corresponds to the difference in interaction score between two successive ranks. The reliability score represents the fraction of bootstrapping replicates where the ranking down to this domain remained identical to the one obtained without bootstrapping (see "Materials and Methods"). The receiver domains belonging to the GacS cluster are marked in bold.  Table 2). The fact that only three out of seven chimeras whose receiver is phylogenetically close to that of GacS lead to restoration of wild-type strain phenotypes, suggests that the observed phenotypes are not only the result of the high local concentration but also specificity, because four chimeras were ineffective despite their local concentrations likely being high (although we cannot exclude the possibility of misfolding or a high level of dephosphorylation for non-functional chimeras). Taken together, these results suggest that the most phylogenetically similar domains retain the capacity for intramolecular phosphotransfer, a principle we call 'phylogenetic promiscuity' .

GacS chimeric proteins are capable of both intra-and intermolecular signal transduction.
As an unorthodox hybrid HK, phosphorylated GacS transfers phosphoryl group intramolecularly to its C-terminal HPt domain, and then intermolecularly to its cognate response regulator GacA. To determine whether this still happens for chimeras that completely (3082, 4122) or partially (1633) restored wild-type phenotypes to the gacS mutant (Table 2), GacA-dependence was tested by expressing each of the three chimeras in a gacA mutant. This resulted in phenotypes comparable to the control gacA mutant (Fig. 3), demonstrating that phosphotransfer to GacA is required for the 1633, 3082 and 4122 chimeras to restore wild-type phenotypes to a gacS mutant. As the expression of rsmX is completely controlled by the GacS-GacA system 23 , we examined its expression, by quantitative reverse transcription-PCR (qRT-PCR). The rsmX expression of the mutant gacS harbouring each of the seven chimeras belonging to the GacS cluster was compared to that of the wild-type and the gacS mutant. The expression of rsmX is significantly higher in the wild-type and 3082-4122 chimeras, than in the gacS mutant strain and the four remaining chimeras including 1633, which only partially restored the wild-type phenotypes (Fig. 4, Supplementary Fig. S3).  www.nature.com/scientificreports/ Potentially, GacS chimeric proteins might not be fully functional, interacting with, and receiving phosphoryl group from the hybrid HK that had donated the receiver domain to the chimera. This possibility was tested for 3082 by introducing the GacS-3082_3 chimera into the gacS-3082 double mutant, which restored wild-type phenotypes (Fig. 5).
These observations suggest that functional GacS chimeras are able to perform both the intramolecular phosphotransfer reactions (from kinase to receiver to HPt domains) and also the intermolecular phosphotransfer to GacA, as performed by wild-type GacS.
Transplanted receiver domains in GacS chimeric proteins can be phosphorylated by donor hybrid HKs. Additionally, it is possible that phosphorylation of the transplanted receiver domain in a GacS chimera also occurs via cross-talk from another TCS (as indeed the native GacS receiver domain can). To test this possibility, we generated a 'kinase-dead' GacS-3082 chimera in which the phosphoaccepting histidine residue (H294) in the kinase domain was substituted by alanine, preventing autophosphorylation. Expression of this construct in a gacS mutant resulted in restoration of wild-type phenotypes (Fig. 6), suggesting cross-talk between the GacS chimera and another TCS protein. Indeed, expression of the GacSH294A-3082 chimera in the gacS-3082 double mutant did not restore wild-type phenotypes (Fig. 6), implying that it is 3082, which transfers phosphoryl group to GacS-3082 chimeras.  To test whether such cross-talk involves the aspartate residue of the transplanted receiver domain and/or the histidine residue of the GacS HPt domain, a mutant version of the GacS-3082 was engineered in which the aspartate residue in position 718 was substituted with an alanine residue. The GacS-3082D718A construct did not restore wild-type phenotypes to the gacS mutant (Fig. 7), likely indicating that the GacS-3082_3 chimera receives phosphoryl group onto the transplanted receiver domain from the 3082 HK.
These observations raised the question of whether the 3082 kinase is involved in the GacS-GacA system, under physiological conditions. To examine this possibility, we constructed a 3082 mutant, which displayed the same phenotypes as the wild-type. This is in accordance with the work of Lee et al. 35 on Pseudomonas alkylphenolica, which showed that the expression of rsmZ, one of the final targets of the GacS-GacA system, was not significantly altered in a bmsA (orthologous to the 3082 gene) mutant, and overexpression of bmsA in a gacA mutant did not produce the wild-type phenotype. This does not exclude the possibility that the 3082 gene, and by extension 1633 and 4122 genes, participates indirectly to the GacS-GacA TCS. Indeed, the genomic context of the 3082 gene, for instance, includes three genes encoding CheR (3081), CheB (3080) and a hybrid histidine kinase (3079). This cluster, as well the GacS-GacA system, have been shown to be involved in common processes such as biofilm formation and motility within the Pseudomonas genus [35][36][37] , probably through common mechanisms that have yet to be elucidated.

Discussion
Phylogenetic promiscuity. Evolutionary analysis suggested that hybrid HKs were not derived from a common ancestor, and their origin and expansion were achieved by lateral recruitment of a receiver domain into an HK molecule and then duplication as one unit 20 . For example, a comparative study of six species of Xanthomonas have shown that the individual domains of a hybrid histidine kinase in one species, originated directly from a gene fusion event that combined cognate HK and RR from closely related species. Such event likely occurs through a mutation of the stop codon, resulting in read-through of the open reading frame 38 . In comparison to orthodox HKs, hybrid HKs have special evolutionary characters, manifested by a higher level of DNA polymorphism and faster evolutionary rate, and frequent gene fusion or fission and duplication 38 .
Consequently, the expansion of hybrid HKs by gene duplication raises the question of their potential crosstalk. Although available data indicate that cross-talk between TCSs generally decreases TCS signaling activity, it cannot be ruled out that such systems could tolerate the presence of more than one interaction partner 39 .
It has been postulated that phylogenetic proximity and the sequence similarity of hybrid HKs are elements that may indicate the degree to which a set of HKs cross-talk or not 10 . The squid symbiont Vibrio fischeri uses a signaling network composed of two interacting hybrid HKs to promote biofilm formation and colonization 17 . The receiver domain of hybrid HK RscS and the HPt domain of hybrid HK SypF, interact to phosphorylate two downstream RRs 17 . A second example shows intra-and interprotein phosphotransfer between two hybrid HKs to control the developmental program in Myxococcus xanthus 14 . Other examples of interacting hybrid HKs have been described in Pseudomonas spp., including the RcsC/PvrS 16 , and GacS multikinase networks. Through GacA activity, the GacS network controls the transcription of several sRNAs, which orchestrate diverse functions in gammaproteobacteria 24,25 . In all these cases, phylogenetic trees of hybrid HK kinase and receiver domains in   Fig. S4). In P. brassicacearum, similar phylogenetic clustering of hybrid HK kinase and receiver domains is observed, suggesting that they may interact with each other in a multikinase network.
Interconnection of hybrid HKs. Current knowledge of hybrid HKs, stipulates that a spatial tethering of kinases to their substrates relaxes evolutionary constraints on specificity [6][7][8] , implying that a covalently linked receiver domain can be functional even in the absence of specific interactions. Nevertheless, using chimeras transplanting the receiver domain of GacS with receiver domains from donor hybrid HKs, we showed that phylogenetic proximity reflects whether a transplant retains biological activity, suggesting interactions were specific (although it is possible non-functional chimeras were misfolded or had high dephosphorylation rates). Interestingly, identification of transmitter-receiver partnerships was more successful for hybrid HKs if domains from hybrid HKs were used as the training set, than if domains from prototypical TCS were used (see "Materials and Methods"). This also suggests that intramolecular interactions within hybrid HKs require specificity, not just tethering of domains to one another.
In addition, the expression of chimeras (with and without mutated kinase domains), in single and double mutant backgrounds, demonstrated that the 3082 third receiver domain can receive signal from both the GacS and 3082 kinase domains, i.e. both intra-and intermolecularly. These observations suggest that 1633, 3082 and 4122 are likely targets to be phosphorylated by GacS in vivo.
The experiments described in this work investigated flow of signal through the GacS-GacA TCS with domains transplanted into GacS from other TCS proteins. The use of chimeras in mutant backgrounds precluded us from directly defining which/whether specific phosphotransfer interactions occur within/between wild-type proteins. However, in our experiments the signaling domains under study were maintained in the same protein context in which they are found in the wild-type proteins, and the use of chimeras allowed assessment of physiological phenotypes, which bypassed many of the problems associated with in vitro studies involving the phosphorylation of purified proteins 40 .
In P. aeruginosa, the hybrid HK RetS attenuates GacS signaling via three mechanisms, including phosphotransfer from the GacS kinase to the second receiver (Rec2) domain of RetS 19 . However, in P. brassicacearum, a GacS chimera carrying the Rec2 of RetS (625_2), does not restore wild-type phenotypes to a gacS mutant ( Table 2). This can be explained by the difference in phylogenetic distance between the two sets of receiver domains. While in P. brassicacearum the receivers of the two proteins are in two distinct clusters (Fig. 1), the Rec2 domain of RetS in P. aeruginosa is the closest receiver to that of GacS ( Supplementary Fig. S4B).
The avoidance of unwanted cross-talk is a major selective pressure for classical HK-RR two-component pathways, driving the diversification of specificity residues to produce post-duplication insulated pathways 41 . However, duplicated hybrid HKs such as those exemplified by the GacS multikinase network, converge towards a common signal output and biological response functions. This convergence allows horizontal interconnection of hybrid HKs, presumably producing sophisticated decision-making networks able to assimilate multiple sensory inputs into a single response 42 . It is also possible that additionally non-hybrid HKs may influence signaling through the network.
It should be noted that the GacS network comprises unusual hybrid sensor kinases that harbor more than one receiver domain. RetS contains two receiver domains, while 3082 contains three. In each hybrid HK, only one of the receiver domains phylogenetically clusters with GacS, or can function within GacS chimeras, implying separate roles and interaction partners for the different receiver domains, as seen for hybrid HKs in similar and diverse organisms 8,19,43 . It is likely that these proteins have multifunctional roles, potentially monitoring multiple environmental stimuli.
Multikinase network. Through in silico prediction and the use of chimeras, we have identified three proteins (1633, 3082 and 4122) whose receiver domains function interchangeably with that of GacS, enabling signal transduction via GacA and resulting in transcriptional activation of rsmX. A receiver domain of one of these proteins, 3082_3, is shown to be able to receive signal (phosphoryl group) from its cognate kinase domain both intra-and intermolecularly, suggesting that 1633, 3082 and 4122 might all be involved in the GacS network in P. brassicacearum. Further experiments are needed to confirm whether this is the case in vivo, for example by identifying signals which stimulate 1633, 3082 and 4122 signaling and testing for a GacS response to those signals. Our 'phylogenetic promiscuity' approach can be used to suggest additional hybrid HKs that may be involved in multikinase networks from publicly available data in the P2CS database. For instance, the P. aeruginosa proteins PA_2824 and PA_3462 are likely to be involved in the GacS network 44 , the VF_A0072 protein of V. fischeri is likely to interact with the RscS-SypF network 17 , and we would also predict that MXAN_2386 and MXAN_0314 of M. xanthus are part of the EspB/EspC network 14 .
The use of evolutionary relationships to predict signaling interactions also implies that signaling relationships can act as indicators of evolutionary heritage. For instance, our results would support a model of GacS network evolution in P. brassicacearum by duplication of an ancestral GacS (producing the ancestors of 4122 and 1633) and recruitment of a copy of the GacS receiver domain by another hybrid HK (the ancestor of 3082).
In summary, our results show that the phylogenetic relationships between kinase and receiver domains can be used to guide the rational 'rewiring' of communications between and within the HKs forming multikinase networks. This opens possibilities for engineering sophisticated signaling networks by simple domain transplantation, as well as investigating novel functional associations.

Materials and methods
Bacterial strains, plasmids and growth conditions. The bacterial strains and plasmids used in this study are listed in Supplementary Table S1. P. brassicacearum NFM421 and its mutants were grown at 30 °C in tenfold-diluted tryptic soy broth (TSB 1/10) or in 20-fold-diluted tryptic soy broth (TSB 1/20). For detection of extracellular protease activity, bacteria were plated on TSB 1/10 agar plates containing 1% skimmed milk. Pseudomonas agar F (PAF) (Difco) was used to reveal colony morphology. Escherichia coli strains GM2163, TOP10, and DH5α were grown in lysogeny broth (LB) at 37 °C. For growth on plates, media were solidified with 15 g/l agar (Sigma).

Construction of mutants.
Mutant of gacA was generated by deletion mutagenesis as previously described 23 and gacS mutant was developed according to the same procedure, using primers listed in Supplementary Table S2. The mutants obtained by deletion were tested for their phenotypes and then complemented, confirming the expected phenotypes. To construct the 3082 mutant and the gacS-3082 double mutant, the plasmid pCM184 45 , unable to replicate in strain NFM421, was used. To generate a vector carrying only kanamycin resistance, pCM184-Km r was created by eliminating ampicillin and tetracycline resistances from pCM184 through ScaI/XmnI and EcoRV/PshA1 digestion and re-ligation of the blunt cuts. The plasmid pCM184-Km r was then engineered by inserting an internal fragment of the target gene. Integration of the resulting plasmid into the chromosomal copy disrupts the target gene leading to the production of truncated non-functional proteins. A 0.78 kb fragment, between positions 1190 and 1977 of 3082 gene (Supplementary Table S1), surrounding the kinase domain and including the upstream region of the HATPase domain, was PCR amplified from genomic DNA with the Platinum Pfx Polymerase (Thermo Fisher Scientific), using specific primers (Supplementary  Table S2) and cloned into the pCR-XL-Topo vector (Thermo Fisher Scientific). After sequence checking, the internal fragment was digested with EcoRI, ligated into the EcoRI site of pCM184-Km r , and used to transform E. coli strain GM2163 that does not methylate plasmid DNA. The gacS-3082 double mutant is then obtained by triparental conjugation (GM2163 with pCM184-Km r plasmid as a donor, E. coli strain DH5α pRK2013 as a helper and the wild-type or the gacS mutant as a recipient strain), and screened on TSB 1/10 agar plates supplemented with 100 µg/ml ampicillin (P. brassicacearum is naturally ampicillin resistant) and 50 µg/ml kanamycin. Mutation of the 3082 gene was confirmed by PCR amplification between the plasmid Km r gene and the chromosomal regions flanking the 3082 gene, then sequencing the PCR amplification products.

GacS chimeras synthesis and expression. All chimeras consisted of full-length hybrid histidine kinases
including transmembrane regions. The GeneArt service (Invitrogen) was used for construction of chimeric protein expression vectors. Briefly, synthetic gene chimeras were assembled from synthetic oligonucleotides and/or PCR products. Synthetic genes were inserted into pMA-RQ (ampR), and verified by sequencing before inserting the chimeric genes into the expression vector pME6032 46 . Plasmids were transformed into E. coli GM2163 to avoid DNA methylation and then introduced into recipient P. brassicacearum strains by tri-partite conjugation (GM2163 with chimeric protein expression plasmids, helper DH5α pRK2013, and the P. brassicacearum strain). Potential mutants were obtained by growth on TSB 1/10 agar plates and screening for tetracycline (and kanamycin for the gacS-3082 double mutant) resistant cells.
Sequence analysis. The software MAFFT (https:// mafft. cbrc. jp/ align ment/ softw are/) was used to align kinase and receiver domain sequences from HK proteins (http:// www. p2cs. org). A threshold of 40% identity across domains was used to identify conserved amino acid residues. The multiple sequence alignment is then visualized using Jalview (https:// www. jalvi ew. org).
qRT-PCR analyses. All the strains that had received the pME6032 plasmids were grown on TSB 1/20 supplemented with isopropyl β-d-1-thiogalactopyranoside (IPTG, 1 mM) and tetracycline (20 µg/ml). Total cellular RNA from 0. Phylogenetic analysis of kinase and receiver domains and interaction prediction. The generation of phylogenetic trees was carried out as previously described 10 . Briefly, the kinase and receiver domains of TCS proteins were identified as previously described 47 and used to construct pairwise alignments using TULIP 1.5 48 , which uses the Smith-Waterman algorithm, with 1000 sequence shuffles, to estimate pairwise Z-values and infer a distance matrix. In the R environment, a hierarchical cluster analysis was undertaken using this distance matrix and the hclust function with 'Ward' as a linkage method. An in-house R function then plotted dendrograms to display the hierarchical relationship between domains. www.nature.com/scientificreports/ To predict potential interaction partners among paralogs of hybrid HKs in P. brassicacearum, we built a direct coupling analysis (DCA) model for hybrid HK kinase and receiver domains using intra-protein pairs between such domains from the P2CS database. Unorthodox proteins were included, but the set of pairs used to construct the model was restricted to those proteins that comprise just one kinase domain and one receiver domain, in order to avoid any ambiguous cases. The resulting training set comprised 12,462 domain pairs. The mean-field approximation was employed to compute DCA couplings between amino acid sites [32][33][34] , and we used the same alignment methods and parameters as in 34 . Note that in principle we could have used a training set that also included prototypical cognate HK-RR pairs, or a training set based only on prototypical cognate HK-RR pairs, as described previously 49 . However, we found that such a model was less good at predicting intra-protein domain pairs from hybrid HKs than the model constructed using hybrid domains only (the former predicts 29% of all true positives, compared to 96% for the latter), which presumably reflects differences in composition and coevolution between prototypical and hybrid TCSs. The couplings obtained from our DCA model were then employed to compute interaction scores (effective interaction energies) between the P. brassicacearum GacS kinase domain and all the receiver domains involved in P. brassicacearum hybrid HKs, as previously defined 34 . All the hybrid HKs included in our study were predicted with a 'response_reg' receiver domain, except for HK 2650 which carries a REC domain (data available on www. p2cs. org). This precluded its inclusion in the DCA analysis. To assess the statistical significance of the computed scores, we performed 1000 bootstrapping replicates of the predictions by randomly resampling sequences from the training set (with replacement). This allowed us to compute the variance of each interaction score. For each receiver domain ranked by increasing interaction score with the GacS kinase domain, we further calculated the fraction of bootstrapping replicates where the ranking down to this domain remained identical to the one obtained without bootstrapping. This fraction is indicative of the reproducibility of the ranking by interaction scores, and is called "reliability" in our results.

Data availability
Almost all data generated or analysed during this study are included in this published article (and its Supplementary Information files). Upon request raw data would be made available.