Evolutionary expansion of signaling pathway families often underlies the evolution of regulatory complexity. Expansion requires the acquisition of a novel homologous pathway and the diversification of pathway specificity. Acquisition can occur either vertically, by duplication, or through horizontal transfer, while divergence of specificity is thought to occur through a promiscuous protein intermediate. The way by which these mechanisms shape the evolution of rapidly diverging signaling families is unclear. Here, we examine this question using the highly diversified Rap-Phr cell–cell signaling system, which has undergone massive expansion in the genus Bacillus. To this end, genomic sequence analysis of >300 Bacilli genomes was combined with experimental analysis of the interaction of Rap receptors with Phr autoinducers and downstream targets. Rap-Phr expansion is shown to have occurred independently in multiple Bacillus lineages, with >80 different putative rap-phr alleles evolving in the Bacillius subtilis group alone. The specificity of many rap-phr alleles and the rapid gain and loss of Rap targets are experimentally demonstrated. Strikingly, both horizontal and vertical processes were shown to participate in this expansion, each with a distinct role. Horizontal gene transfer governs the acquisition of already diverged rap-phr alleles, while intralocus duplication and divergence of the phr gene create the promiscuous intermediate required for the divergence of Rap-Phr specificity. Our results suggest a novel role for transient gene duplication and divergence during evolutionary shifts in specificity.
The evolution of signaling complexity often occurs by diversification and repeated utilization of signal transduction pathways [1–6]. This generally requires two processes: the acquisition of homologous copies of the pathway' s components and the co-diversification of interacting components to ensure specificity of interaction within a pathway while avoiding cross-talk between pathways (Fig 1A) [7,8]. Bacteria have a multitude of signal transduction pathways, which have undergone evolutionary expansion and divergence of specificity, such as two-component systems [6,8–10], antisigma-sigma factors [11], and toxin-antitoxin systems [12,13]. The large number of available bacterial genomes allows for high-resolution analysis of evolutionary expansion, rendering bacterial signal transduction a favorable model system for studying diversification. While eukaryotes can only acquire paralogous genes through duplications, bacteria can acquire them either by gene duplication (Fig 1C, right) or by horizontal transfer (Fig 1C, left) [14]. Previous works on the prevalence of these two processes in the acquisition of bacterial two-component signal transduction pathways have indicated that it is dominated by gene duplication, but it is also affected by horizontal transfer [15–17]. However, the coarse-grained resolution of these studies prevents the distinction between vertical acquisition and horizontal transfer between closely related strains [18,19]. The second requirement for paralogous expansion is the divergence of interaction specificity between pathways (Fig 1B). This is generally thought to evolve using a promiscuous form of one of the interacting components, which can interact with both variants of its partner (Fig 1B, top) [2,4, 12,20–22]. The promiscuous form can be the ancestral state, subsequently evolving into two states of different specificity [4,20], or it can be an evolutionary intermediate between the ancestral specific state to a novel state [22,23]. The ability to distinguish between these two diversification scenarios typically depends on our capacity to infer and analyze the ancestral state from phylogenetic data [2,20,21]. A recent work used deep mutational scanning to show the abundance of promiscuous bacterial intermediates in the evolution of a bacterial toxin-antitoxin family [12]. This approach, however, cannot distinguish whether the promiscuous form is ancestral or intermediate or determine the evolutionary relevance of the identified diversifying trajectories. The modes by which rapidly diversifying signaling families expand are therefore still unclear. The Rap-Phr cell–cell signaling system of Bacilli can serve as a model system to study bacterial modes of expansion and diversification [24–26]. The cytoplasmic Rap receptor can bind, and sometimes dephosphorylate, its target, leading to inhibition of target activity [27,28]. The cognate phr gene codes for a pre-polypeptide, which undergoes multiple cleavage events during its secretion, resulting in the release of a mature penta- or hexa-peptide Phr autoinducer [25,29–31]. The mature Phr peptide is transported into the cytoplasm through the oligopeptide permease system, where it can interact with Rap receptors [26], subsequently leading to major conformational changes in the Rap protein and preventing Rap from repressing its target [27,28,32,33]. Rap-Phr systems have mostly been studied in the B. subtilis 168 lab strain. This strain encodes for eight paralogous rap-phr loci, each coding for a different Phr autoinducer. In addition, it encodes for three orphan rap genes that lack a cognate phr locus [33–35]. Despite the genomic expansion of paralogous Rap-Phr systems, they all have the same overall structural organization and most have a redundant function in repressing either Spo0F or ComA, two key response regulators of the Bacillus stress response network [31,36–40]. We recently demonstrated how social selection can explain the acquisition of additional Rap-Phr systems, despite their redundant regulation of the same target [41]. Some rap-phr loci are encoded by mobile genetic elements [31,39,42–47], and while many mobile-element-associated Rap systems maintain their repressive effect on Spo0F or ComA, some also play a direct role in controlling the mobility of their associated mobile genetic elements [44,47]. To study the expansion and diversification of the Rap-Phr family, we combined computational mining of available Bacillus genomes and experimental characterization of the target and autoinducer specificity of multiple Rap-Phr systems. We found that at the organismal level, acquisition of a novel Rap-Phr paralogous system occurred by horizontal gene transfer (Fig 1C, left). At the locus level, diversification of Rap-Phr specificity was facilitated by phr gene duplication or intragenic duplication of the Phr autoinducer coding sequence, followed by diversification of the autoinducer sequence. We show that the diverged duplicated phr form can serve as a promiscuous intermediate between two states of specificity (Fig 1B, bottom). Therefore, the extreme diversity of the Rap-Phr system results from a combination of horizontal and vertical processes operating at two different levels of genetic organization.
To understand the extent to which Rap-Phr systems are prevalent in the Bacillus genus, we downloaded 413 whole-genome sequences of strains from this genus from the National Center for Biotechnology Information (NCBI) (S1 Data). The species association of these genomes is heavily biased towards genomes from the B. subtilis (127 strains) and B. cereus (216 strains) groups of species (S1 Fig). The conserved Rap structure, which includes a three-helix N-terminal and tetra-tricopeptide repeat C-terminal domain [24,32,33], allowed us to search for Rap homologs in all the genomes using the basic local alignment search tool for translated DNA (BLAST tblastn program). Following identification of Rap homologs, we searched for candidate phr genes, relying on the known organization of annotated phr genes (S2 Fig) —short open reading frames with a secretion signal sequence, located immediately downstream of the Rap gene in the same direction (see Methods) [24]. Our final database contained ~2,700 functional rap genes (S1 and S2 Data). rap genes were identified in all strains of the B. subtilis and B. cereus groups as well as in two evolutionarily distinct Bacillus species—B. halodurans and B. clausii (S1 Fig). Notably, all strains harboring at least one rap gene encoded for multiple rap paralogs (Fig 2A, S5 Data). The number of rap genes differed between groups, averaging 11 ± 2 (mean ± standard deviation [st. dev. ]) in the B. subtilis group and 6 ± 3 in the B. cereus group. In B. subtilis 168, there are three orphan rap genes not accompanied by an adjoining phr gene, a scenario typical for B. subtilis strains, which have an average 2. 7 ± 1 orphan raps per strain (mean ± st. dev.). In contrast, almost all (>95%) of the rap genes from the B. cereus group had an adjoining putative phr gene. We next performed a phylogenetic analysis of the ensemble of rap genes (see Methods for details of the phylogenetic analysis and S3 and S4 Data). Although the overall divergence of Rap homologs was large, the family was clearly divided into two groups, corresponding to the division between the B. cereus and B. subtilis groups of species (Fig 2B). This suggests that the divergence of rap genes in each of these groups occurred after their evolutionary separation, with no horizontal transfer between groups. The phylogeny of the B. clausii and B. halodurans Rap proteins suggests that they acquired their rap genes by one or two horizontal gene transfer events, respectively, from B. subtilis group isolates, followed by intraspecific diversification and accumulation, as seen in the two major groups (Fig 2B). No evidence of divergence of the Rap protein through recombination of different Rap homologs was found (Methods). These observations suggest that diversification and paralogous expansion occurred independently multiple times during the evolution of the Rap-Phr system. To gain further insight into the population genetics underlying the diversification of the Rap-Phr family, we focused on the B. subtilis group, in which multiple Rap-Phr systems have been previously characterized. The known Phr autoinducer sequences and the patterns of phr sequence conservation along the Rap phylogenetic tree were used to identify putative penta or hexa-peptide autoinducers and to cluster the Rap proteins (Fig 2C). All together, we defined 102 clusters with 81 unique Phr autoinducer peptides. To the best of our knowledge, this extreme autoinducer diversity is much greater than that observed in any other family of quorum-sensing systems. In order to identify the mode of acquisition of a novel Rap-Phr system into a genome, we analyzed the level of horizontal gene transfer of Rap-Phr systems. We used two independent measures to estimate this trait—guanine-cytosine (GC) -content analysis and abundance analysis. First, because mobile element-related genes in B. subtilis typically have a significantly lower GC-content as compared to the rest of the genome [48], we characterized Rap-Phr as mobile if their GC-content was significantly lower than the average GC-content of their respective strain (Methods, S3 Fig). We found that 75% of Rap-Phr clusters were mobile (Fig 2C, bottom right). In parallel, mobile (or accessory) genes can be identified by their intermittent appearance within strains of a given species. Thus, we constructed an association matrix, in whcih each Rap cluster was marked as either present or absent in each of the genomes of B. subtilis group isolates (S4 Fig). With few exceptions, raps identified as core genes by their GC-content appeared in the great majority of isolates from a given species, whereas mobile Rap systems appeared in only a few strains (S5 Fig, S5 Data), demonstrating a good correlation between the two measures of mobility. These results suggest that the acquisition of a novel Rap-Phr into a genome is dominated by horizontal gene transfer. To determine whether duplication, as the alternative mode of acquisition, occurred as well, we searched for cases in which two Rap-Phr systems from the same cluster were coded in the same genome (S4 Fig). Only five such cases were identified, three of which occurred in clusters that were categorized as mobile by both criteria above and may result from a recent duplicated introduction of a single mobile element. Therefore, there is little evidence for direct duplication events in Rap-Phr that belong to the “core” genome. We noted that different species in the B. subtilis group have different core Rap paralogs (S4 Fig). The observed diversity pattern fits a slow ongoing process of fixation of Rap-Phr systems, in which some Rap variants (e. g. , RapA and RapC) are fixed in multiple related species, while others are fixed only in a single species within the group. Interestingly, all orphan Rap systems belonged to the core group, by both modes of assessment, with RapD present in all but one species (B. licheniformis). Although mobile Rap-Phr systems dominated the population diversity, most Rap paralogs in any given strain (60% ± 10% mean ± st. dev.) belonged to the core group (Fig 2C, top right). Despite the abundance of available genomic data, we do not observe a saturation of Rap-Phr diversity with strain number (S6 Fig). The large diversity is also evident from the fact that several rap-phr clusters were found only on separately sequenced plasmids [49–52]. Current data indicate that despite the large sequence and autoinducer diversity, many of the Rap receptors target either Spo0F, ComA, or both. The evolutionary rate of target specificity shift is unclear. To experimentally determine the specificity of multiple Rap systems, we used two isolates (marked in S4 Fig), B. amyloliquefaciens FZB42 and B. licheniformis ATCC 14580, as templates for the cloning of ten novel rap genes, most of them core genes of their respective species (Fig 3 and S4 Fig, S1 Table). These newly cloned Rap genes belong to previously unexplored branches of the Rap phylogeny. Correspondingly, these Rap homologs almost double the phylogenetic diversity [53] of characterized Rap proteins (from ~6% to ~10% of the total phylogenetic diversity of Rap proteins in the B. subtilis group). In addition, seven B. subtilis-associated rap genes (rapC, F, I, P, J, B, and D) were cloned under the control of the inducible hyper-spank promoter [54]. All genes were introduced into the genome of strain PY79. To prevent interference by the endogenous Phr product, the rapC, F genes were introduced into a strain with a deletion of these two systems (S7 Fig, S5 Data). Finally, an available phrA deletion was used to assay the effect of RapA. The different Rap proteins were assayed for their effects on the Spo0A and ComA pathways by measuring their impact on sporulation efficiency and expression of the ComA-regulated srfA promoter using a yellow fluorescent protein (YFP) reporter [43], respectively (Fig 3A, S5 Data, Methods). Because ComA activity is indirectly affected by the Spo0A pathway [56], we introduced a spo0A deletion into each of the YFP-reporting strains. Five out of the ten novel Rap proteins affected both sporulation efficiency and srfA expression, while RapBL4 affected only sporulation. Four of the novel rap overexpression constructs did not strongly affect either pathway. The eight B. subtilis-associated Rap proteins had the expected, previously characterized effect, with RapA, I, B, and J affecting Spo0A [26,33], RapF, C, and D affecting ComA [34,37], and RapP affecting both pathways [43]. These data allow us to better estimate the rate at which target choices change along the evolution of the Rap lineage. Based on our results and those reported by others, we assembled a phylogenetic tree of 25 Rap variants whose targets have been at least partially characterized (Fig 3B). We used the GLOOME program [55] to estimate the rate of gains or losses of regulation of the Spo0A and ComA pathways. We found the most parsimonious switching model to include 12 gain and loss events (Fig 3B), starting with an ancestral strain that regulated spo0A activity. This ancestral strain acquired the ability to control ComA in multiple independent events (see Discussion). The interactions between Rap proteins with the aforementioned targets were recently analyzed at the structural level, allowing for the identification of specific Rap residues that directly interact with each target [27,28]. Upon analysis of the conservation of these residues in the characterized Rap proteins according to their functional targets (S8 Fig), we found that the amino acid residues, where RapF interacts with its target ComA, were not conserved in many other ComA-interacting Rap proteins. In contrast, the RapH amino acid residues at the interface with Spo0F were highly conserved in characterized Rap proteins, irrespective of whether they regulate the Spo0A pathway or not. The high level of conservation of Spo0F-interacting residues and low level of conservation of ComA-interacting residues were also demonstrated upon analysis of all B. subtilis group-associated Rap proteins using the ConSurf program (S9 Fig) [57]. These results further support the ancestral origin of the interaction between Rap proteins and Spo0F and the independent gain of ComA interaction by multiple sub-lineages of Rap proteins, as suggested by the parsimony analysis (Fig 3B). Anecdotal experiments in strain 168 have indicated that divergent Rap-Phr pairs are orthogonal—a receptor from one Rap-Phr strain will predominantly respond only to its cognate autoinducer [33,35,38,41]. This notion has not been studied systematically. It is also unclear whether divergent Rap-Phr systems encoded on different chromosomes would maintain orthogonality. We took advantage of the large collection of inducible Rap systems to thoroughly analyze these points. Fourteen custom-made putative autoinducer peptides were assayed for their ability to restore gene expression in the presence of different inducible Raps (Fig 3C). We used a peptide concentration of 10 μM, a level that exceeds both the measured affinity of Phr peptides to cognate Raps and the physiological levels of Phr (Methods) [25,30,43]. The interactions between peptides and Rap proteins were monitored using either the PsrfA-YFP or PspoIIG-YFP reporter constructs, depending on whether the Rap targets the ComA or Spo0A pathways, respectively (Fig 3C, S5 Data). Rap proteins that affect both pathways were assayed only once. We found that the repressive effect of all Rap proteins on gene expression was alleviated by addition of saturating amount of their respective cognate Phr peptide. One exception to this rule was RapBL4, which did not interact with its putative Phr pentapeptide (with amino-acid sequence GRAIF). We also found that the orphan RapB, J proteins were not affected by any Phr but that a 10-fold higher concentration of PhrC did activate them, in accordance with previous works that suggested this weak interaction (Fig 3C) [33,35]. We observed strict maintenance of orthogonality of Rap-Phr systems residing on the same genome. In two cases, cross-talk between two systems encoded by different strains was detected, with RapBL5 responding to both its cognate PhrBL5 autoinducer and to the related PhrBA1 autoinducer, and RapBA2 responding to its cognate PhrBA2 and more weakly to PhrBL6. Notably, the RapBL5 and RapBA1 proteins were only weakly divergent (Fig 3B), but RapBA2 and RapBL6 were unrelated. Our data therefore support the notion of strong orthogonality of Rap-Phr systems encoded in the same genome and some orthogonality between divergent Rap-Phr systems encoded by different genomes. Thus far, our results suggest that the Phr peptides coevolve with their cognate Rap receptors to maintain the specificity of interaction, while Rap receptor affinity to its main two targets can change. To understand the evolution of the Phr peptides, we studied the diversity of the phr sequences in further detail. All Rap-Phr systems analyzed to date have a single phr gene, coding for a single Phr autoinducer penta- or hexa-peptide. In contrast, we found multiple cases in our database where peptide autoinducer coding regions were duplicated (Fig 4, S10 Fig). In some cases, all putative autoinducer repeats were identical (Fig 4A and S10E Fig), while in others, a single phr gene coded for multiple similar, but nonidentical, putative peptide autoinducers (Fig 4B and S10A–S10D Fig). For example, the Phr prepeptides of a group of closely homologous Rap proteins (related to RapH) contain multiple varying repeats of the motif [S/I][D/I/N/Y]RNT[T/I] (S10B Fig). We also identified two subclusters of the B. subtilis Rap-Phr systems, in which the entire phr gene had undergone a duplication event (S10C and S10D Fig). The putative peptide autoinducers of the two phr genes had also diverged. We also observed sequence duplications (either intragenic or full-gene) events in rap-phr loci of other Bacilli (S11A–S11C Fig). A similar analysis of the related NprR-NprX quorum-sensing family [58,59] showed duplications in some nprX genes as well (S11D Fig, Methods). These results indicate that autoinducer duplications are abundant and that putative autoinducer sequences diverge after duplication. Autoinducer duplication may facilitate the coevolution of Rap-Phr pairs by allowing a duplicated and diverged phr gene to serve as a transient promiscuous intermediate (Fig 1B). To experimentally examine this possibility, we analyzed a subset of Rap-Phr systems of the RapK/RapG/RapBL3 cluster (Fig 4B) [37]. The phr gene of three closely related systems in this cluster encodes for a pre-peptide with two putative autoinducer peptides. One system, designated RapK2-RR, encodes twice for the putative penta- or hexapeptide ERPVG (T). The second system (RapK2-KR) encodes for the putative autoinducers ERPVG (T) and EKPVG (T), while the third (RapK2-KK) encodes twice for the putative autoinducer EKPVG (T). RapK2 variants (S1 Table) were cloned under the control of the hyper-spank promoter and monitored for the effect of their overexpression on a PspoIIG-YFP reporter, as described above (Fig 4C, S5 Data). spoIIG promoter activity was repressed to background levels in all three overexpression strains. spoIIG promoter activity was restored in strains overexpressing either RapK2-RR or RapK2-KK upon addition of their cognate hexapeptides (ERPVGT and EKPVGT, respectively), but not when their non-cognate hexapeptide was added (Fig 4C). RapK2-KR, whose cognate Phr encodes both type of peptides, responded only to the addition of ERPVGT. None of the strains responded to the addition of the relevant pentapeptides. The specificity shift between the different RapK2 variants may therefore provide an example for the role of duplication in such an event.
The diversity and functional orthogonality of diverging signal transduction paralogs in bacteria are well characterized [9,11,12]. However, the way by which new paralogs are acquired and diversified and, specifically, the roles of horizontal and vertical events in this process are not clear. In this work, we showed that both horizontal and vertical processes are crucial for the expansion of the Rap-Phr quorum-sensing system but operate at different levels of organization. Acquisition of a novel Rap-Phr system was shown to be facilitated by horizontal gene transfer, while diversification was facilitated by phr duplications within the diversifying locus (Fig 5). A key novelty of our finding is exposure of the role of duplications in the divergence of Rap-Phr system specificity. The prevalence of intragenic and whole-gene phr duplications and the experimental analysis of the RapK2-PhrK2 variants (Fig 4 and S10 and S11 Figs) suggest that transient Phr duplication and divergence play a role during evolutionary shifts in Rap-Phr specificity (Fig 4D). The ancestral copy of Phr interacts with the ancestral Rap form, while the duplicated and diverged autoinducer copy has the potential to interact with a coevolved receptor. Importantly, this mechanism differs in two aspects from the common views of pathway diversification. First, divergence of a signaling pathway is typically linked with a promiscuous protein that can interact with the two forms of its partner [2,12]. Here, the promiscuous form is the duplicated phr and not a single Phr peptide, which interacts with both Rap variants. Second, duplications are typically only considered important if both diverged duplicates survive over evolutionary timescales [3]. In contrast, Phr duplication and divergence are evolutionarily crucial for a specificity shift, but to complete the shift it has to be transient—with either divergence or duplication itself being lost. In the specific case we examined, the duplication persisted, while duplicate diversity was transient (Fig 4D). We found that Rap acquisition and divergence occurred independently in multiple evolutionary lineages, indicating that it is an intrinsic feature of the function of this system. In the B. subtilis group alone, we identified dozens of putative Phr autoinducer peptides, rendering it the largest known quorum-sensing family. While the high orthogonality of a significant number of pairs was experimentally verified (Fig 3B), further experimental work will be required to explore the level of orthogonality between all clusters. In fact, some cases of cross-interactions were detected (Fig 3C). Notably, strong cross-interactions between Rap-Phr pairs encoded in the same genome were not observed, while weak interactions, as seen between PhrC and the orphans RapB and J (Fig 3C) [33,35], or RapF [41], were noted. In general, the functional importance of nonspecific interactions is unclear. More specifically, the interaction between orphan Raps and non-cognate Phrs may be physiologically irrelevant, given the low affinity of these interactions (~100 μM) compared with the physiological concentration of Phrs (~100 nM) [25]. Whether orphan Raps interact with other signaling molecules remains to be determined. Notably, formation of orphan Raps is rare, and all major B. subtilis orphan Raps are anciently fixed in their genome (Fig 2C and S4 Fig). Rap proteins also diverge with regards to the targets they regulate. Our data indicate that the distinction between targets is not the result of ancient diversification but rather is the result of an ongoing process of multiple events of gain and loss of target regulation (Fig 3A and 3B). The GLOOME parsimony analysis indicated that the ancestral Rap receptor regulated the Spo0A pathway and that the regulation of ComA by Rap proteins has been gained multiple times within the B. subtilis group. This is in agreement with the low sequence conservation between different ComA-regulating Rap variants (S7 and S8 Figs) and the absence of ComA homologs in the majority of B. cereus strains. In addition, the mechanism of ComA regulation differs across Raps. RapF blocks comA binding to DNA through direct competition for the DNA binding domain [27]. In contrast, Rap60 does not block ComA DNA binding but prevents the DNA-bound ComA from activating transcription [46]. Phr diversification is accompanied by coevolution of the Rap receptor. A key future challenge is to identify the amino-acid positions of Rap that are crucial for its coevolution and the underlying structural principles of peptide-receptor specificity. Our results suggest that even key conserved features of this interaction can be lost during diversification. Specifically, arginine at the second position of the Phr autoinducer is highly conserved due to a salt-bridge with a conserved negatively charged residue on the Rap protein (Fig 2) [32,33]. While substitution to lysine (as in PhrG or PhrK2-KK) does not dramatically interfere with this interaction, this residue is substituted with the uncharged leucine in PhrBL3. Correspondingly, the conserved Rap aspartate residue is substituted by the non-charged glutamine residue in RapBL3, suggesting that the electrostatic interaction has been replaced by another type. The mobile nature of the majority of Rap-Phr systems (Fig 2C and S3 and S4 Figs) and their functional adaptive role in the transfer of mobile elements [47,52] and in social interactions [41] indicate that they act as “kind-discrimination” systems [62]. Such systems mediate discriminative interactions between bacteria or between their genetic parasites. Kind-discrimination can operate through various mechanisms such as cell–cell signaling [60], intracellular toxin-antitoxin [63], aggregation [64,65], surface exclusion [66], bacteriocin-immunity [67], or contact-mediated toxins [68,69]. Most kind-discrimination systems are two-gene systems with a high divergence of specificity between interacting pairs. Like the Rap-Phr system, some of these systems tend to accumulate in large numbers within bacteria [70]. Notably, the social nature of kind-discrimination implies that alleles may strongly interact even if they are not encoded in the same bacterium. This presumably increases selection pressure for specificity (Fig 5). Intralocus duplication events can yield a transient promiscuous intermediate in other kind-discrimination systems. A recent analysis showed that toxin-antitoxin systems can shift specificity through promiscuous toxin intermediates, which can mediate interactions with two different antitoxins [12]. However, this does not rule out the possibility of duplication as an alternative mechanism. Further analysis of natural variation will be required to further assess these phenomena. One difference between Rap-Phr and other kind-discrimination systems is the short length of both the phr gene and the mature autoinducer peptide. This may promote duplication and neofunctionalization in peptide-based quorum-sensing systems in comparison to other systems where both interacting partners are larger globular proteins. Interestingly, fungi mating pheromones show a striking similarity to the Phr diversity, with both genic and intragenic duplications of the pheromone peptide coding sequences as well as some cases where there is sequence variability between duplicates [71]. Duplications may arise by sexual selection [72], and it has been suggested, but not proven, that they may facilitate diversification. Taken together, the data from this work and previous works [41,47] show how duplication and rapid horizontal transfer can work together to rapidly expand a signaling pathway family operating at multiple levels of selection. Further work will be needed to determine the generality of this phenomenon.
Many molecular pathways are found multiple times in a given organism, where they are often reutilized for different functions. Such expansion of a family of pathways requires two main evolutionary processes—acquisition of additional copies of the pathway' s genes and divergence of interaction specificity to prevent cross-talk between pathways while preserving interactions within each copy of the pathway. In bacteria, acquisition can occur horizontally, by transfer between different lineages, or vertically, by duplication within the lineage. Interaction specificity is thought to diverge through a promiscuous intermediate component that prevents loss of interaction during the process. In this work, we study the mechanisms underlying the extreme expansion of the Rap-Phr cell–cell signaling family in the Bacillus genus. Specificity of Rap-Phr interaction is critical for guiding preferential action towards kin. We find that horizontal transfer and not duplication guides the acquisition of an already divergent Rap-Phr variant. Surprisingly, duplication still has a key role during expansion, as duplication and subsequent divergence of the signaling molecule gene provide the promiscuous intermediate state needed for divergence of specificity. We therefore identify two complementary roles for horizontal and vertical processes in the evolution of social bacterial pathways.
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Transient Duplication-Dependent Divergence and Horizontal Transfer Underlie the Evolutionary Dynamics of Bacterial Cell–Cell Signaling