The evolution of signal transduction pathways is constrained by the requirements of signal fidelity, yet flexibility is necessary to allow pathway remodeling in response to environmental challenges. A detailed understanding of how flexibility and constraint shape bacterial two component signaling systems is emerging, but how new signal transduction architectures arise remains unclear. Here, we investigate pathway remodeling using the Firmicute sporulation initiation (Spo0) pathway as a model. The present-day Spo0 pathways in Bacilli and Clostridia share common ancestry, but possess different architectures. In Clostridium acetobutylicum, sensor kinases directly phosphorylate Spo0A, the master regulator of sporulation. In Bacillus subtilis, Spo0A is activated via a four-protein phosphorelay. The current view favors an ancestral direct phosphorylation architecture, with the phosphorelay emerging in the Bacillar lineage. Our results reject this hypothesis. Our analysis of 84 broadly distributed Firmicute genomes predicts phosphorelays in numerous Clostridia, contrary to the expectation that the Spo0 phosphorelay is unique to Bacilli. Our experimental verification of a functional Spo0 phosphorelay encoded by Desulfotomaculum acetoxidans (Class Clostridia) further supports functional phosphorelays in Clostridia, which strongly suggests that the ancestral Spo0 pathway was a phosphorelay. Cross complementation assays between Bacillar and Clostridial phosphorelays demonstrate conservation of interaction specificity since their divergence over 2.7 BYA. Further, the distribution of direct phosphorylation Spo0 pathways is patchy, suggesting multiple, independent instances of remodeling from phosphorelay to direct phosphorylation. We provide evidence that these transitions are likely the result of changes in sporulation kinase specificity or acquisition of a sensor kinase with specificity for Spo0A, which is remarkably conserved in both architectures. We conclude that flexible encoding of interaction specificity, a phenotype that is only intermittently essential, and the recruitment of kinases to recognize novel environmental signals resulted in a consistent and repeated pattern of remodeling of the Spo0 pathway.

Survival in a changing world requires signal transduction circuitry that can evolve to sense and respond to new environmental challenges. The Firmicute sporulation initiation (Spo0) pathway is a compelling example of a pathway with a circuit diagram that has changed over the course of evolution. In Clostridium acetobutylicum, a sensor kinase directly activates the master regulator of sporulation, Spo0A. In Bacillus subtilis, Spo0A is activated indirectly via a four-protein phosphorelay. These early observations suggested that the ancestral Spo0A was directly phosphorylated by a kinase in the earliest spore-former and that the Spo0 phosphorelay arose later in Bacilli via gain of additional proteins and interactions. Our analysis, based on a much larger set of genomes, surprisingly reveals phosphorelays, not only in Bacilli, but in many Clostridia. These findings support a model wherein sporulation was initiated by a Spo0 phosphorelay in the ancestral spore-former and the direct phosphorylation Spo0 pathways, which are observed in distinct sets of Clostridial taxa, are the result of convergent, reductive evolution. Further, our evidence suggests that these remodeling events were mediated by changes in kinase specificity, implicating flexible pathway remodeling, potentially combined with the recruitment of kinases, in Spo0 pathway evolution.

Competing interests: N. Luisa Hiller is an Adjunct Assistant Professor at the Center of Excellence in Biofilm Research at the Allegheny Health Network. This organization has no financial or other interests in this manuscript or its findings; no other competing interests exist.

Funding: This work was supported by the Human Frontiers Science Program (Grant RGP0043/2013 to D.D. and M.T.L., http://www.hfsp.org/ ) and the National Science Foundation (Grant No. DBI-1262593 to D.D., https://www.nsf.gov/ ). M.T.L. is an Investigator of the Howard Hughes Medical Institute ( http://www.hhmi.org/ ). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2018 Davidson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Regardless of the status of the ancestral pathway, some combination of gains and losses of interaction must have occurred to produce the distinct pathway architectures observed in present day species. We took advantage of the dramatic increase in the number of sequenced Firmicutes genomes available to investigate these remodeling events. Our results challenge the prevailing hypothesis. In silico analyses, combined with in vitro experimental verification of a Clostridial phosphorelay, reveal that phosphorelay architectures are present throughout the Firmicutes. Further, we demonstrate that interaction specificity of representative Bacillar and Clostridial phosphorelays is functionally conserved. In contrast to the prevailing model, our results support a scenario in which the ancestral Spo0 pathway in the Firmicutes ancestor was a phosphorelay. The phylogenetic distribution of Spo0 architectures is patchy, consistent with several independent transitions from phosphorelay to direct phosphorylation architecture. Our results further suggest that these transitions were mediated via changes in sensor kinases, while Spo0A specificity is conserved across the Firmicutes phylum. Our findings provide a framework for reasoning about the forces that act to maintain signaling fidelity in complex signal transduction pathways with multiple interactions.

Considering that these two different signal transduction architectures both orchestrate the initiation of sporulation through the phosphorylation of an orthologous regulator, they likely arose from a common ancestral pathway. How, then, did different signaling architectures evolve in present day species? The prevailing view is that the ancestral Spo0 pathway had a two-component direct phosphorylation architecture and the more complex phosphorelay observed in B. subtilis is a derived state [ 32 – 34 ]. This hypothesis was inspired by the apparent lack of Spo0F and Spo0B orthologs in the first Clostridium genome sequenced [ 32 ]. The simplicity of the direct phosphorylation architecture and the similarly anaerobic lifestyles of the ancestral Firmicutes and present-day Clostridia, taken together, provided further support for predictions that the original Spo0 pathway also functioned through direct phosphorylation [ 33 ]. It was further proposed that the phosphorelay likely arose in the Bacillar lineage, possibly as the result of duplication of a cognate HK-RR pair [ 35 ], and that the additional points of control associated with a phosphorelay may have contributed to adaptation to rising oxygen levels in early Bacilli [ 36 ].

Strikingly, a comparison of the Spo0 pathways in the type species of the two Firmicutes classes, Bacillus subtilis [ 13 ] and Clostridium acetobutylicum [ 24 ], reveals that the outputs of these pathways are conserved [ 25 ], but the inputs and the signal transduction architectures are not. Spo0A, the terminal component of the pathway in both species, initiates spore development upon phosphorylation [ 26 , 27 ] and is encoded by all known sporulators [ 22 ]. Spo0A is a canonical response regulator protein in its domain composition, including a REC domain [ 28 ] and a highly conserved, DNA-binding output domain, Spo0A_C [ 29 ]. Unlike Spo0A, which is likely orthologous in these distantly related species, the upstream signal transduction architectures are different. In contrast to the B. subtilis multi-input phosphorelay Spo0 architecture, C. acetobutylicum and other closely related species possess a multi-input architecture in which Spo0A is directly phosphorylated by multiple kinases [ 24 , 30 , 31 ] ( Fig 1C ).

To explore this issue, we present here an analysis of the evolution of the Spo0 pathway. The Spo0 pathway controls entrance into a developmental program that produces stress-resistant, dormant endospores. The ability to produce endospores is a common feature of the Firmicutes phylum, observed in numerous species throughout two anciently related classes, the Bacilli and Clostridia, suggesting that this survival mechanism is ancient [ 19 , 20 ]. These two classes are predicted to have diverged 2.7 billion years ago, coinciding with the atmospheric rise of oxygen during the great oxidation event [ 21 ]. The ancestral Firmicute was likely an obligate anaerobe, a trait that has been preserved in the present-day Class Clostridia, whereas the Bacilli are typically facultative aerobes. Many taxonomic families in both classes include both sporogenous and asporogenous species, suggesting that the ability to sporulate is frequently lost [ 22 ] through adaptation to a stable niche where sporulation is unnecessary for survival [ 23 ].

The maintenance of signal fidelity in these more complex pathways entails additional constraints on the genetic determinants of specificity because a single protein must support multiple interactions. The interaction requirements of the Spo0 phosphorelay necessitate precise molecular recognition to allow both Spo0F and Spo0A to interact with Spo0B, but only Spo0F to accept a phosphoryl group from sporulation kinases ( Fig 2B ). The balance of flexibility and constraint that shapes molecular recognition in these complex architectures is not well understood.

Histidine-aspartate phosphotransfer also admits more complex signal transduction architectures. Examples include multiple-input architectures [ 9 ], multiple-output architectures [ 10 ], and so-called phosphorelays comprising a sequence of phosphotransfer interactions [ 11 – 13 ]. For example, the sporulation initiation (Spo0) pathway is a multi-input phosphorelay characterized extensively in B. subtilis [ 13 – 15 ] and also observed in closely related species [ 16 – 18 ]. In this architecture, multiple sensor kinases phosphorylate Spo0F, a protein possessed of a REC domain, but lacking an output domain; subsequently, that phosphoryl group is transferred via Spo0B, an intermediate histidine phosphotransferase, to Spo0A, the master regulator of sporulation ( Fig 1B ).

(A) Molecular recognition maintains signaling fidelity between cognate histidine kinase–response regulator pairs and prevents phosphotransfer between non-cognate proteins encoded within the same genome (e.g., two-component signaling systems shown at left). Each response regulator is capable of recognizing multiple histidine kinase specificity signatures. The set of kinase specificity signatures recognized by the response regulator is represented qualitatively as a spectrum (right). Selection likely acts to separate the specificity spectra of response regulators encoded within the same genome, resulting in little or no overlap between spectra. Each histidine kinase must occupy a non-overlapping region of the specificity spectrum of its cognate response regulator. (B) The requirements of signaling fidelity exert greater constraints on the specificity signatures of a phosphorelay. The phosphorelay sporulation kinase (HK) must interact with Spo0F and not Spo0A, while Spo0B must interact with both Spo0F and Spo0A (left). The phosphorelay interaction pattern requires that the spectra (right) for Spo0F (blue) and Spo0A (green) must overlap and that Spo0B (orange dot) be located in the overlapping region. Additionally, sporulation kinases (e.g., teal dot) must be located in the Spo0F specificity spectrum, but outside of the region that overlaps with the Spo0A spectrum.

A set of non-contiguous, co-evolving residues at the interface of HK and RR proteins, six in the HisKA domain and seven in the REC domain, ensure specific interaction within each cognate pair [ 3 – 6 ]. These specificity residues are partially degenerate: multiple sets of kinase specificity residues permit phosphotransfer to the same receiver (and vice versa [ 7 ]), such that each receiver has a spectrum of kinase specificity with which it can interact ( Fig 2A ). To prevent deleterious crosstalk between non-cognate proteins [ 8 ], selection acts to separate the spectra of two-component signaling pathways encoded in the same genome. Acquisition of novel pathways (e.g., through duplication or horizontal gene transfer) can cause conflicts in interaction space. The degeneracy of these interactions allows for repositioning in interaction space to eliminate crosstalk via mutational trajectories involving compensatory mutations in the cognate pair. However, in the absence of a perturbation, pathways likely remain in the same region of interaction space over the course of evolution [ 8 ].

(A) A canonical two-component signaling system consists of a histidine kinase and a response regulator, wherein a signal is transmitted by transfer of a phosphoryl group from a conserved histidine in the HisKA domain (teal oval) to a conserved aspartate in the REC domain in the response regulator (red rectangle). (B) The B. subtilis Spo0 pathway is a phosphorelay. Signal transduction is initiated by activation of one of the five sensor histidine kinases that are associated with this pathway. The phosphoryl group is transferred from the HisKA domain in the kinase to the Spo0F REC domain (blue rectangle), then to the phosphotransferase Spo0B (orange oval), and finally to the REC domain of Spo0A (green rectangle). Spo0F lacks an output domain; Spo0A has the domain architecture of a typical response regulator, including a REC domain and a DNA-binding domain. (C) The C. acetobutylicum Spo0 pathway has a direct phosphorylation architecture, wherein multiple sporulation kinases are capable of direct transfer of a phosphoryl group to Spo0A.

Responses to changing environmental conditions are mediated by signal transduction pathways that recognize a signal, convey that signal into the cell, and initiate an appropriate cellular response. In bacteria, two-component signaling systems, typically comprised of a histidine kinase (HK) and a cognate response regulator (RR), are a primary mechanism of environmental response ( Fig 1A ). Signal recognition by the N-terminal sensor region of the HK leads to the autophosphorylation of a conserved histidine residue in the so-called HisKA domain by the catalytic (HK_CA) domain. The signal is then transduced by phosphotransfer from the autophosphorylated HK to a conserved aspartate residue in the N-terminal receiver (REC) domain of the RR [ 1 ]. Phosphorylation of the REC domain activates the C-terminal output domain of the RR, initiating a response to the recognized signal. Bacteria typically encode 20 to 30 two-component signaling pathways per genome [ 2 ].

Results

The recent increase in the number of sequenced Firmicutes genomes available offers an unprecedented opportunity to investigate Spo0 pathway evolution using a comparative approach. We assembled a set of 84 whole genome sequences that are representative of the two major sporogenous Firmicutes Classes, the Clostridia and the Bacilli (S1 Table). Genomes from Class Bacilli were selected to obtain a broad representation of the taxa in this class [37]. Within the Clostridia, most of the Clostridial clusters defined by Collins et al. [38] are represented by at least one species. The taxonomic nomenclature within this phylum is currently in flux [39, 40]; here, we use the taxonomic nomenclature that is currently associated with the genomes in the NCBI genome database [41, 42].

To investigate the phylogenetic distribution of genes encoding Spo0 pathway components, we constructed a maximum likelihood phylogeny for these 84 representative genomes from a concatenated alignment of 50 ribosomal proteins (Materials and methods). Ribosomal proteins have largely congruent phylogenetic signal in the Firmicutes [43], and phylogenies constructed from concatenated ribosomal protein sequences provide robust relationships in this phylum [39, 43–45]. The resulting phylogeny (Fig 3, S1 Fig) supports early divergence of Classes Bacilli and Clostridia. Further, divergence of the orders and families within each class is consistent with other phylogenies based on ribosomal proteins [43, 44]. See S1 Text and S2 and S3 Figs for a comparison of the inferred phylogenies.

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larger image TIFF original image Download: Fig 3. Phylogenetic distribution of predicted Spo0 pathway proteins. Cladogram of Firmicutes species used in this study, annotated with colored dots indicating predicted Spo0 pathway proteins: one or more orphan kinases (cyan); Spo0F (blue); Spo0B (orange); Spo0A (green). A filled cyan dot indicates a genome that encodes at least one orphan kinase with a PAS domain. The number of orphan kinases in each genome is given in S5 Table. Stars indicate genomes used in the in vitro phosphotransfer assays reported in this study. Phylogeny constructed from the concatenated alignments of 50 ribosomal protein families using RAxML with the CAT model and 100 bootstrap replicates (branch support values greater than or equal to than 50 are shown). Colored branches indicate species that are known to sporulate in Class Bacilli (blue) and Class Clostridia (red) (see also S1 Table). Species in which sporulation has not been reported are shown in grey. Tree representation created using ITOL [71]. See S1 Fig for the corresponding phylogram showing the outgroup used to root the tree and support values on all branches. https://doi.org/10.1371/journal.pgen.1007470.g003