Orthology assignment of putative function for all reported genes was performed using the HHPRED web service (HHpred, RRID:SCR_010276) with default options and an e-value threshold of 10⁻³⁰ on the TIGRFAM (JCVI TIGRFAMS, RRID:SCR_005493), PFAM (Pfam, RRID:SCR_004726), and COG (COG, RRID:SCR_007139) databases (Tatusov et al., 2000; Söding et al., 2005; Haft et al., 2013; Finn et al., 2016). eggNOG identifiers, categories, and descriptions were retrieved from the eggNOG database (eggNOG, RRID:SCR_002456) using HMMER (Hmmer, RRID:SCR_005305; Eddy, 2011; Powell et al., 2014). Taxonomical identification numbers (TaxID) for all species analyzed in the manuscript were retrieved from the NCBI Taxonomy server (NCBI Taxonomy, RRID:SCR_003256; Federhen, 2012).

Protein sequences for V. spinosum DSM 4136 [TaxID: 240016] LexA [WP_009959117], RecA [WP_009965965], and ImuA [WP_009959308] were obtained from the NCBI RefSeq database [RefSeq, RRID:SCR_003496; Pruitt et al., 2012]. The promoter regions ([–250, 0] with respect to the translational start site) for orthologs of these sequences in Verrucomicrobia genome and metagenome assemblies were downloaded from the Integrated Microbial Genomes (IMG) resource [IMG, RRID:SCR_007733; Markowitz et al., 2014], after identifying orthologs through the IMG BLAST service using a 10⁻²⁰ e-value threshold and the V. spinosum sequences as queries (Altschul et al., 1997). Downloaded promoter sequences were used as input for the MEME motif discovery tool on the MEME Suite server [MEME Suite—Motif-based sequence analysis tools, RRID:SCR_001783], requesting palindromic motifs between 8 and 20 bp long and otherwise default options (Bailey et al., 2006). Sequence logos were generated using the Weblogo sequence service (WEBLOGO, RRID:SCR_010236; Crooks et al., 2004).

A sequence model for the Verrucomicrobia α3 helix of the LexA N-terminal helix-turn-helix motif was obtained through multiple sequence alignment of available LexA protein sequences for this phylum, using the information in P0A7C2 (UniProtKB, RRID:SCR_004426) on the E. coli LexA crystal structure (Zhang et al., 2010) to define penalty masks for CLUSTALW profile alignment mode (Thompson et al., 1994). The Verrucomicrobia LexA α3 helix motif was compared to previously compiled LexA α3 helix motifs for different phyla (Sanchez-Alberola et al., 2015) using the TomTom service of the MEME suite (Gupta et al., 2007). Amino acid property plots and differential analysis of the Verrucomicrobia LexA α3 helix motif with respect to the Betaproteobacteria, Actinobacteria, and Firmicutes α3 helix motifs were generated with the iceLogo web service (iceLogo, RRID:SCR_012137; Colaert et al., 2009).

Experimentally validated LexA-binding motifs were downloaded from the CollecTF database (CollecTF, RRID:SCR_014405; Kiliç et al., 2014). Whole genome shotgun assemblies for Verrucomicrobia species with total sequence lengths larger than or equal to the smallest complete Verrucomicrobia genome (Methylacidiphilum infernorum V4, TaxID: 481448; 2,287,145 bp) were obtained from the NCBI RefSeq database (RefSeq, RRID:SCR_003496). LexA-binding motif searches on individual genomes were performed using xFITOM (xFITOM, SCR_014445) with the sequence information content (R_i) scoring method and default parameters (Schneider, 1997; Bhargava and Erill, 2010). Comparative genomics analyses were performed with CGB, a collection of Python scripts implementing a computational pipeline for comparative genomics of regulatory transcriptional networks in bacterial genomes. The pipeline is based on previous work (Sanchez-Alberola et al., 2015) and is available under a GPL license on GitHub (http://www.github.com/erilllab/cgb). Given a set of genome assemblies, a transcription factor (TF) and its known binding motif, the pipeline first searches for binding motif instances in the promoter region of all genes (−250, +50 of TLS). Genes in directons with intergenic distance below the mean intergenic distance of each genome are considered to form operons and TF-binding sites identified in the lead operon gene are assigned accordingly to all operon members. The presence of genes with high-scoring TF-binding sites within predicted operons is used to revise operon predictions. Orthologs across all analyzed species are detected as best reciprocal BLAST hits using a 10⁻²⁰ e-value threshold. The pipeline summarizes analysis results using a heatmap with species clustered using a distance-based TF tree and a color scheme indicating the presence/absence of orthologs and the score of detected TF-binding sites in the corresponding operon.

E. coli (DH5α and BL21; Thermo Fisher Scientific, RRID:SCR_013270) and V. spinosum DSM 4136 strains were grown at 37°C in LB (Green and Sambrook, 2012) and at 30°C in M13 media [DSMZ 607; German Collection of Microorganisms and Cell Cultures, RRID:SCR_001711], respectively. Antibiotics were added to the cultures at reported concentrations (Green and Sambrook, 2012).

Plasmid isolation, restriction digestion, DNA ligation, transformation, DNA extraction, and PCR were carried out using standard protocols, as described elsewhere (Green and Sambrook, 2012). Restriction enzymes, T4 DNA ligase, DNA polymerase, and the DIG-DNA labeling and detection kit were from Roche (Roche NimbleGen, RRID:SCR_008571). The oligonucleotides used for this work are listed in Supplementary Material 1 and were purchased from Invitrogen (Molecular Probes, RRID:SCR_013318). Mutants of the V. spinosum recA promoter (VSP_RS32310) were obtained using oligonucleotides carrying designed substitutions (Supplementary Material 1). The DNA sequence of generated fragments was verified by sequencing (Macrogen, RRID:SCR_014454).

V. spinosum DSM 4136 DNA was extracted from phosphate buffered (50 mM) saline (pH 8.0)-washed pellets containing cells using the easy-DNA^TM DNA isolation kit (Molecular Probes, RRID:SCR_013318). The V. spinosum DSM 4136 was amplified using suitable primers (Supplementary Material 1) and cloned into a pET15b vector (Millipore, RRID:SCR_008983). The O. terrae PB90-1 lexA was obtained by chemical synthesis (GeneArt; Thermo Fisher Scientific, RRID:SCR_013270) and cloned into a pET15b vector. Overexpression and purification of the V. spinosum DSM 4136 and O. terrae PB90-1 and B. subtilis LexA proteins was performed as described previously for other LexA proteins (Cambray et al., 2011; Cornish et al., 2014). DNA probes for electro-mobility shift assays (EMSA) were generated using two complementary synthetic oligonucleotides centered on the target LexA-binding sites (Supplementary Material 1). The dsDNA synthetic fragments were ligated into pGEMT vector (Roche NimbleGen, RRID:SCR_008571) and transformed into E. coli DH5α (Thermo Fisher Scientific, RRID:SCR_013270). In all cases the plasmids were confirmed by sequencing and DNA probes were obtained by PCR using M13 forward and reverse digoxigenin-labeled oligos (Supplementary Material 1). EMSAs were performed as described previously (Sanchez-Alberola et al., 2012), using 20 ng of each digoxigenin-marked DNA probe in the binding mixture and adding the corresponding LexA protein (from 80 to 400 nM). Samples were loaded onto 6% non-denaturing Tris-glycine polyacrylamide gels and digoxigenin-labeled DNA-protein complexes were detected using the manufacturer's protocol (Roche NimbleGen, RRID:SCR_008571).

The presence of core SOS response operons (lexA [VSP_RS04780], recA [VSP_RS32310], and imuA-imuB-dnaE2 [VSP_RS05590-VSP_RS05595-VSP_RS05600]) in the genome of the representative Verrucomicrobia species V. spinosum DSM 4136 indicates that this phylum might possess a functional LexA regulatory network. However, computational searches using known LexA-binding motifs did not yield putative LexA-binding sites upstream of any SOS related genes in V. spinosum. Taking advantage of the availability of multiple genome and metagenome assemblies for the Verrucomicrobia phylum, we compiled 116 promoter sequences from 59 different assemblies corresponding to orthologs of the V. spinosum DSM 4136 lexA, recA, and imuA genes through the JGI-IMG service (Supplementary Material 2). We then used MEME to identify overrepresented motifs in these sequences. The most significant motif identified by MEME (Figure 1A) is a 14 bp palindromic motif with consensus sequence (TGTTC-N4-GAACA). This motif was identified in the promoter region of 27 lexA genes, 25 recA genes, and 3 imuA genes, corresponding to 36 different genome and metagenome assemblies and spanning all three major groups of Verrucomicrobia (Supplementary Material 3). A computational search also identified instances of this motif in the promoter sequences of the V. spinosum DSM 4136 lexA, recA, and imuA-imuB-dnaE2 operons (Figure 1B). The TGTTC-N4-GAACA motif is reminiscent of the LexA-binding motif (GAAC-N4-GTTC) previously reported in the Firmicutes, Actinobacteria, and Gallionellales (Davis et al., 2002; Au et al., 2005; Sanchez-Alberola et al., 2015). Together with its structural similarity to previously reported LexA-binding motifs, the presence of this motif in the promoter region of multiple orthologs for three core components of the SOS response strongly suggested that the identified TGTTC-N4-GAACA motif is the LexA-binding motif of the Verrucomicrobia.

To validate that the palindromic motif identified in silico was the LexA-binding motif of V. spinosum, we purified the V. spinosum DSM 4136 LexA protein [WP_009959117] and performed electro-mobility shift assays (EMSA) with wild-type and mutant versions of the V. spinosum DSM 4136 recA promoter containing single-nucleotide substitutions at each position of the predicted LexA-binding motif. The results of this site-directed mutagenesis analysis (Figure 1C) are in broad agreement with the motif predicted in silico, confirming that V. spinosum LexA targets a spaced dyad motif with consensus sequence TGTTC-N4-GAACA. Single-nucleotide mutations to the bases of the inverted repeat regions (TGTTC and GAACA) of the V. spinosum LexA-binding motif systematically abolish LexA binding in the recA promoter context, indicating that these conserved elements likely correspond to the monomer binding site and are therefore essential for LexA binding activity (Groban et al., 2005). In contrast, the 4 bp spacer region and 3 bp flanking regions tolerate single-nucleotide mutations, suggesting that they are predominantly involved in indirect readout and DNA bending (Zhang et al., 2010).

Previous work has established that the α3 helix of the N-terminal helix-turn-helix motif is responsible for the majority of the specific contacts with monomer binding sites of LexA-binding motifs (Oertel-Buchheit et al., 1990; Ottleben et al., 1991; Thliveris and Mount, 1992; Groban et al., 2005; Zhang et al., 2010). Comparison of the α3 helix sequence in Verrucomicrobia LexA proteins with previously reported LexA α3 helix motifs (Sanchez-Alberola et al., 2015) shows that the Verrucomicrobia LexA α3 helix is most closely related to those of the Betaproteobacteria, Firmicutes, and Actinobacteria. As shown in Figure 2, the majority of the changes observed in the α3 helix of Verrucomicrobia localize to the N-terminal part of the helix, affecting residues that change sequence specificity through direct readout, but that are not essential for DNA bending and structural motif recognition (Oertel-Buchheit et al., 1990; Thliveris et al., 1991; Thliveris and Mount, 1992; Groban et al., 2005; Zhang et al., 2010). Furthermore, the overall distribution of hydrogen donors and hydrophobic residues is preserved across the entire α3 helix (Supplementary Material 4). These observations suggest that the structural similarities between Firmicutes, Actinobacteria, Betaproteobacteria, and Verrucomicrobia LexA-binding motifs are the result of an evolutionary process in the LexA DNA-binding motif that has modified the specific readout of monomer sites without altering the recognition of the overall motif structure.

Close inspection of the V. spinosum lexA promoter reveals a poorly conserved LexA-binding site immediately downstream (1 bp) of the putative LexA-binding site identified in silico (Figure 3). To confirm that both these putative motif instances are involved in LexA binding, we performed EMSA with purified V. spinosum LexA protein on the lexA promoter. The results shown in Figure 4A revealed the distinct formation of two retardation bands on the lexA promoter at low protein concentrations, corresponding to LexA binding at either one or the two LexA-binding sites identified in the lexA promoter. Further, increasing protein concentration resulted in a single retardation band corresponding to LexA recognizing both LexA-binding sites. Taken together, these results indicate that the two identified LexA-binding sites in the promoter region of the V. spinosum lexA gene are bound cooperatively by LexA. A systematic analysis of the promoter regions of V. spinosum lexA gene orthologs in the Verrucomicrobia revealed that more than half of the lexA ortholog promoters with predicted LexA-binding sites display similar tandem site configurations (Supplementary Material 5). Most of these tandem arrangements involve a conserved TGTTC-N4-GAACA motif instance followed by a degenerate site in which only the first TGTTC element is conserved, but a tandem site arrangement with both conserved sites can be observed in at least two species (Figure 3).

In Opitutus terrae PB90-1, there are two fully conserved Verrucomicrobia LexA-binding motifs in the promoter region of a putative lexA-imuA-imuB-dnaE2 operon [OTER_RS20480-OTER_RS20475-OTER_RS20470-OTER_RS20465] separated by 2 bp. This arrangement generates an instance of the canonical GAAC-N4-GTTC LexA-binding motif of Firmicutes and Actinobacteria. Using purified O. terrae and B. subtilis LexA proteins, we performed EMSA to validate the functionality of this tandem arrangement in O. terrae (Figure 4). EMSA with O. terrae LexA [WP_012376858] reveals two retardation bands at low protein concentration, confirming that this protein also binds to both Verrucomicrobia LexA target sites in the lexA-imuA-imuB-dnaE2 promoter (Figure 4B). Mobility assays with incremental concentrations of B. subtilis LexA [WP_003238209] show that B. subtilis LexA binds a unique element in the O. terrae lexA-imuA-imuB-dnaE2 promoter, yielding a single retardation band similar to the one observed on the B. subtilis recA [BSU16940] promoter (Figure 4C). These results suggest that B. subtilis LexA binds the Firmicutes-like LexA-binding motif instance generated by the tandem arrangement of Verrucomicrobia LexA-binding sites.

Having established the LexA-binding motif of the Verrucomicrobia, we performed a comparative genomics analysis of the LexA regulon in this phylum. We compiled 15 whole-genome shotgun assemblies for members of all the major classes of Verrucomicrobia (Opitutae, Spartobacteria, and Verrucomicrobiae) presenting a V. spinosum LexA homolog and searched for putative LexA-binding sites in the promoter region [−250, +50] of predicted operons. The results of the comparative genomics analysis (Figure 5; Supplementary Materials 6, 7) reveal a core LexA regulon present in all classes of the Verrucromicrobia phylum and composed of three operons: lexA, splB, and imuA-imuB-dnaE2. The lexA gene displays high-scoring sites in all the analyzed species, except for Verrucomicrobium sp. 3C (TaxID: 1134055), Verrucomicrobia bacterium LP2A (TaxID: 478741), and Pedosphaera parvula Ellin514 (TaxID: 320771). The LexA proteins of these species display significant changes to the α3 helix of the LexA DNA-binding domain, suggesting that they may target a divergent LexA-binding motif. Consistent with this result, the genomes of these organisms do not reveal any instance of the Verrucomicrobia LexA-binding motif in the promoter regions of previously documented SOS genes (Erill et al., 2007). The promoter region of the splB gene shows evidence of LexA regulation in several Verrucomicrobiae, one Opitutaceae (O. terrae) and the only available assembly of a Spartobacteria species (Chthoniobacter flavus Ellin428; TaxID: 497964). The product of the splB gene contains a radical SAM domain (PFAM04055) and has homology to COG1533 (ENOG4105DCH), classified as a DNA repair photolyase. Members of this orthologous group have been reported to be regulated by LexA in the Actinobacteria, the Gammaproteobacteria, the Betaproteobacteria, and the Alphaproteobacteria (Davis et al., 2002; Cirz et al., 2006; Sanchez-Alberola et al., 2012, 2015; Ulrich et al., 2013), suggesting that it may be a previously unrecognized core component of the SOS response. Lastly, the promoter region of the imuA-imuB-dnaE2 operon presents Verrucomicrobia LexA-binding motif instances in C. flavus and the same Verrucomicrobiae species as splB. As noted above, O. terrae presents a putative lexA-imuA-imuB-dnaE2 with verified O. terrae LexA binding in its promoter region (Figure 4). Even though the intergenic distance between lexA and imuA is larger than the genomic average for this species (264 bp), the prevalence of lexA-imuA-imuB-dnaE2 arrangements across the Bacteria domain suggests that this direction constitutes a functional operon in O. terrae (Erill et al., 2006).

The results of the comparative genomics analysis reveal remarkable variation in the size and composition of the inferred LexA regulon. In the Verrucomicrobiae, the predicted regulon ranges from one operon (Verrucomicrobia bacterium IMCC26134; TaxID: 1637999) to over 14 (V. spinosum), with several species displaying intermediate sizes [2 operons in Rubritalea marina DSM 17716 (TaxID: 1123070) or 5 in Haloferula sp. BvORR071 (TaxID: 1396141)]. The only available Spartobacteria representative (C. flavus) shows a moderate regulon size (5 operons). In contrast, the LexA regulon appears to have shrunk noticeably in the Opitutaceae, where it encompasses at the most two operons. Two members of this family [O. terrae and Opitutaceae bacterium TAV5 (TaxID: 794903)] present a duplication of the lexA gene. The products of the two Opitutaceae TAV5 lexA genes (OPIT5_RS22040 and OPIT5_RS25725) present 91% identity and their promoter regions contain almost identical LexA-binding sites. The lexA genes in O. terrae (OTER_RS20480 and OTER_RS11645) have diverged substantially (42% protein sequence identity) and only the promoter of the lexA1 gene (OTER_RS20480) presents Verrucomicrobia LexA-binding motif instances, following the tandem arrangement discussed above (Figure 3).

The overall composition of the inferred Verrucomicrobia LexA regulon is in broad agreement with experimental and computational descriptions of the SOS regulatory network in other phyla (Erill et al., 2007; Sanchez-Alberola et al., 2012, 2015). Beyond the core regulon described above (lexA, splB, imuA-imuB-dnaE2), the Verrucomicrobia LexA regulon encompasses genes coding for the recombination protein RecA (COG0468; ENOG4105C68), the excinuclease ABC subunits A (COG0178; ENOG411DGUH) and B (COG0556; ENOG4105CCW), two DNA helicase RecQ homologs (COG0514; ENOG4107QS5 and ENOG410QKP1) and two homologs of the error-prone DNA polymerase IV (COG0389; ENOG4105CCW and ENOG4105CQ3). In addition to these previously established SOS genes, the Verrucomicrobia LexA regulon shows evidence of regulation for an operon encoding proteins matching the TIGR03916 (ENOG4105ES7) and TIGR03915 (ENOG4105T12) models. These models are present in about 20% of sequenced bacterial genomes, arranged always in operon configuration, and are thought to constitute a DNA base excision repair system involving a uracil-DNA glycosylase (UDG) domain that is conserved in all Verrucomicrobia TIGR03915-matching homologs.

To validate the predictions of the comparative genomics approach and further establish the LexA regulon of the Verrucomicrobia, we performed EMSA with purified V. spinosum and O. terrae proteins on the promoter region of several genes with predicted LexA-binding sites in these organisms and evidence of regulation in at least three different genomes. The results, shown in Figure 6, confirm that LexA binds to the promoter region of the splB gene in O. terrae (OTER_RS07185) and V. spinosum (VSP_RS12190). V. spinosum LexA also binds the imuA-imuB-dnaE2 operon promoter, the promoters of genes coding for DNA polymerase IV (VSP_RS08510) and RecQ (VSP_RS32195) homologs, and the recA (VSP_RS04780) and uvrA (VSP_RS32650) promoters. Together with the comparative genomics analysis, these results confirm the existence of a conserved core LexA-regulon in the Verrucomicrobia and demonstrate that, in some Verrucomicrobia species, LexA controls a network of similar size and function to those reported in well-studied bacterial phyla, using a novel LexA-binding motif.

Phylogenetic and protein signature analyses have firmly established the Verrucomicrobia as one of the major phyla in the PVC super-phylum, an ancient bacterial group estimated to have diverged from other bacterial clades almost two billion years ago (Gupta et al., 2012; Kamneva et al., 2012; Lagkouvardos et al., 2014). The analysis of the LexA regulon performed in this work hence provides for the first time insights into the organization of a complex transcriptional system in this large bacterial clade. Our results show evidence of a functional LexA protein targeting the same LexA-binding motif in all three major Verrucomicrobia classes (Opitutae, Spartobacteria, and Verrucomicrobiae), suggesting that a functional LexA regulatory network was present in the ancestor of the Verrucomicrobia (Figure 1). However, the Verrucomicrobia also display substantial heterogeneity in the size of their predicted LexA regulons (Figure 5). Some families, such as the Methylacidiphilaceae do not present LexA homologs, while many members of the Verrucomicrobiales display small (1–3 operon) regulons, a setup that appears to be the rule in the Opitutales. Small SOS regulatory networks have been experimentally reported for several species, but mostly in association with drastic changes in the LexA-binding motif (Jara et al., 2003; Campoy et al., 2005; Mazon et al., 2006). In these instances, the LexA regulon is typically constrained to the regulation of translesion synthesis polymerases (Erill et al., 2007). Conversely, moderately large (10–40 genes) LexA regulons incorporating several DNA repair pathways have been documented in the Gammaproteobacteria, the Betaproteobacteria, the Actinobacteria, and the Firmicutes (Fernandez De Henestrosa et al., 2000; Davis et al., 2002; Au et al., 2005; Ulrich et al., 2013; Sanchez-Alberola et al., 2015). These findings have substantiated the notion that translesion synthesis is the primordial function of the SOS response, and the identification of a translesion synthesis operon (imuA-imuB-dnaE2) in the core LexA regulon of the Verrucomicrobia confirms the ancestral role of this mechanism in the SOS response. Nonetheless, the presence of a putative photolyase (splB) in the core Verrucomicrobia LexA regulon, with documented LexA-regulated orthologs in several bacterial clades (Davis et al., 2002; Cirz et al., 2006; Sanchez-Alberola et al., 2012, 2015; Ulrich et al., 2013), suggests that photoreactivation might have played an essential DNA repair role in the primordial SOS response.

Beyond the presence of a putative photolyase, the SOS response of the Verrucomicrobia presents several interesting differences with the canonical SOS response of E. coli and B. subtilis. The Opitutae, for instance, show a consistent absence of LexA regulation for the recA gene. The lack of recA regulation by LexA has been reported in several bacterial groups, such as the Acidobacteria and the Deltaproteobacteria (Jara et al., 2003; Campoy et al., 2005; Mazon et al., 2006). Loss of recA regulation is often associated with small LexA regulons and lexA gene duplication, which are both features of the Opitutae LexA network inferred in this work. Another distinct feature of the Verrucomicrobia LexA regulon is the regulation of multiple RecQ homologs (ENOG4107QS5 and ENOG410QKP1; Figure 5). One of these RecQ homologs (ENOG4107QS5) shares functional domains with B. subtilis RecS and RecQ proteins and therefore likely fulfills similar repair functions. The other RecQ homolog (ENOG410QKP1) lacks DNA-binding HRDC (Helicase and RNase D C-terminal) and RecQ-C-terminal (RQC) domains and presents weaker evidence of homology with B. subtilis RecS and RecQ proteins (Fernández et al., 1998). RecQ helicases are involved in the initiation and reversal of recombination and are known to act in concert with the product of SOS genes (recA, ssb), and to facilitate the onset of the SOS response (Heyer, 2004; Nakayama, 2005). Although SOS regulation of RecQ homologs has not been documented to date, LexA regulation of other DNA helicases (UvrD, PcrA, DinG) is a well-established feature of the SOS response in several organisms (Fernandez De Henestrosa et al., 2000; Au et al., 2005; Abella et al., 2007). These helicases do not appear to be regulated in the Verrucomicrobia, suggesting that the putative LexA regulation of RecQ homologs might be fulfilling a complementary role in this phylum. Our analysis also provides evidence of LexA regulation for an operon encoding radical SAM and uracil-DNA glycosylase domain-containing proteins (ENOG4105ES7 and ENOG4105T12; Figure 5), presumed to function as a DNA base excision repair system. SOS-regulated error-prone polymerases have been shown to have poor sugar discrimination, leading to the frequent misincorporation of ribonucleotides (Schroeder et al., 2015). Misincorporated ribonucleotides are usually removed by RNase HII-mediated ribonucleotide excision repair, but SOS-regulated nucleotide excision repair has also been shown to address ribonucleotide incorporation (Vaisman et al., 2013). The presence of putative LexA-regulated translesion synthesis polymerases in the Verrucomicrobia (Figures 5, 6) hence suggests that the regulation of this base excision repair operon by LexA may play a role in addressing uracil misincorporation resulting from SOS induction in this phylum.

In those bacterial phyla where the SOS response has been experimentally documented, the LexA-binding motif shows evidence of high conservation, punctuated by periods of rapid divergence and further stabilization (Erill et al., 2007). In the Firmicutes and the Actinobacteria, LexA targets a conserved GAAC-N4-GTTC LexA-binding motif that is monophyletic for both clades (Cornish et al., 2012), and variations of this motif are also seen in other bacterial groups, such as the Cyanobacteria or the Chloroflexi (Fernandez de Henestrosa et al., 2002; Mazon et al., 2004b). In the Proteobacteria, however, LexA shows an extraordinary diversity of binding motifs (Erill et al., 2007). In Proteobacteria classes with abundant sequence information (Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria), the LexA-binding motif has been found to be extremely well-conserved, but exceptions to the canonical LexA-binding motif of Gammaproteobacteria and Betaproteobacteria have been reported in several subgroups (Campoy et al., 2002; Abella et al., 2007; Sanchez-Alberola et al., 2015). These exceptions are associated with duplications of the lexA gene, suggesting a model for LexA-binding motif evolution (Figure 7) in which lexA duplication leads to progressive divergence in the LexA-binding motif of the duplicated lexA, until the primary lexA gene is deleted and the divergent LexA takes control of the regulon (Abella et al., 2007; Yang et al., 2008; Sanchez-Alberola et al., 2015). While this model provides a causal mechanism for LexA-motif divergence, it does not address how a divergent LexA can swiftly take control over a regulatory network defined, up to the deletion event, by LexA-binding sites matching the primary LexA-binding motif. Furthermore, the model does not provide a mechanistic explanation for the recurrence of very similar LexA-binding motifs in distantly related bacterial clades, such as the Firmicutes and the Gallionellales, recognized through seemingly unrelated LexA α3 helices (Sanchez-Alberola et al., 2015).

Many bacterial transcription factors bind cooperatively to tandem sites (Barnard et al., 2004). The existence of tandem sites for LexA was first reported in the promoter region of the E. coli lexA gene (Brent, 1982) and then shown to be a common feature of lexA genes in the Gammaproteobacteria (Garriga et al., 1992), the Betaproteobacteria, and Alphaproteobacteria (Sanchez-Alberola et al., 2012), and the Firmicutes and Actinobacteria (Cornish et al., 2012). These arrangements feature highly conserved and spatially close tandem sites (1–10 bp apart). Tandem LexA-binding sites have also been experimentally reported for other SOS genes, such as the ydjM gene of E. coli (Fernandez De Henestrosa et al., 2000) or the umuDC-like operon (yqjW-yqzH) of B. subtilis (Au et al., 2005). Furthermore, the use of cooperative LexA-binding to enhance repression has been experimentally demonstrated for several colicin genes, which display a tandem arrangement with a strong and a weak LexA-binding site overlapping at their terminal positions (Gillor et al., 2008). In the Verrucomicrobia, there is evidence of a recent lexA duplication in the Opitutaceae and tandem LexA-binding sites separated by short distances appear to be a conserved feature of the lexA promoter (Figure 3). The ability of the Verrucomicrobia LexA to cooperatively bind degenerate sites and the fact that at least in one of these species the tandem arrangement generates a functional B. subtilis LexA-binding motif (Figure 4) suggest that tandem site arrangements can yield a simple mechanistic process for the evolution of LexA-binding motifs.

In the tandem site model (Figure 7), LexA binds consecutive sites in its own promoter and in the promoter of key SOS genes that need to be tightly regulated (Gillor et al., 2008). Upon lexA duplication, the site generated by the tandem arrangement provides the secondary LexA with a conserved target for motif divergence. This allows the secondary LexA to maintain cross-regulation with the primary LexA and a subset of its regulon, while incorporating novel elements to its network. After deletion of the primary lexA gene, the secondary LexA is hence already in control of a core LexA regulon, and can rapidly evolve sites on other target genes by exploiting its partial overlap, and presumable weak binding affinity, with primary lexA sites. The tandem site model therefore provides a conservative mechanism for the evolution of LexA-binding motifs that is capable of addressing outstanding questions regarding the complex evolutionary history of the SOS response. On the one hand, the conservative nature of the model provides a natural explanation for the persistence of conserved SOS response networks under divergent LexA-binding motifs, without the need for a strong selective process driving convergent evolution of similar networks (Sanchez-Alberola et al., 2015). On the other hand, the implicit reuse of LexA monomer-binding sites in the tandem model helps explain the observation of many LexA-binding motifs involving the rearrangement of similar monomer binding sites on different motif structures (Mazon et al., 2004a; Sanchez-Alberola et al., 2015).

Several lines of evidence provide indirect support for a tandem site-based model of LexA-binding motif evolution. As mentioned above, the prevalence of such arrangements in the promoter of lexA and other SOS genes has been documented in several bacterial clades. Furthermore, lexA duplications targeting identical and divergent motifs have also been experimentally reported, and cross-regulation between duplicated lexA genes has been demonstrated in these systems (Jara et al., 2003; Abella et al., 2007; Yang et al., 2008). Lastly, previous work has shown that LexA can bind to degenerate sites that partially match other LexA-binding motifs, indicating that transitional stages of LexA divergence in which the secondary LexA could partially bind the original and tandem-generated motifs are possible (Mazon et al., 2004a). Due to its broad distribution in several phyla, the Firmicutes and Actinobacteria LexA-binding motif has long been assumed to represent the ancestral motif of LexA. The mirror image relationship between Firmicutes and Verrucomicrobia LexA-binding motifs, and the generation of functional B. subtilis LexA-binding sites from tandem Verrucomicrobia LexA-binding sites, hence suggest that the Verrucomicrobia LexA-binding motif might have originated after the duplication of a lexA gene targeting a tandem arrangement of Firmicutes-like LexA-binding sites in a common ancestor of these lineages. The analysis of the α3 helix of the Verrucomicrobia LexA DNA-binding domain (Figure 2) supports this hypothesis, revealing overall conservation of amino acid properties and a substitution pattern consistent with changes in the specific readout of monomer sites, but not in overall motif recognition, as expected in the tandem site evolution model.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.