Table 1.
The identification of ExoR orthologs.
Fig 1.
ExoR, ExoS, and ChvI proteins interact to create the RSI Invasion Switch in S. meliloti.
ExoR is composed of Sel1 repeats, is secreted to the periplasm as ExoRm (lacking the signal peptide), and is cleaved to release its suppression of ExoS. The domain architectures of ExoS and ChvI, along with phosphorylation sites, are illustrated here. Signal transmission is achieved via a phosphorylation cascade and ChvI completes the “switch” by binding to DNA to alter gene expression [96]. Our current model suggests that the levels of ExoRm in the periplasm are maintained through the combination of biosynthesis and proteolysis. Proteolysis is possibly sensitive to host signals or to changes in environmental conditions. These mechanisms allow for (1) ExoRm levels to be dramatically reduced in the presence of host signals, (2) the turning “ON” of the RSI invasion switch, (3) the activation of host-invading genes, and (4) the suppression of free-living genes.
Fig 2.
Evolutionary history of ExoR is congruent with speciation history of Rhizobiales.
ML and Bayesian phylogenetic patterns support the existence of an ExoR ancestor that arose prior to diversification among the Rhizobiales and five Rhodobacterales species identified in our genomic searches. The ExoR gene was maintained among these species, giving rise to the ortholog set. Phylogenetic reconstruction of ExoR orthologs agrees with currently accepted speciation patterns.
Fig 3.
Concordant evolution of ExoS and ExoR.
The overall evolutionary rates and branching pattern of the ExoS phylogeny agree with those of ExoR (see S3 Fig for mirrored comparisons). Although not all ExoS orthologs resolve strictly with family speciation patterns, most differences are minor. The Phyllobacteriaceae orthologs are, in fact, the most significant difference in the predicted histories of the ExoR and ExoS sets. The comparison of the ExoS reconstruction to the ExoR (Fig 2) supports the assertion that the evolution of the two ortholog sets was concordant.
Fig 4.
Conserved sequence evolution of ChvI in Rhizobiales and selected Rhodobacterales.
As a cognate TCS pair, ChvI and ExoS sequences are expected to diversify at similar rates. However, the ChvI phylogeny suggests that it is under strong purifying selection with the lowest amino-acid substitution rate among the three components. It is possible that the functional role of ChvI orthologs (i.e., DNA binding) limits diversification.
Fig 5.
RSI Synteny analysis within Rhizobiales and ancestral Alphaproteobacteria.
Clusters of orthologous gene (COG) groups for representative Proteobacteria are presented for comparison to the prototype, the loci of Rm1021. Each COG, as defined in the IMG database, is represented by an arrow of different color. For the exoR-exoS-chvI genes, a lack of homologs or orthologs is presented as an ORF with a solid or dotted slash, respectively. The sizes and positions of the ORFs are approximate, representing relative expansions/deletions. The ExoR ortholog found in Azorhizobium caulinodans, although annotated as an exopolysaccharide regulator, is predicted to encode transmembrane helices and is a structural outlier as compared to ExoR-like molecules found in other Rhizobiales. Lifestyles and host type are given by A (animal pathogen), P (plant pathogen), or S (symbionts). Gene loss/gain is indicated with-/+, respectively. COG# color key (L to R): Dark yellow, 0499; green, 1925; light blue, 2893; dark violet, 1493; blue (exoS/chvG), 0642; orange (chvI), 0745; light violet, 1866; pale green, 1186; grey, 1652; white, 3145; red (exoR), 0790; purple, 0708; magenta, 1502; bright yellow, 0232; turquoise (sporulation-domain encoding). In Rm1021 RSI loci are exoS (bp49252–51039), chvI (51419–52141), and exoR (1637310–1638116) on the SMc chromosome.
Fig 6.
Site-specific evolutionary rates of ExoR (A) and ExoS (B).
Substitution rates (y-axis) at amino-acid positions (x-axis, numbers based on (A) the periplasmic region of S. meliloti ExoS and (B) full-length ExoR) were obtained using Rate4Site [94]. While variable and conserved residues are evenly dispersed in ExoR (B), two variable residues are notable and specific to two regions in ExoS (A). We hypothesize, subject to experimental verification, that these two variable regions form binding domains to ExoR and are the molecular basis of host-specific responses among Rhizobiales species.
Fig 7.
Two host-associated radical amino-acid substitutions in ExoS.
Sequences within the two hypervariable regions (Fig 6) of ExoS are displayed according to a phylogeny of ExoS sequences. Based on parsimony reasoning, an asparagine residue (N131of the ExoS periplasmic region) is an evolutionarily derived state that is conserved among plant- or dinoflagellate-associated taxa. A tryptophan residue (W61of the ExoS periplasmic region) is derived from a leucine ancestor and conserved among animal-infecting species with the exception of Rhizobium leguminosarum (a plant pathogen).