Fig 1.
Distribution and conservation of competence pathways in Streptococcus spp.
(Top) The Type-I ComRS pathway is found in the Salivarius group (yellow shading) and the ComCDE pathway in the Anginosus and Mitis groups (blue shading). The Type-II ComRS pathway is present in Bovis, Mutans, and Pyogenic groups of Streptococcus (red shading). The Suis group, or Type-III ComRS pathway (violet), contains attributes distinguishable from Type-I and Type-II. Underlined species indicate the demonstration of natural competence under laboratory conditions. (Bottom) The putative XIP sequences from the Type-II ComRS pathway demonstrate a conserved double tryptophan motif. Represented sequences were derived from small ORFs directly downstream of ComR and include the C-terminal 7 or 8 amino acids predicted to encode the mature pheromone. aThe ComR derived from S. agalactiae NEM316 was not investigated, however this species does encode a putative Type-II XIP. bThe S. suis 05ZYH33 XIP represents a Type-III XIP encoding two tryptophan residues (in green) interrupted by glycine and threonine. cThe S. thermophilus LMD-9 Type-I XIP, lacking a WW-motif, is included for reference.
Fig 2.
The S. mutans ‘test-bed’ to characterize ComR and cognate XIP activity of Bovis, Mutans, and Pyogenic species of Streptococcus.
(A) The test-bed strain ES1 (comRS::spec) was used to host a plasmid containing an intact comR locus, including the comR gene (with 500 bp upstream), the conserved ComR DNA-binding site, and the comS promoter, from the selected Streptococcus strain. The comS promoter is positioned upstream from the luxAB luciferase reporter. (B) Luciferase activities of ES1 harboring the test plasmid containing the S. mutans UA159 ComR locus treated with synthetic UA159 (cognate) XIP-C7 or S. pyogenes MGAS5005 (non-cognate) XIP-C8. The dashed line indicates the EC50 value (88 nM) for UA159 XIP-C7. (C) Luciferase induction activity from cognate ComR–XIP treatments of each test strain, determined as maximum light activity responses to 50 μM cognate XIP compared to untreated cultures. (D) Cognate XIP EC50 values calculated from dose-responsive curves of each ComR (S1 Fig).
Fig 3.
Type-II ComRs expressed in the ES1 (ΔcomRS) test-bed strain exposed to heterologous XIPs can be grouped into strict, intermediate or promiscuous ComRs based upon relative activity profiles.
(A) Type-II ComR proteins were tested for the ability to promote luciferase expression using putative XIP proteins of varying length from both the same and heterologous species. The normalized test-bed activities for each ComR variant is defined as 100% for luminescence activity stimulated by the cognate XIP; activities from heterologous pairings are calculated by dividing the activity from the cognate XIP by the maximum luminescence attained from the heterologous XIP and represented as (++++) for >90%, (+++) for 70–89%, (++) for 50–69%, and (+) 25–49%, no observable induction is denoted by (-). Color indicates species. (B-D) Five representative strains that exhibit different ComR activity profiles: strict, intermediate, and promiscuous. (B) Strict ComRs, S. mutans UA159 (circles) and the S. suis 05ZYH33 (squares), exclusively respond to cognate XIPs, UA159 C7 XIP (yellow) and suis C7 (red), respectively. (C) The intermediate ComR of S. pyogenes MGAS5005 (circles) exhibits reporter activity when treated with the cognate GAS M1 C8 XIP (purple), UA159 C7 XIP (yellow), and the S. bovis SboB C7 XIP (orange) with different EC50 values. No activity is observed when treated with the suis C7 XIP (red). (D) The promiscuous ComR of S. bovis SboB (circles) exhibits strong activity upon treatment with the cognate SboB C7 XIP (orange) with an apparent EC50 of <1 nM. The SboB ComR also exhibits dose-responsive reporter activity upon treatment with the UA159 C7 XIP (yellow), the GAS M1 C8 XIP (purple) and Suis C7 XIP (red), which does not contain the WW-motif.
Fig 4.
Overall structure of apo-ComR from Streptococcus suis.
(A) Cartoon representation of the asymmetric unit of the native crystal form compared to the biological unit of Rgg2 (PDB code 4YV6). Each monomer is colored and the approximate boundary between the DNA binding domain (DBD) and the tetratricopeptide repeat domain (TPR) is indicated. (B) The biological unit of the apo-form of ComR. The DBD domain and TPR domains are shaded in grey with primary sequence boundaries listed in parenthesis with the extended loop that links the domains indicated by a blue line. The XIP binding region in the TPR is highlighted with a blue oval, in addition to the interface region between the DBD and TPR with a green oval. Each secondary structure element is labeled in by number order from N to C terminus (α = α-helix) (C) Alignment of the S. suis TRP (red) and Rgg2 TPR (teal). Black arrows and dots indicate a rotation of α-helices and orange arrows and dots show a translation of α-helices. The regions shaded in grey and the C-terminal CAP helix show significant differences.
Table 1.
X-ray crystallographic data collection and refinement statistics
Fig 5.
The molecular surface properties of apo-ComR S. suis.
(A) Solvent accessible surface representation colored by amino-acid conservation using the Consurf server and the bacterial strains used in this study. Darker red indicates increased conservation with dark red showing completely conserved positions. Unconserved residues are shown in grey, with dark grey as least conserved. The XIP binding site consists of a conserved surface and a variable surface. Residues of interest are indicated by position and labeled. The inset shows the DBD and TPR domain interface that consists of highly conserved residues, with hydrogen bonds indicated by a dashed green line. (B) The solvent accessible hydrophobic residues of the ComR S. suis surface are highlighted in orange (C) Electrostatic surface potential as calculated by APBS with a contour of -10 kT/e to 10 kT/e. (D) Alignment of ComR S. suis with all species studied in this work. Secondary structure is annotated as Fig 4. Conserved and homologous residues are highlighted in red with the XIP variable face residues in a gray box. The alignment was generated by Clustal Omega and Espript3
Fig 6.
Structural comparison of the XIP binding pocket between different peptide recognition types.
(A) Residues from strict and promiscuous ComR proteins are compared and labeled by color. S. suis in purple, S. mutans in pink, MGAS5005 in green, and S. bovis 83 in orange. (B) Crystal packing artifact of the N-terminus (yellow) may mimic initial ComR peptide recognition. Residues making van der Waals contacts and/or hydrogen bond with the peptide are shown in purple, hydrogen bonds are represented by a dashed green line.
Fig 7.
Structural comparison of ComR S. suis and ComR S. thermophilus.
Alignment of ComR monomers using the TPR domain. (A) Alignment of the two apo-structures, (B) as (A) including the monomer from the ComR-ComS-DNA ternary complex. (C) Is panel (B) looking at the TPR from the activated DBD conformation (rotated y, -90° and x, 45° from the middle panel) (D) Sequence conservation and homology between ComR S. suis and S. thermophilus. Conserved residues are boxed in red with white lettering and homologous residues in pink with black lettering. The DNA binding domain (DBD) and TPR are labeled and helices labeled by number. The alignment was generated by PDBeFold and Espript3.
Fig 8.
Luciferase reporter assay results of ComR S. suis mutants stimulated with Suis C7 XIP.
(A) Point-mutations were introduced into ComR S. suis in the DBD (triangles R19A, Q28A, T42A), the DBD-TPR interface (hexagons Q40A, R43A), or in the conserved (K260A, R103A) or variable surfaces (N220A) of the XIP binding pocket (square symbols). Wild-type S. suis ComR activity with Suis C7 XIP is depicted as circles. (B) Circular dichroism spectra of purified variant proteins of interest displaying decreased transcriptional activity compared to wild-type, plotted as mean residue ellipticity.
Fig 9.
Isothermal titration calorimetry of ComR variants and XIP.
(A) Heats from control titrations are shown. Buffer was titrated into wild-type ComR S. suis and each variant protein, whereas XIP was titrated into buffer. The bottom panel shows the titration of S. suis XIP-C7 and the conserved surface variant K260A. (B) Titration curve for S. suis wild-type and cognate XIP-C7. (C) Titration of the variable face variant N220A with cognate XIP C7. (D) Titration of DBD and TPR interface variant Q40A with cognate XIP-C7. (E) Titration of S. suis wild-type with S. mutans XIP C7. (F) Titration of the variable face variant N220A with S. mutans XIP C7. Binding constants and thermodynamic values are presented in Table 2.
Table 2.
Isothermal titration calorimetry of ComR S. suis variants and XIP
Fig 10.
Designed XIP agonists of ComR S. agalactiae 2603.
XIP peptides found to be ineffective signals for ComR S. agalactiae 2603 were redesigned to satisfy hypothetical ComR-activating criteria. Designed synthetic peptides were titrated to strain ES1 carrying ComR S. agalactie 2603 reporter and maximum relative luciferase activities were recorded.