A novel pheromone quorum-sensing system controls the development of natural competence in Streptococcus thermophilus and Streptococcus salivarius

Abstract

In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor sigma(X), which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci.

FIG. 1.

Growth (OD₆₀₀) and luciferase activities (RLU/OD₆₀₀) of LMD-9 reporter strains and their isogenic mutants used in the microarray experiments. (A) Growth (black) and luciferase activities (gray) of P_comX-luxAB fusions of strains CB001 (ComX⁺ Ami⁺; triangles), CB002 (ComX⁻ Ami⁺; squares), and LF116 (ComX⁺ Ami⁻; circles). (B) Growth of strains LMD-9 (wild type [WT]; open triangles), CB003 (LMD-9 ComX⁻ Ami⁺; open squares), and LF115 (LMD-9 ComX⁺ Ami⁻; open circles).

FIG. 2.

Analysis of the ster_0316-shp0316 locus in strain LMD-9. (A) Sequence alignment of the upstream regions of comX, shp0316, blpA, blp_orf4 (orf4 of the blp locus), and ster_1720. The consensus sequence is shown below the alignment: nucleotides present in each of these sequences are in uppercase, and nucleotides occurring in 3 or 4 of the sequences are in lowercase. The following conserved elements are boxed: inverted repeats (ECom box) (arrows), followed by a sequence rich in T (T tract) and a putative −10 box (−10). The numbers (in parentheses) indicate positions relative to the translational start codon. (B) Schematic representation of the locus from ster_0316 to ster_0319 (3,470 bp) and sequence analysis of the intergenic region (ster_0316-ster_0317) coding for Shp0316. Genes encoding proteins or peptides with predicted functions are represented by arrows: Rgg-like transcriptional regulator (black; ster_0316), small hydrophobic peptide (white; shp0316), and inactive peptide ABC exporter (gray; pseudogene ψster_0317-319). The following elements are annotated in the ster_0316-ster_0317 intergenic region: the ORF for Shp0316 (24 amino acids), start and stop codons (lowercase, boldface, and underlined), Shine-Dalgarno sequences (boldface and italics), the conserved inverted repeat (arrows), and the putative −10 box (uppercase and underlined). The deletion-replacement regions of the P32-cat cassette of ster_0316 (ster_0316::P32-cat), IR_shp0316 (IR_shp0316::P32-cat), and shp0316 (shp0316::P32-cat) are indicated by brackets.

FIG. 3.

Role of the ster_0316-shp0316 locus of S. thermophilus LMD-9 in the activation of the comX promoter. (A) Role of ster_0316 encoding a Rgg-like regulator. Shown are the growth (OD₆₀₀; black) and luciferase activities (RLU/OD₆₀₀; gray) of strains CB001 (control strain; triangles) and LF117 (Ster_0316⁻; diamonds). (B) Role of shp0316. Shown are the growth and luciferase activities of strains CB001 (control strain; triangles) and LF118 (Shp0316⁻; diamonds). (C) Role of the inverted repeat (IR_shp0316) located upstream of shp0316. Shown are the growth and luciferase activities of strains CB001 (control strain; triangles) and LF119 (IR_shp0316::P32-cat; circles).

FIG. 4.

Role of the C-terminal region of Shp0316 in the activation of the comX promoter and transformation rate of LF118 (Shp0316⁻). (A) Alignment of Shp0316 of S. thermophilus (S. th.) LMD-9 and its orthologue identified in the S. salivarius SK126 (S. sa.) genome. Identical amino acids are shown in gray, the positively charged amino acids are shown in boldface, the hydrophobic region is underlined, and the conserved Cys residue of a putative lipobox is in italics. The sequence used to generate the synthetic peptides is indicated with a thick black line. (B) Sequences of the different synthetic peptides derived from the C-terminal region of Shp0316 (left) and mean transformation rate ± standard deviations (triplicates) of strain LF118 (Shp0316⁻) in the presence of 1 μM of each synthetic peptide (right). The numbers in parentheses indicate amino acid positions relative to the Shp0316 sequence. The heptapeptide required for activation is indicated in gray. ND, transformation rate below the detection limit (<1 × 10⁻⁸). (C) Growth (OD₆₀₀; black) and luciferase activities (RLU/OD₆₀₀; gray) of strain CB001 (control strain; triangles) and strain LF118 (Shp0316⁻) in response to increasing concentrations of Shp0316_18-24 (nM): 0 (diamonds), 10 (+), 100 (circles), 500 (squares), 1,000 (X), and 2,500 (bars). The RLU/OD₆₀₀ obtained with 0 and 10 nM Shp0316_18-24 constantly decreased during growth, and the corresponding curves overlap. A 1.5-fold increase in luciferase activity in the presence of 100 nM Shp0316_18-24 was measured directly after peptide addition. (D) Transformation rate of strain LF118 (Shp0316⁻) in response to increasing concentrations of Shp0316_18-24 (nM): 0, 10, 100, 500, 1,000, and 2,500. Mean values ± standard deviations were calculated from triplicates. The substrate DNA was plasmid pGIUD0855ery (1 μg). The transformation rates at 0 mM and 10 mM were arbitrarily fixed below the detection limit (<1 × 10⁻⁸).

FIG. 5.

Model of quorum-sensing activation of comX of S. thermophilus LMD-9 in CDM. (1) Under CDM growth conditions, the transcription of comS (shp0316) is triggered and the precursor pre-ComS is produced. (2) Pre-ComS is secreted, probably through the general secretory system. (3) Pre-ComS undergoes one or more maturation steps, and its mature form, ComS*, accumulates in the extracellular medium. (4) At a critical concentration, ComS* is recognized by the oligopeptide-binding protein AmiA3 and internalized by the oligopeptide Ami transporter (AmiCDEF). (5) Intracellular ComS* interacts with and activates the transcriptional regulator ComR (Ster_0316). (6) ComR binds to its operator sequence (ComR box; ECom box) located upstream of the −10 box of comS (shp0316) and comX, resulting in the autoamplification loop and activation of the late competence genes, respectively. Black arrowheads, promoters; +, activation by ComR.