Control of the transcription of a short gene encoding a cyclic peptide in Streptococcus thermophilus: a new quorum-sensing system?

Abstract

Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three S. thermophilus strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by S. thermophilus LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.

FIG. 1.

Chromatographic separation profiles of S. thermophilus culture supernatants. Analysis by RP-HPLC of supernatants of cultures stopped at the early stationary phase (S1) for the strains CNRZ1066, LMG18311, and LMD-9. The numbers indicate the peaks detected in the supernatants and sequenced by Edman degradation. The numbers with asterisks indicate the peaks detected in the supernatants of the three strains.

FIG. 2.

Analysis by RP-HPLC of culture supernatants of S. thermophilus LMD-9 and its mutants, LMD-9ΔSTER_1357, LMD-9ΔSTER_1358, LMD-9Δami, and LMD-9Δshp, in mid-exponential phase (E2). The arrow indicates the peak corresponding to Pep1357C detected in the supernatant of the wild-type strain and sequenced by Edman degradation.

FIG. 3.

MALDI-TOF spectrum of the peptide Pep1357C produced by S. thermophilus strain LMD-9. After internal calibration, the MALDI spectrum of the peptide indicated a major peak at m/z 989.48, corresponding to the ion [M+H]⁺. [M+H]⁺_m, measured mass; [M+H]⁺_t, theoretical mass.

FIG. 4.

IT-MS spectra of the linear form and modified Pep1357C showing the localization of the modification. (A) CID-MS/MS spectrum of the linear peptide, showing all ion series, b and y types, corresponding to the complete sequence of the peptide. (B) CID-MS/MS spectrum of Pep1357C, a cyclic peptide produced by S. thermophilus strain LMD-9, showing the loss of 2 Da for ion series [b6-b8] and y8, indicating the localization of the modification on the fragment (2-6) of Pep1357C. In each spectrum, only b and y ions are labeled; mass range, 135 Da to 1,000 Da.

FIG. 5.

Growth kinetics of S. thermophilus LMD-9 (▪) and comparison with production of the peptide Pep1357C in the culture medium (CDM) (gray bars). AU, arbitrary units normalized against the OD₆₀₀ of the cultures.

FIG. 6.

(A) Schematic representation of the genes potentially involved in the production of Pep1357C, as deduced from the S. thermophilus LMD-9 genome. The arrows indicate open reading frames and the proposed directions of transcription, with the gene name below: shp, short hydrophobic peptide; STER_1358, transcriptional regulator of the Rgg family; STER_1357, Pep1357C; rsp, radical SAM superfamily protein; ptr, predicted transporter. The broken arrows indicate the three predicted promoters. The amino acid sequence of the secreted peptide Pep1357C is shown in boldface type. (B) Double-stranded nucleotide sequence of the intergenic region between the genes encoding the short hydrophobic peptide (SHP) and the Rgg regulator. The arrows followed by the names of the genes indicate the directions of transcription. The ribosomal binding sites are underlined, and the start codons of each gene are in boldface. The boxes with a broken arrow above or below indicate the putative −10 and −35 boxes of the promoters predicted using BPROM prediction of bacterial promoters Softberry software. RBS, ribosomal binding site.

FIG. 7.

General schematic representation of the quorum-sensing mechanism involved in the production of Pep1357C in S. thermophilus LMD-9. A short hydrophobic peptide (SHP) is synthesized and exported via an unknown mechanism. It accumulates in the supernatant, and when its concentration reaches a certain threshold, it is reimported back into the cell via the oligopeptide permease (Opp) system. In the cell, it activates the Rgg transcriptional regulator, which in turn induces the expression of STER_1357. The product of the gene is Pep1357, which is probably modified by the Sam enzyme Rsp and secreted by Ptr.