Rgg-associated SHP signaling peptides mediate cross-talk in Streptococci.

We described a quorum-sensing mechanism in the streptococci genus involving a short hydrophobic peptide (SHP), which acts as a pheromone, and a transcriptional regulator belonging to the Rgg family. The shp/rgg genes, found in nearly all streptococcal genomes and in several copies in some, have been classified into three groups. We used a genetic approach to evaluate the functionality of the SHP/Rgg quorum-sensing mechanism, encoded by three selected shp/rgg loci, in pathogenic and non-pathogenic streptococci. We characterized the mature form of each SHP pheromone by mass-spectrometry. We produced synthetic peptides corresponding to these mature forms, and used them to study functional complementation and cross-talk between these different SHP/Rgg systems. We demonstrate that a SHP pheromone of one system can influence the activity of a different system. Interestingly, this does not seem to be dependent on the SHP/Rgg group and cross-talk between pathogenic and non-pathogenic streptococci is observed

Lactococcus lactis Gene yjgB Encodes a γ-d-Glutaminyl-l-Lysyl- Endopeptidase Which Hydrolyzes Peptidoglycan▿ †

YjgB is one of five peptidoglycan hydrolases previously identified in Lactococcus lactis. Analysis of its amino acid sequence revealed that YjgB contains an NlpC/P60 domain, whereas no specific cell wall binding domain or motif could be identified. The NlpC/P60 family is characterized by three conserved residues, a cysteine, a histidine, and a polar residue. In agreement with the presence of a Cys residue in the catalytic site of YjgB, its enzymatic activity was enhanced in the presence of dithiothreitol. Peptidoglycan-hydrolyzing activity of YjgB was detected in growing cells of an L. lactis strain overexpressing YjgB, as revealed by the presence of disaccharide (DS)-dipeptide in the muropeptide composition of the overexpressing strain. YjgB hydrolyzes the peptide chains of L. lactis muropeptides between γ-d-Gln and l-Lys residues. Its hydrolytic activity was detected on DSs with tetra- and pentapeptide chains, whereas hydrolytic activity was very low on DS-tripeptides. Thus, we demonstrated that YjgB is an endopeptidase which cleaves γ-d-Gln-l-Lys bonds in peptide chains of L. lactis peptidoglycan

Peptidoglycan Structure Analysis of Lactococcus lactis Reveals the Presence of an l,d-Carboxypeptidase Involved in Peptidoglycan Maturation

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has l,d-carboxypeptidase activity and is involved in peptidoglycan maturation

Fragmentation spectra of the ions of mature forms of SHP1299, SHP1555 and SHP1509.

<p>Fragmentation of the ions m/z 1018.56 (A) and m/z 564.28 (B) identified in the supernatant of cultures of <i>S. thermophilus</i> strain LMD-9. Fragmentation of the ions m/z 799.49 (C) identified in the supernatant of cultures of <i>S. agalactiae</i> strain NEM316 and m/z 872.5 (D) identified in the supernatant of cultures of <i>S</i>. <i>mutans</i> strain UA159. All ions were analyzed in the linear ion trap.</p

Plasmids used in this study.

a<p>Ts indicates that the plasmid encodes a thermosensitive RepA protein.</p>b<p>Km and Erm indicate resistance to kanamycin and erythromycin, respectively.</p

Cross-complementation of the <i>shp/rgg</i> loci with synthetic SHP pheromones.

<p>Maximum relative luciferase activities of the reporter strains TIL1052 (<i>shp1299</i>::<i>erm blp</i>::P<i>_shp1299-luxAB aphA3</i>), TIL1200 (Δ<i>shp1358 blp</i>::P<i>_shp1358-luxAB</i>), TIL1382 (<i>blp</i>::<i>gbs1555</i>::P<i>_shp1555</i>-<i>luxAB aphA3</i>) and TIL1384 (<i>blp</i>::SMU.1509::P<i>_shp1509</i>-<i>luxAB aphA3)</i> grown in the absence (grey) or in the presence of synthetic SHP peptides added at the beginning of the culture to a concentration of 1 µM: EGIIVIVVG (green), DILIIVGG (red), DIIIIVGG (blue), ETIIIIGGG (purple), DIIIFPPFG (yellow). The legitimate SHP synthetic peptide associated to the locus studied is hatched in each case.</p

Description of strains containing P<i>_shp</i>-<i>luxAB</i> transcriptional fusions in various genetic backgrounds.

<p>These strains were constructed in <i>S. thermophilus</i> strain LMD-9 and used to study the expression of the <i>shp</i> genes of <i>S. agalactiae</i> strain NEM316 (<i>shp/gbs1555</i> locus) and <i>S. mutans</i> strain UA159 (<i>shp/</i>SMU.1509 locus) in the presence and absence of the corresponding <i>shp</i> and <i>rgg</i> genes and in the presence and absence of the <i>ami</i> genes of <i>S. thermophilus</i> strain LMD-9.</p

Bacterial strains used in this study.

a<p>Km and Erm indicate resistance to kanamycin and erythromycin, respectively.</p>b<p>Arrows indicate construction by transformation with chromosomal DNA or plasmid.</p>c<p><i>shp1299</i> is annotated <i>ster_1298</i> in Genbank.</p

Growth and luciferase activities of strains containing P<i>_shp</i>-<i>luxAB</i> fusions in various genetic backgrounds.

<p>Growth curves (OD₆₀₀) are presented in gray and relative luciferase activities (RLU/OD₆₀₀) in black. Growth and relative luciferase activities of derivatives of <i>S. thermophilus</i> strain LMD-9 grown in CDM and containing P<i>_shp-luxAB</i> fusions of the loci <i>shp/gbs1555</i> of <i>S. agalactiae</i> (A), <i>shp/</i>SMU.1509 of <i>S. mutans</i> (B) and <i>shp/ster_1299</i> of <i>S. thermophilus</i> strain LMD-9 (C). The genetic backgrounds are indicated as follows: (•) the <i>shp</i> and <i>rgg</i> genes of the locus tested and the <i>ami</i> gene <i>of S. thermophilus</i> are present (▴) the cognate <i>shp</i> gene of the locus studied is not present, (▪) the cognate <i>rgg</i> gene of the locus studied is not present and, (<b>×</b>) the <i>ami</i> genes of <i>S. thermophilus</i> are not present. Experiments were done at 30°C for the <i>shp/gbs1555</i> and the <i>shp/</i>SMU.1509 loci and at 42°C for the <i>shp/ster_1299</i> locus. Data shown are representative of three independent experiments.</p

The <i>shp/rgg</i> loci used in this study.

a<p>Group number of the SHP-associated Rgg according to the classification described in Fleuchot <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066042#pone.0066042-Fleuchot1" target="_blank">[21]</a>.</p>b<p>The <i>shp</i> gene is followed by the Genbank id of the <i>rgg</i> genes.</p>c<p>The <i>shp</i> genes are not annotated in Genbank but were identified using BactgeneSHOW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066042#pone.0066042-Ibrahim2" target="_blank">[20]</a>, except for the <i>shp</i> gene associated with <i>ster_1299</i>, which is annotated <i>ster_1298</i> in the genome of <i>S. thermophilus</i> strain LMD-9. Consequently, all the <i>shp</i> gene products are indicated with the term “SHP” followed by the number of the cognate <i>rgg</i> gene in Genbank. To unify the nomenclature, the <i>ster_1298</i> gene product was renamed SHP1299.</p>d<p>The sequences of the synthetic peptides used in this study are underlined.</p