Mass Spectrometry Analysis of the Extracellular Peptidome of <i>Lactococcus lactis</i>: Lines of Evidence for the Coexistence of Extracellular Protein Hydrolysis and Intracellular Peptide Excretion

We report here the use of a peptidomic approach to revisit the extracellular proteolysis of <i>Lactococcus lactis</i>. More than 1800 distinct peptides accumulate externally during growth of the plasmid-free protease-negative strain <i>L. lactis</i> IL1403 in a protein- and peptide-free medium. These peptides mainly originate from cell-surface- and cytoplasmic-located proteins, despite the fact that no cell lysis could be evidenced. Positioning each identified peptide on its parental protein sequence demonstrated the involvement of exo- and endopeptidase activities. The endopeptidases responsible for the release of surface and cytoplasmic peptides had distinct specificities. The membrane-anchored protease HtrA was responsible for the release of only a part of the surface peptides, and its preference for branched-chain amino acids in the N-terminal side of the cleaved bond was established in situ. Other yet uncharacterized surface proteases were also involved. Several lines of evidence suggest that surface and cytoplasmic peptides were produced by different routes, at least part of the latter being most likely excreted as peptides from the cells. The mechanism by which these cytoplasmic peptides are excreted remains an open question, as it is still the case for excreted cytoplasmic proteins

Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci-1

<p><b>Copyright information:</b></p><p>Taken from "Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci"</p><p>http://www.biomedcentral.com/1471-2180/7/36</p><p>BMC Microbiology 2007;7():36-36.</p><p>Published online 2 May 2007</p><p>PMCID:PMC1876236.</p><p></p

Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci-0

<p><b>Copyright information:</b></p><p>Taken from "Positive role of cell wall anchored proteinase PrtP in adhesion of lactococci"</p><p>http://www.biomedcentral.com/1471-2180/7/36</p><p>BMC Microbiology 2007;7():36-36.</p><p>Published online 2 May 2007</p><p>PMCID:PMC1876236.</p><p></p

Immunolocalization of the YhhB basal pilin.

<p>Negative staining of <i>L. lactis</i> strains was performed using phosphotungstic acid. Strains were immobilized on Formvar-carbon-coated nickel grids and pilins were detected using as primary antibodies, guinea-pig anti-YhhB polyclonal antibodies. The preparations were treated with secondary antibodies consisting of anti-guinea-pig conjugated to 15 nm gold beads. The negatively stained pili are indicated by black arrows and the YhhB pilin is indicated with purple arrowheads. Control refers to <i>L. lactis</i> IL1403 strain harboring pIL253 plasmid and IL pPil to <i>L. lactis</i> IL1403 strain in which the <i>pil</i> operon is overexpressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>). (Scale bars, 200 nm).</p

Pili display and related auto-aggregation and biofilm phenotypes in <i>L. lactis</i> IL pPil and derivatives.

*<p>for strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>.</p

Immunolocalization of the YhgE backbone pilin by TEM.

<p>Negative staining of <i>L. lactis</i> strains was performed using phosphotungstic acid. Strains were immobilized on Formvar-carbon-coated nickel grids and pilins were detected using as primary antibodies, guinea-pig anti-YhgE and rabbit anti-YhgD polyclonal antibodies. The preparations were treated with secondary antibodies consisting of anti-guinea-pig conjugated to 5 nm gold beads for YhgE and anti-rabbit conjugated to 15 nm gold beads for YhgD. Red arrowheads, YhgE pilin subunits present in the pilus fibers or in the cell wall; blue arrows, YhgD pilin subunits. Control refers to <i>L. lactis</i> IL1403 strain harboring pIL253 plasmid and IL pPil to <i>L. lactis</i> IL1403 strain in which the <i>pil</i> operon is overexpressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>). (Scale bars, 200 nm).</p

Auto-aggregation phenotype of <i>L. lactis</i> cultures.

<p>Strains over-expressing all or parts of the <i>pil</i> operon as indicated above the pictures were grown overnight under static conditions. Control refers to <i>L. lactis</i> IL1403 strain harboring the pIL253 plasmid. For strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>.</p

Western blot analysis of cell wall-anchored proteins of <i>L. lactis</i> strains using anti-YhgE antibodies.

<p>Equivalent protein amounts from <i>L. lactis</i> control strain and from derivatives expressing all or parts of the <i>pil</i> operon were separated on 3–8% gradient Tris-acetate Criterion XT SDS-PAGE gel and were detected by immunoblotting. Control refers to <i>L. lactis</i> IL1403 strain harboring pIL253 plasmid. For strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>. The positions of molecular mass standards (in kilodaltons) are indicated and the YhgE monomer is shown by a black arrowhead.</p

Maximum height of biofilms obtained with <i>L. lactis</i> strains.

<p>Strains that yielded biofilms whose maximum height measured at 4 and 15 h of growth was significantly different (<i>P</i><0.05) to that of control <i>L. lactis</i> IL1403 are marked by asterisks. Standard error is indicated. For strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>. Indicated values are the mean of 3 determinations per experiment.</p

Bacterial strains and plasmids used in this study.

*<p>ColE1 and pAMβ1 refer to the replicon; Tet^r, tetracycline resistance; Em^r, erythromycin resistance; Kan^r, kanamycin resistance; <i>srtC</i>*, mutated <i>srtC</i> gene encoding an inactive sortase C; plasmid and strain designations used in the text are indicated in parentheses.</p>$<p>Christine Delorme, INRA, Micalis-UMR1319, F78350-Jouy-en-Josas.</p