Additional file 2: Figure S2. of The MarR-like protein PchR (YvmB) regulates expression of genes involved in pulcherriminic acid biosynthesis and in the initiation of sporulation in Bacillus subtilis

Effect of iron starvation on the expression of cypX and yvmB. The BFA815 (cypX::lacZ) and BSAS108 (pyvmB’-lacZ) strains were cultivated in LB medium until OD600 of 1. Samples of 2 ml of the cultures were spread onto solid LB medium containing 20 μg.ml−1 X-gal. A drop of 10 μl 10 mM bipyridyl was deposited at the center of each plate. Blue rings corresponded to expression of the fusion in cells around the inhibition zone of bipyridyl drops. (PDF 75 kb

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

The B₁₂-Radical SAM Enzyme PoyC Catalyzes Valine C_β‑Methylation during Polytheonamide Biosynthesis

Genomic and metagenomic investigations have recently led to the delineation of a novel class of natural products called ribosomally synthesized and post-translationally modified peptides (RiPPs). RiPPs are ubiquitous among living organisms and include pharmaceutically relevant compounds such as antibiotics and toxins. A prominent example is polytheonamide A, which exhibits numerous post-translational modifications, some of which were unknown in ribosomal peptides until recently. Among these post-translational modifications, C-methylations have been proposed to be catalyzed by two putative radical <i>S</i>-adenosylmethionine (rSAM) enzymes, PoyB and PoyC. Here we report the <i>in vitro</i> activity of PoyC, the first B₁₂-dependent rSAM enzyme catalyzing peptide C_β-methylation. We show that PoyC catalyzes the formation of <i>S</i>-adenosylhomocysteine and 5′-deoxyadenosine and the transfer of a methyl group to l-valine residue. In addition, we demonstrate for the first time that B₁₂-rSAM enzymes have a tightly bound MeCbl cofactor that during catalysis transfers a methyl group originating from <i>S</i>-adenosyl-l-methionine. Collectively, our results shed new light on polytheonamide biosynthesis and the large and emerging family of B₁₂-rSAM enzymes

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

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

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

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