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

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

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

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

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

Image4.TIFF

<p>Pseudomonas aeruginosa is a severe opportunistic pathogen and is one of the major causes of hard to treat burn wound infections. Herein we have used an RNA-seq transcriptomic approach to study the behavior of P. aeruginosa PAO1 growing directly on human burn wound exudate. A chemical analysis of compounds used by this bacterium, coupled with kinetics expression of central genes has allowed us to obtain a global view of P. aeruginosa physiological and metabolic changes occurring while growing on human burn wound exudate. In addition to the numerous virulence factors and their secretion systems, we have found that all iron acquisition mechanisms were overexpressed. Deletion and complementation with pyoverdine demonstrated that iron availability was a major limiting factor in burn wound exudate. The quorum sensing systems, known to be important for the virulence of P. aeruginosa, although moderately induced, were activated even at low cell density. Analysis of bacterial metabolism emphasized importance of lactate, lipid and collagen degradation pathways. Overall, this work allowed to designate, for the first time, a global view of P. aeruginosa characteristics while growing in human burn wound exudate and highlight the possible therapeutic approaches to combat P. aeruginosa burn wound infections.</p

Image2.TIFF

<p>Pseudomonas aeruginosa is a severe opportunistic pathogen and is one of the major causes of hard to treat burn wound infections. Herein we have used an RNA-seq transcriptomic approach to study the behavior of P. aeruginosa PAO1 growing directly on human burn wound exudate. A chemical analysis of compounds used by this bacterium, coupled with kinetics expression of central genes has allowed us to obtain a global view of P. aeruginosa physiological and metabolic changes occurring while growing on human burn wound exudate. In addition to the numerous virulence factors and their secretion systems, we have found that all iron acquisition mechanisms were overexpressed. Deletion and complementation with pyoverdine demonstrated that iron availability was a major limiting factor in burn wound exudate. The quorum sensing systems, known to be important for the virulence of P. aeruginosa, although moderately induced, were activated even at low cell density. Analysis of bacterial metabolism emphasized importance of lactate, lipid and collagen degradation pathways. Overall, this work allowed to designate, for the first time, a global view of P. aeruginosa characteristics while growing in human burn wound exudate and highlight the possible therapeutic approaches to combat P. aeruginosa burn wound infections.</p

Table2.PDF

<p>Pseudomonas aeruginosa is a severe opportunistic pathogen and is one of the major causes of hard to treat burn wound infections. Herein we have used an RNA-seq transcriptomic approach to study the behavior of P. aeruginosa PAO1 growing directly on human burn wound exudate. A chemical analysis of compounds used by this bacterium, coupled with kinetics expression of central genes has allowed us to obtain a global view of P. aeruginosa physiological and metabolic changes occurring while growing on human burn wound exudate. In addition to the numerous virulence factors and their secretion systems, we have found that all iron acquisition mechanisms were overexpressed. Deletion and complementation with pyoverdine demonstrated that iron availability was a major limiting factor in burn wound exudate. The quorum sensing systems, known to be important for the virulence of P. aeruginosa, although moderately induced, were activated even at low cell density. Analysis of bacterial metabolism emphasized importance of lactate, lipid and collagen degradation pathways. Overall, this work allowed to designate, for the first time, a global view of P. aeruginosa characteristics while growing in human burn wound exudate and highlight the possible therapeutic approaches to combat P. aeruginosa burn wound infections.</p