Promoter and terminator consensus sequences found among 258 <i>M. tuberculosis</i> sRNAs by bioinformatics approach.

<p>Sequence inspection of the −50/+15 region at the predicted 5′end for sigA consensus sequences and of the region −20/+200 at the 3′ end for termination signals. Presence (POS); Absence (NEG).</p

Classification of validated sRNAs according to their genomic position.

<p>UTR: untranslated region; AS: antisense.</p>*<p>5′ UTR: 51; 3′ UTR: 8; both 5′ and 3′ UTR: 25.</p

Competition between <i>P. aeruginosa</i> and <i>S. aureus</i> strains in a murine model of pneumoniae.

<p>Planktonic <i>S. aureus</i> strain Newman and <i>P. aeruginosa</i> clinical isolates AA2 and AA43 and reference strain PA14 were used to infect C57BL/6NCrlBR mice at a ratio of 1∶1. After 18 hours of acute infection lungs homogenates were plated on selective plates to determine <i>S. aureus</i> and <i>P. aeruginosa</i> CFU. Each circle represents the CI for a single animal in each group. A CI value equal to 1 indicates equal competition of the two species; a CI value significantly <1 indicates a competitive advantage of <i>S. aureus</i> that outcompetes <i>P. aeruginosa</i>; a CI value significantly >1 indicates a competitive advantage of <i>P. aeruginosa</i> that outcompetes <i>S. aureus</i>. Wilcoxon signed rank test of the null hypothesis that the distribution of CI is symmetric about 1 was performed. Statistically significant differences are indicated by symbols when present: *: p<0.05; **: p<0.01. The data are pooled from two or three independent experiments.</p

<i>S. aureus</i> and <i>P. aeruginosa</i> planktonic and sessile cells in single and dual cultures.

<p>Bacteria were grown overnight in 96-well flat-bottom microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio. CFU counts were determined in both planktonic and sessile fractions. Panel A: planktonic (left) and sessile (right) cells of <i>S. aureus</i> strain Newman in pure culture and in co-culture with <i>P. aeruginosa</i> strains PA14, AA2 and AA43. Statistically significant differences are referred to Newman in pure culture. Panel B: planktonic (left) and sessile (right) cells of <i>P. aeruginosa</i> strains PA14, AA2 and AA43 in pure culture and in co-culture with <i>S. aureus</i> strain Newman. The values represent the means of three independent experiments, and the bars indicate standard deviation. Statistically significant differences in non-parametric Mann–Whitney test are indicated by symbols when present: **: p<0.01; ***: p<0.001.</p

Heat-map representing the expression profiles of microarrays.

<p>As positive controls we used reference <i>sigA</i>, <i>rrs</i> and the short ribosomal 5S RNA. Normalized control genes showed a mean expression level of 7.16±1.57. Expression level: yellow to blue (highly expressed to lowly expressed). Statistics: red = p-value <0.05; green = p-value >0.05.</p

Validation of 23 selected candidates by Northern blot.

a<p>Length of the principal bands visualized by Northern blot analysis.</p>b<p>The presence of a −10 sigA consensus sequence about thirty-eight nucleotides upstream of the predicted 5′end suggests that the sRNAs length is in accordance with the band observed by Northern blot.</p>c<p>Type C candidate.</p>d<p>Negative control.</p>e<p>Northern blot tested in <i>M. tuberculosis</i> H37Rv, exponential and stationary growth phases.</p>f<p>Northern blot tested in <i>M. tuberculosis</i> H37Rv and <i>M. tuberculosis</i> H37Ra (exponential growth phase).</p>g<p>considering 3′-end mapping according to Northern blot results (175 nucleotides downstream the predicted 3′-end).</p>h<p>considering the 5′-end mapping according to the 5′-end primer extension.</p

Northern blot results for selected candidates.

<p>The RNAs were extracted as indicated in M&M: exp: <i>M. tuberculosis</i> H37Rv in exponential growth phase; stat: <i>M. tuberculosis</i> H37Rv in stationary growth phase; Rv: <i>M. tuberculosis</i> H37Rv; Ra: <i>M. tuberculosis</i> H37Ra. Arrows indicate the predicted length.</p

Single and dual species batch growth curves and competition index values.

<p><i>S. aureus</i> strain (Newman) and <i>P. aeruginosa</i> strains (PA14 and two clinical early and late isolates from a CF patient AA2 and AA43) were grown for 24 hours in BHI in single culture and in co-culture after inoculation at equal ratio from mid-exponential phase pure cultures. Growth rate was monitored by colony count after plating on selective media for both species. Results are represented as the mean of values obtained from three independent experiments. The error bars indicate the standard deviations. A nonlinear mixed-effect model was fitted, using a four-parameters logistic regression function. Panel A: growth curves of Newman in pure culture and in co-culture with PA14; Panel B: Competition index (CI) and Relative Increase Ratio (RIR) calculated from single and dual cultures of Newman and PA14; Panel C: growth curves of Newman in pure culture and in co-culture with AA2; Panel D: CI and RIR calculated from single and dual cultures of Newman and AA2; Panel E: growth curves of Newman in pure culture and in co-culture with AA43; Panel F: CI and RIR calculated from single and dual cultures of Newman and AA43. Each value represents the mean of CI and RIR values from three independent experiments and the bars indicate standard deviation. Statistically significant differences in Student's t test and in nonlinear mixed-effect model are indicated by symbols when present: *: p<0.05; **: p<0.01; ***: p<0.001.</p

<i>In vitro</i> growth inhibition of <i>S. aureus</i> and <i>P. aeruginosa</i>.

<p>Twenty-four <i>P. aeruginosa</i> isolates were collected from eight individuals with CF (SG, NN, BT, AA, TR, MF, KK, BST) at the onset of chronic colonization (numbered 1-2) or after 4.5–16.3 years of colonization (numbered 43-83). PAO1 and PA14 were included as reference strains. 5 µl spots of <i>P. aeruginosa</i> overnight cultures, normalized to 0.5 OD, were added to <i>S. aureus</i> lawn (normalized to 0.5 OD) on Mueller-Hinton agar and incubated overnight at 37°C. The table summarizes the results obtained: “weak inhibition” indicates an inhibition halo ≤15 mm; “strong inhibition” indicates an inhibition halo >15 mm and ≤25 mm; “very strong inhibition” indicates an inhibition halo >25 mm; “no inhibition“ indicates absence of inhibition halo (9 mm is the diameter of the <i>P. aeruginosa</i> spot).</p><p>* Indicates mucoid phenotype.</p>#<p>Indicates hypermutable phenotype. For statistical analysis see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089614#s2" target="_blank">Results</a>”.</p

Functional enrichment of <i>cis-</i>regulated genes.

<p>Functional annotation of <i>M. tuberculosis</i> genes was obtained from different databases; Fisher’s exact test, P<0.05 (see Methods).</p><p>GO: gene ontology; mtu: KEGG pathway.</p