Effect of <i>hcp and tssC</i> gene mutations on the maturation of <i>P</i>. <i>fluorescens</i> MFE01 biofilms.

<p>Biofilms were grown on a glass surface for 48 h at 28°C, under a flow of LB medium. Bacteria were visualised with the Syto 9® green fluorescent nucleic acid stain. A 3D shadow representation and a side-view projection are shown at the top and bottom, respectively, for each strain. Images show representative data from at least five independent biofilm assays. Bars, 10 μm.</p

Effect of MFE01 and T6SS mutants on MFP05 biofilm formation.

<p>Biofilms were grown on a glass surface, for 48 h at 28°C, under a flow of LB medium. Biovolumes of fluorescent bacteria were determined by COMSTAT analysis after confocal laser scanning microscopy observation. <i>P</i>. <i>fluorescens</i> MFP05 bearing pSMC2.1 <i>gfp</i>, encoding green fluorescent protein, was co-cultured alone or with MFE01 or derivatives, in a 1:5 ratio. Each histogram represents the biovolume of fluorescent MFP05, relative to that of fluorescent MFP05 when MFP05-<i>gfp</i> is cultivated alone. Comparisons were made with the control MFP05-<i>gfp</i>; ** <i>p</i>-value < 0.01; * <i>p</i>-value < 0.05; ns = non-significant; <i>n</i> = 6 (the error bars represent the standard error of the mean).</p

Detection of AHLs with biosensor strains.

<p><i>P</i>.<i>a</i> indicates <i>Pectobacterium atrosepticum 6276</i> strain, which produces C_8- NAHLs, used as positive control. <i>P</i>.<i>aΔexp1</i> means <i>Pectobacterium atrosepticum</i> 6276 mutant strain, which does not produce AHLs, used as negative control. <b>A.</b> Detection of short-chain AHLs with <i>Chromobacterium violaceum</i> CV026 (CV026). CV026 indicates <i>C</i>.<i>violaceum CV026</i> strain, which produces vioalacein in contact with C₄-C₈ NAHLs, used as biosensor strain. Black arrow indicates violacein production by <i>C</i>.<i>violaceum CV026</i> strain. LB plates were incubated for 72 h at 37°C (<i>n</i> = 3). <b>B.</b> Detection of long-chain AHLs with <i>Agrobacterium tumefaciens</i> NTI (ANTI1). ANT1 means <i>Agrobacterium tumefaciens</i> NT1 strain, which produces β-galactosidase in contact with C₆-C₁₂ NAHLs, used as biosensor strain. Red arrow indicates β-galactosidase production by <i>Agrobacterium tumefaciens</i> strain. LB plates containing X-Gal (40 μg/mL) were incubated for 72 h at 28°C (<i>n</i> = 3).</p

Effect of MFE01 and T6SS mutants on MFP05 biofilm formation.

<p>Biofilms were grown on a glass surface, for 48 h at 28°C, under a flow of LB medium. Biovolumes of fluorescent bacteria were determined by COMSTAT analysis after confocal laser scanning microscopy observation. <i>P</i>. <i>fluorescens</i> MFP05 bearing pSMC2.1 <i>gfp</i>, encoding green fluorescent protein, was co-cultured alone or with MFE01 or derivatives, in a 1:5 ratio. Each histogram represents the biovolume of fluorescent MFP05, relative to that of fluorescent MFP05 when MFP05-<i>gfp</i> is cultivated alone. Comparisons were made with the control MFP05-<i>gfp</i>; ** <i>p</i>-value < 0.01; * <i>p</i>-value < 0.05; ns = non-significant; <i>n</i> = 6 (the error bars represent the standard error of the mean).</p

Genomic organisation of the T6SS core component locus and the <i>hcp3</i> locus in MFE01.

<p><b>A. Genomic organisation of the T6SS core component locus in MFE01.</b> Genes are represented as arrows, indicating the direction of transcription. The sequences of the T6SS core component genes have been deposited in GenBank under the following accession numbers: <i>vgrGc1</i>: KX941475, <i>tssA</i>: KX941476, <i>tssB</i>: KX941477, <i>tssC</i>: KX941478, <i>tssE</i>: KX941479, <i>paar-motif</i>: KX941480, <i>unkown1</i>: KX907122, <i>unknown2</i>: KX941481, <i>tssF</i>: KX941482, <i>tssG</i>: KX941483, <i>tssH</i>: KX941484, <i>sfa2</i>: KX941485, <i>tagU</i>: KX941486, <i>tagH</i>: KX941487, <i>tssJ</i>: KX941488, <i>tssK</i>: KX941489, <i>tssL</i>: KX941490, <i>tssM</i>: KX941491, <i>pppA</i>: KX941492, <i>ppkA</i>: KX941493, <i>vgrGc2</i>: KX941494. <b>B. Genomic organisation of the <i>hcp3</i> locus.</b> Genes are represented as arrows, indicating the direction of transcription. The sequences of the <i>hcp3</i> locus genes have been deposited in GenBank under the following accession numbers: <i>hcp3</i>: KX941495, <i>vgrG3</i>: KX941496, <i>arginine-decarboxylase</i>: KX941497, <i>sui1</i>: KX941498, <i>nudix-hydrolase</i>: KX941499, <i>duf2333</i>: KX941500, <i>amidase</i>: KX941501, <i>tec3</i>: KX941502.</p

Effects of <i>hcp and tssC gene</i> mutations on biofilm biovolume in <i>P</i>. <i>fluorescens</i> strain MFE01.

<p>Biofilms were grown on a glass surface, for 48 h at 28°C, under a flow of LB medium. Bacteria were visualised with the Syto 9® green fluorescent nucleic acid stain. Biovolumes were determined by COMSTAT analysis, after confocal laser scanning microscopy observation. The values shown are biofilm biovolumes relative to that of wild-type MFE01. The data presented are the mean values for at least five independent experiments and the error bars represent the standard error of the mean. Statistical analyses were performed with non-parametric Mann-Whitney tests (two-tailed): ns indicates no significant difference in biovolume (<i>p</i>-value > 0.05) relative to the MFE01 biofilm, * and ** indicates significant difference in biovolume relative to the MFE01 biofilm: *<i>p</i>-value < 0.05, ** <i>p</i>-value < 0.01. ⧫ indicates a significant difference in biovolume (<i>p</i>-value < 0.05) relative to the MFE01Δ<i>tssC</i> biofilm. EV means empty pPSV35 (plasmid control).</p

Oligonucleotides used for this study.

<p>Oligonucleotides used for this study.</p

The absence of SigX modulated <i>P</i>. <i>aeruginosa</i> virulence in the <i>C</i>. <i>elegans</i> model.

<p>Kaplan-Meier survival plots of <i>C</i>. <i>elegans</i> nematodes fed with the wild type strain <i>P</i>. <i>aeruginosa</i> H103 (n = 210), the <i>sigX</i> mutant PAOSX (n = 257), and the SigX-complemented mutant strain PAOSX+ (n = 201). Each value reported for the assay is the mean of measurements of eight samples from three independent experiments. Pairwise strain comparisons (log rank test) were as follows: H103 <i>versus</i> PAOSX, <i>p</i>-value< 0.0001; PAOSX <i>versus</i> PAOSX+, <i>p</i>-value< 0.0001; H103 <i>versus</i> PAOSX+, <i>p</i>-value <0.001. Four independent experiments were performed. </p

Altered levels of (A) exotoxinA, (B) pyocyanin and (C) siderophores in the culture supernatants of the <i>sigX</i> mutant.

<p>H103 (black), PAOSX (white) and PAOSX+ (grey) culture supernatants were obtained from overnight cultures in (A) LB, (B) King A or (C) King B media. The relative amounts of exotoxin A, pyocyanin and the total siderophores were assayed at least three times independently for each strain and means and standard deviations are presented. For the Chrome Azurol S (CAS) assay, the haloes around the wells in the CAS plate show siderophore production in sample supernatant. Statistics were done by pairwise strain comparisons (<i>t</i> test). *<i>p</i>-value<0.05, **<i>p</i>-value<0.01, ***<i>p</i>-value<0.001, **** <i>p</i>-value<0.0001, NS no significant difference.</p

Functional classes of SigX-regulated genes identified by expression profiling on DNA array.

<p>All 307 genes that had a significant difference in expression between wildtype and mutant strains (Fold change ≥2,<i>p</i>-value ≤0.05 as determined by Empirical Bayes) were included and classified according to their function. Functional classes were determined using the <i>Pseudomonas</i> Genome Project website (<a href="http://www.pseudomonas.com" target="_blank">www.pseudomonas.com</a>; Winsor et al., 2011), among which these framed in grey were discussed.</p