Identification of <i>tspO</i> genes in fluorescent <i>Pseudomonas</i>.

<p>PCR amplification of <i>tspO</i>-encoding sequences in the biovar V strains <i>Pseudomonas fluorescens</i> MF37 and MF0, the clinical biovar II strain <i>Pseudomonas fluorescens</i> MFY70, the biovar B strain <i>Pseudomonas putida</i> MFN3597, and the biovar I strains, <i>Pseudomonas fluorescens</i> MFY162 and MFN1032. The biovar II strain, <i>Pseudomonas fluorescens</i> MFY63, lacks <i>tspO</i>-related sequences. Std: Molecular mass standards.</p

Expression of TSPO in fluorescent <i>Pseudomonas</i>.

<p>(a) Western blot analysis of total proteins extracts from the biovars V <i>Pseudomonas fluorescens</i> MF37 and MF0, the clinical strains of <i>Pseudomonas fluorescens</i> MFY70, MFY162, and MFN1032, and <i>Pseudomonas putida</i> MFN3597. (b) Alignment of the fragments obtained by ESI-MS/MS analysis of the TSPO immunoreactive bands extracted from MF37 and showing 92% recovery with the theoretical sequence of the protein. Std: Molecular mass standards.</p

Effect of PK 11195 on the cytotoxicity of <i>Pseudomonas fluorescens</i> MF37 and its <i>OprF</i>-deficient (373) and complemented <i>OprF</i>-deficient (373O) mutants.

<p>(a) Effect of PK 11195 (10⁻⁵ M) on the apoptotic-like activity of the bacteria determined by measuring nitrite production (NO₂⁻) resulting from NO biosynthesis by eukaryotic cells. (b) Effect of PK 11195 (10⁻⁵ M) on necrotic-like effect of <i>P. fluorescens</i> MF37, 373 and 373O determined by lactate dehydrogenase (LDH) activity released by eukaryotic cells. ***<i>P</i><0.001. NS: not significant.</p

Binding of PK 11195 to bacterial TSPO of <i>Pseudomonas fluorescens</i> MF37.

<p>(a) Demonstration of the binding of [³H] PK 11195 at the position of the TSPO-immunoreactive band visualized by western blots in <i>P. fluorescens</i> MF37 extracts. (b) Scatchard plot showing the binding characteristics of PK 11195 to TSPO of <i>P. fluorescens</i> MF37. The maximal binding potential (Bmax) is 0.087 pmole.µ g⁻¹ bacterial protein and the Kd is 0.92 nM.</p

Comparison of the putative topological organization of bacterial and mitochondrial TSPO.

<p>(a) <i>Pseudomonas fluorescens</i> MF37 bactTSPO. (b) BactTSPO from <i>Rhodobacter sphaeroides</i> 2.4.1 recalculated from Yeliseev and Kaplan <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006096#pone.0006096-Yeliseev3" target="_blank">[12]</a>. (c) Mitochondrial TSPO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006096#pone.0006096-JosephLiauzun1" target="_blank">[13]</a>. <i>P. fluorescens</i> MF37 bactTSPO has a typical five transmembrane helix structure, a larger L1 intra-cytoplasmic loop, and an extended cytoplasmic C-terminal end. In contrast, the extracellular N-terminal end is absent, and a peptidoglycane binding domain signature is present between the L2 loop and H3 transmembrane domain.</p

Effect of PK 11195 on <i>Pseudomonas fluorescens</i> MF37 adhesion to glass or eukaryotic cells, and on biofilm formation on PVC.

<p>Results are expressed as percentages of the values measured in the absence of treatment (100%). These changes were not associated with variations in the global surface polarity of bacteria. ***<i>P</i><0.001.</p

Comparison of <i>tspO</i> genomic environments among <i>Pseudomonas</i>.

<p>Physical maps of the regions upstream and downstream of <i>tspO</i> are shown for <i>P. fluorescens</i> strains MF37, SBW25, and Pf0-1 as well as <i>P. syringae</i> strains 1448A (pathovar phaseolicola), DC3000 (pv. tomato), and B728a (pv. syringae). ORF: non-identified putative protein, ¤ Data obtained from Blast, * Protein putative function. Black areas represent intergenic regions.</p

Amino acid sequence cladogram showing the relationships between bacterial TSPO.

<p>Bacterial names, accession numbers, and taxonomic groups are indicated for each branch. Bold characters indicate γ-proteobacteria, and the black arrow indicates the position of TSPO expressed in human mitochondria. The two dotted boxes indicate fluorescent <i>Pseudomonas</i> strains containing a bactTSPO gene (<i>P. fluorescens</i> and <i>P. syringae</i>). The degree of statistical support for branches was determined with 1000 bootstrap replicates. All branches showed less than 30% divergence.</p

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

<i>hcp2</i> mutation inhibits VgrG secretion.

<p>Concentrated culture supernatants for wild-type MFE01 and mutant MFE01Δ<i>hcp2</i> were analyzed by SDS-PAGE and Coomassie Blue staining, for bacteria in stationary growth phase after incubation at 28°C. MFE01Δ<i>hcp2</i> displayed much lower levels of Hcp secretion (indicated by an arrow) than MFE01. The residual band was analysed by mass spectroscopy and identified as an Hcp-like protein. The second arrow indicates the absence of the band corresponding to the VgrG-like protein in MFE01Δ<i>hcp2</i> supernatant.</p