Cytoskeleton modifications of HeLa cells infected with <i>S. meliloti</i> queuosine biosynthesis mutants.

<p>HeLa cells were incubated with <i>S. meliloti queC</i> (A, E), <i>queF</i> (B, F), <i>tgt</i> (C, G), <i>queA</i> (D, H), the wild type 1021 strain (I) and the <i>queF</i> complemented strain (GMI11186) (J) in 0.5% FCS culture medium alone (A–D, I, J) or supplemented with preQ1 (E–H). HeLa cells were stained with phalloidin-Texas red and observed by fluorescence microscopy 48 hpi. Scale bar: 10 µm.</p

Bacteria-induced cytoskeleton modifications of HeLa cells.

<p>HeLa cells untreated (A), inoculated with <i>S. meliloti</i> (B), <i>R. leguminosarum</i> (C), <i>A. caulinodans</i> (D), <i>C. taiwanensis</i> (E), <i>B. tuberum</i> (F), <i>C. crescentus</i> (G) and <i>E. coli</i> (H). HeLa cells were stained with phalloidin-Texas red and observed by fluorescence microscopy 48 hours after bacterial inoculation. Arrow: stress fiber.</p

Bacterial strains and plasmids.

a<p>The location of plasmid insertions (number of nucleotides after the start codon) is indicated between brackets.</p

The <i>S. meliloti</i> queuosine biosynthetic pathway.

<p>preQ₀: 7-cyano-7-deazaguanine, preQ1: 7-(aminomethyl)-7-deazaguanine, AdoMet: S-adenosyl-L-methionine, EpoxyQ: epoxyqueuosine, Q: queuosine. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056043#pone.0056043-IwataReuyl1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056043#pone.0056043-Reader1" target="_blank">[50]</a>.</p

Symbiotic phenotype of <i>S. meliloti</i> queuosine mutants.

<p>Dry weight of <i>M. truncatula</i> seedlings inoculated with <i>S. meliloti</i> 1021, different queuosine-deficient mutants and the <i>queF</i> complemented (GMI11186) strain at 40 dpi. Statistical significance (P<0.01) is shown with respect to strain 1021(*) and the <i>queF</i> mutant (^#), respectively. (B, C) Sections of <i>M. truncatula</i> 21–day old nodules induced by 1021 (B) and the <i>queF</i> isogenic mutant (C). (D, E) Electron micrographs of nodule cells infected with 1021 (D) or the <i>queF</i> mutant (E). <i>queF</i> mutant bacteroids are randomly organized within the infected cell whereas 1021 bacteroids show a radial organization. (Insert panel in E): arrows point to symbiosome membranes detached from <i>queF</i> bacteria (). Arrowhead, type 4/5 bacteroid. *, starch granules.</p

Determination of GTPases activation state in bacteria-treated HeLa cells.

<p>(A) Representative pull down assays of active Cdc42, Rac1 and RhoA GTPases at 48 hpi in non-inoculated- (control), <i>S. meliloti</i> 1021- and <i>queF</i>-inoculated HeLa cells. (B) Quantification of pull down assays using ImageJ software. Means ± S.D. were calculated from three independent experiments for Cdc42-GTP and mean from two independent experiments for Rac1-GTP and RhoA-GTP. Results were normalized to the corresponding total protein. Statistical significance (P<0.001) is shown (*) with respect to the control. (C) Immunoprecipitation of active and total CdC42 from non-inoculated (control) HeLa cells or cells inoculated with live and heat-killed wild-type bacteria 48 hpi. (D). Kinetics of Cdc42 activation. Actin, total Cdc42, Rac1 and RhoA or active GTP-bound forms of Cdc42, Rac1 or RhoA were detected by immuno-blotting of SDS-PAGE gels.</p