Microarray identification and qRT-PCR validation of <i>S. meliloti</i> genes controlled by RpoE1 and/or RpoE4.

a<p>Genes found to be regulated by RpoE4 in both experiments ii) and iii) are indicated in bold.</p>b<p>All genes with ratio >2 and P value (t test) <0.05 in microarrays are shown, except SMc04051 which is included in i) because it is of interest for the study, and SMc04050 which did not show up in ii).</p>c<p>All genes tested were significantly up-regulated (>2-fold, P<0.05), except SMb21671 in (iii) (P = 0.36).</p><p>ND, not determined. NA, not applicable.</p

Schematic representation of the <i>rpoE1</i> (A) and <i>rpoE4</i> (B) chromosomal regions of <i>S. meliloti</i>.

<p>Grey-colored arrows represent open reading frames. Promoters mapped in the present study (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050768#pone-0050768-g004" target="_blank">Fig. 4</a>) are indicated. In A is shown a comparison of the <i>rpoE1</i> regions in the reference strain 1021 and in strain GMI11495 used in this study (see text).</p

Growth curves of various <i>S. meliloti</i> strains, in the presence of succinate or taurine.

<p>Strains were cultured in Vincent minimal medium supplemented with either 10 mM sodium succinate (VMMS, A) or 100 mM taurine (VMMT, B and C) as sole carbone source. Strains GMI11495 (wt), CBT1022 (Δ<i>rpoE1</i>), CBT997 (Δ<i>rpoE4</i>), and CBT1267 (Δ<i>sorT</i>), carrying or not pMLBAD derivatives, as indicated, were pre-cultured to mid-log phase in VMMS, and were then either diluted in fresh VMMS to OD₆₀₀ = 0.002 or centrifuged and resuspended in VMMT to OD₆₀₀ = 0.1. Growth was monitored by measuring OD₆₀₀ over several days. All media were supplemented with Sm, and with Tmp when strains contained pMLBAD derivatives (C). All strains carrying pMLBAD derivatives displayed similar growth curves in VMMS (not shown). The results shown are the means and standard errors of data from three independent experiments.</p

Model of gene regulation by RpoE1 and RpoE4 in <i>S. meliloti</i>.

<p>A. Sulfite (SO₃²⁻) activates sigma factors (open arrowheads), which then control the transcription of target genes (closed arrowheads). The central box is repeated in part B. B. Interpretation of the data presented in this study. Under the different growth conditions tested (left), various levels of activation of the two sigma factors (indicated by no, dotted, or plain arrows) led to various levels of expression and cross-regulation of the target genes (indicated in the central box), and are interpreted as consequences of the various intracellular concentrations of sulfite present in the different conditions (right).</p

Expression of <i>rpoE1</i> and <i>rpoE4</i> at different growth phases and in various genetic backgrounds.

<p>Expression from the promoter of the <i>rpoE1</i> (A, C) or <i>rpoE4</i> (B, D) operon was estimated by measuring β-galactosidase activity driven from the chromosomal <i>PrpoE1-lacZ</i> fusion in strains CBT1183 (wt), CBT1185 (Δ<i>rpoE1</i>), CBT1191 (Δ<i>rpoE4</i>), (CBT1247 (Δ<i>rpoE1</i> Δ<i>rpoE4</i>), CBT1315 (Δ<i>sorT</i>), CBT1350 (Δ<i>sorT</i> Δ<i>rpoE1</i>), CBT1354 (Δ<i>sorT</i> Δ<i>rpoE4</i>) and CBT1358 (Δ<i>sorT</i> Δ<i>rpoE1</i> Δ<i>rpoE4</i>) or from the chromosomal <i>PrpoE4-lacZ</i> fusion in strains CBT1218 (wt), CBT1220 (Δ<i>rpoE1</i>), CBT1224 (Δ<i>rpoE4</i>), CBT1251 (Δ<i>rpoE1</i> Δ<i>rpoE4</i>), CBT1317 (Δ<i>sorT</i>), CBT1356 (Δ<i>sorT</i> Δ<i>rpoE4</i>), CBT1352 (Δ<i>sorT</i> Δ<i>rpoE1</i>) and CBT1360 (Δ<i>sorT</i> Δ<i>rpoE1</i> Δ<i>rpoE4</i>) grown in Vincent minimal medium with sodium succinate as carbon source either to exponential phase (OD₆₀₀∼0.5; white bars) or stationary phase (∼24–30 h after the previous point; grey bars). The results shown are the means and standard errors of data from four to thirteen independent experiments.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Expression levels of RpoE1 or RpoE4 target genes in the presence of sulfite-generating compounds, in various genetic backgrounds.

<p>A and C. Expression levels of <i>sorT</i>, SMc04050, SMc02156 and SMc01418 were measured by qRT-PCR from strains GMI11495 (wt), CBT997 (Δ<i>rpoE4</i>), CBT1022 (Δ<i>rpoE1</i>) and CBT1064 (Δ<i>rpoE1</i> Δ<i>rpoE4</i>), either grown with sodium succinate (white bars) or taurine (pale grey bars) as carbon source, or with succinate plus 20 mM thiosulfate (dark grey bars). Results are expressed as relative transcript levels, with wt control levels arbitrarily set to 1 for each gene, and are the means and standard errors of data from three to five independent experiments. B and D. Expression from the promoter of the <i>rpoE4</i> or <i>rpoE1</i> operon was estimated by measuring β-galactosidase activity driven from the chromosomal <i>PrpoE4-lacZ</i> fusion in strains CBT1218 (wt), CBT1224 (Δ<i>rpoE4</i>), CBT1220 (Δ<i>rpoE1</i>), and CBT1251 (Δ<i>rpoE1</i> Δ<i>rpoE4</i>), or from the chromosomal <i>PrpoE1-lacZ</i> fusion in strains CBT1183 (wt), CBT1191 (Δ<i>rpoE4</i>), CBT1185 (Δ<i>rpoE1</i>), and CBT1247 (Δ<i>rpoE1</i> Δ<i>rpoE4</i>), either grown with sodium succinate (white bars) or taurine (pale grey bars) as carbon source, or with succinate plus 20 mM thiosulfate (dark grey bars). The results shown are the means and standard errors of data from three to seven independent experiments.</p

Promoter regions of various genes.

<p>A. Transcription start sites (+1) in promoter regions of <i>S. meliloti</i> genes controlled by RpoE1 and/or RpoE4, as determined from 5′RACE experiments in the present study (underlined) or deduced from Illumina- or 454-based RNAseq analyses (bold; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050768#pone.0050768-Schlter1" target="_blank">[41]</a>; Brice Roux, unpublished results). The deduced −10 and −35 sequences recognized by the sigma factors are highlighted in grey. Distance (in nucleotides) to the predicted translation start site of each ORF is indicated. B. The 5′ untranslated region of genes encoding known or putative sulfite-oxidizing enzymes from <i>S. meliloti</i> AK83, <i>S. medicae</i> WSM419 (<i>Smed</i>), <i>Xanthobacter autotrophicus</i> Py2 (<i>Xaut;</i> two identical sequences), <i>Starkeya novella</i> DSM506 (<i>Snov</i>), <i>Methylobacterium chloromethanicum</i> (<i>Mchl</i>), <i>Sulfitobacter</i> sp. EE-36 (<i>Sulf</i>; identical sequence in <i>Sulfitobacter</i> sp. NAS-14.1) and <i>Roseovarius nubinhibens</i> ISM (<i>Rnub</i>) are aligned with the <i>sorT</i> promoter region of <i>S. meliloti</i> 1021. Conserved putative −10 and −35 regions are highlighted in grey. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050768#pone.0050768.s004" target="_blank">Fig. S4</a> for further details.</p

Accumulation of the Ole18-CecA fusion protein in rice seeds.

<p>(A) <i>In situ</i> immunolocalization of CecA in <i>pOle18</i>:<i>Ole18-CecA</i> transgenic seeds. Immunoreaction was detected using a fluorescent-labeled secondary antibody. (B) Western blot analysis of OB proteins purified from seeds of the indicated Ariete transgenic lines using anti-cecropin A, anti-oleosin18 and anti-caleosin antibodies (from top to bottom, respectively). Lower panel correspond to the Coomassie Blue stained SDS gel. Molecular weight markers are indicated on the left in kDa. (C) Cecropin A accumulation in seeds from the Ariete (2, 3, 4, 6, 9) and Senia (3.2, 8.1, 8.5, 11.2, 12.1, 16.2) transgenic lines (T3 homozygous) as estimated by immunoblot analysis of OB protein extracts in comparison with known amounts of synthetic cecropin A. Values correspond to the mean of at least 3 independent assays, and bars to the SD.</p

Purification of active cecropin A from <i>pOle18</i>:<i>Ole18-CecA</i> plant seeds.

<p>(A) Schematic purification procedure of CecA. (B) Immunoblot analysis of purified fractions from wild-type (WT) and two independent <i>pOle18</i>:<i>Ole18-CecA</i> transgenic lines (10 seeds per line). As a control, synthetic cecropin A (10 and 50 ng of CecA) was added to one WT F3 fraction. The proteins in the F3 fractions were concentrated by acetone precipitation, and together with the F1 and F2 fraction proteins, were resolved in Tricine-SDS-PAGE and immunodetected with anti-CecA antibodies. (C) MRM chromatograms of CecA synthetic peptide, WT and <i>pOle18</i>:<i>Ole18-CecA</i> (lines #2 and #9) F3 fractions. The MS/MS transitions monitored were 740.9/816.4, 740.9/915.5 and 740.9/1085.6. (D) Amount of CecA in F3 fractions as determined by UV absorbance (280 nm) in comparison with known amounts of synthetic CecA added to wild-type F3 fractions. (E) Antimicrobial activity of purified fractions against <i>Dickeya dadantii</i> bacterial cells. Aliquots (3 μl) of ten-fold serial dilutions of bacterial cells (5 x 10⁴) incubated for 2h with synthetic CecA or with purified fractions (5 μl of F1 or F2, or 50 μl of F3 fractions) from WT or from the indicated <i>pOle18</i>:<i>Ole18-CecA</i> transgenic lines were plated on LB-agar media and grown for 2 days. (F) <i>D</i>. <i>dadantii</i> viable cells after 2h incubation with the indicated amounts of F3 fractions from empty vector (EV) or <i>pOle18</i>:<i>Ole-CecA</i> (line #2) compared to synthetic CecA at indicated concentrations. Mean values of two replicates and SD are shown.</p