Expression of three developmental genes, <i>mrpC</i>, <i>fruA</i> and <i>tps</i>, in copper pre-adapted cells.

<p>WT (solid columns) or Δ<i>corSR</i> (dashed columns) cells harboring <i>mrpC-lacZ</i>, <i>fruA-lacZ</i> and <i>tps-lacZ</i> were grown in the presence of 600 µM copper, concentrated to an OD₆₀₀ of 15, and spotted onto CF medium containing the indicated copper concentrations. Cells were harvested at several time-points to determine specific β-galactosidase activity.</p

<i>In silico</i> analysis of the nine genes of the <i>curA</i> operon.

a<p>MXAN: gene identifiers in <i>M. xanthus</i> genome.</p>b<p>Predicted function inferred from BLASTP, Pfam, ScanProsite, MotifScan, and InterProScan. The Pfam identifiers are in parentheses.</p>c<p>Signal peptides (SP) were deduced from results at SignalP and SOSUI servers.</p>d<p>Transmembrane domains (TM) from TMHMM and SOSUI servers. The numbers of predicted transmembrane domains are indicated in parentheses.</p><p>Programs and databases used are available through ExPASy server (<a href="http://www.expasy.org/" target="_blank">http://www.expasy.org/</a>).</p

Growth of WT and Δ<i>corSR</i> strains in the presence of copper.

<p><b>A.</b> Cells grown in the absence of copper were diluted into CTT liquid media containing the indicated copper concentrations. <b>B.</b> Cells were adapted to grow in the presence of 600 µM of copper prior to dilution in CTT liquid media containing the indicated copper concentrations. OD₆₀₀ in both panels was monitored after 24-h incubation. Error bars indicate standard deviations. WT, blue lines; Δ<i>corSR,</i> red lines.</p

Development of the WT strain and the Δ<i>corSR</i> mutant in the presence of copper.

<p><b>A.</b> Cells grown in the absence of copper were concentrated at an OD₆₀₀ of 15 and spotted onto CF medium with the copper concentrations indicated. <b>B.</b> Cells grown in the presence of 600 µM copper sulfate were spotted onto CF agar plates with the indicated metal concentrations. Photographs in this panel were taken at 72 h of incubation. Bars, 1 mm.</p

Carotenoid accumulation in different strains during growth and development.

<p><b>A.</b> WT, Δ<i>corSR</i>, and Δ<i>corSR</i> Δ<i>carB</i> strains were grown in the absence of copper, concentrated at an OD₆₀₀ of 15, and spotted onto CTT agar plates containing a copper gradient from 0 to 1000 µM. Pictures were taken after 48-h incubation. <b>B.</b> Carotenoid accumulation during development. In these experiments cells grown in the absence of copper were concentrated at an OD₆₀₀ of 15 and spotted onto CF media with 40 µM copper. Pictures were taken at 72 h. Bar represents 100 µm. <b>C.</b> Carotenoid accumulation in the Δ<i>3414</i> and the WT strains during growth in the presence of different copper concentrations. Cells were grown in the absence of copper, concentrated to an OD₆₀₀ of 15, and spotted onto CTT agar plates containing the indicated copper concentrations.</p

The <i>curA</i> operon includes nine genes.

<p><b>A.</b> Strategy used to study the co-expression of the nine genes. cDNA is drawn as dashed line, with the region corresponding to the primer used to synthesize the cDNA represented by an arrow (not to scale). The name of the primer used to synthesize the cDNA is indicated on the left. PCR product is drawn as a solid line above the cDNA used as a template. Primers used for PCR are indicated with numbers, 1 corresponds to ORTF, and 2 corresponds to ORTR. Sequences of all primers are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068240#pone.0068240.s005" target="_blank">Table S1</a>. Vertical lines upstream of genes A, B, C, and G indicate the position of 4 putative σ⁵⁴ promoters (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068240#pone.0068240.s002" target="_blank">Fig. S2</a> for details). <b>B.</b> RT-PCR experiment. Lane 1 contains DNA molecular weight marker VIII (Roche). Lane 2, negative control using total RNA as a template. Lane 3, PCR product obtained using cDNA as a template (see panel A).</p

Measurements of intracellular copper accumulation during growth and development.

<p>Cells from WT, Δ<i>corSR,</i> and Δ<i>3414</i> strains were incubated on CTT (growth) or CF (development) agar plates containing the indicated copper concentrations for 72 h prior to harvesting for copper determinations.</p

Genes regulated by CorSR.

<p><b>A.</b> Qualitative analysis of the CorSR regulated genes. Cells of the different strains were inoculated on rich (growth) or starvation (development) media containing X-gal. Pictures were taken after 48 h of incubation. Bars, 3 mm. <b>B.</b> Quantitative analysis of the expression of <i>MXAN_3421</i>, <i>cuoA</i>, <i>corS,</i> and <i>copA</i> in the WT strain and the Δ<i>corSR</i> mutant. Plasmids containing <i>MXAN_3421-lacZ</i>, <i>cuoA-lacZ</i>, <i>corS-lacZ</i>, and <i>copA-lacZ</i> were introduced into WT (blue lines) or Δ<i>corSR</i> (red lines) backgrounds, and incubated for growth conditions on CTT agar plates containing 600 µM copper. For development, cells were incubated on CF agar plates containing 40 µM copper. Specific β-galactosidase activity in the cell extracts of all the strains was determined as described in Materials and Methods. Note the difference in the scales between panels. Error bars indicate standard deviations.</p