Figure 10

<p>Expression of the <i>Xanthomonas campestris</i> pv. <i>campestris suxA</i> gene in the presence of sucrose, fructose or glucose. The pPr-<i>suxA</i> plasmid carrying the promoterless <i>LacZ</i> reporter gene under the <i>suxA</i> promoter region was used to monitor <i>suxA</i> expression in different genetic backgrounds. Expression was measured in minimal medium and cells were harvested at the indicated times. (A) Expression after 6 hours induction in the presence of different sucrose concentrations, in the wild-type background, the <i>suxR</i> and <i>suxC</i> insertion mutant backgrounds <i>(suxR</i>::pVO and <i>suxC</i>::pVO, respectively), and the <i>suxA</i> and <i>suxB</i> deletion mutant backgrounds <i>(</i>Δ<i>suxA</i> and Δ<i>suxB</i>, respectively). (B) Kinetics of <i>LacZ</i> expression in the wild-type background or in the <i>suxA</i> deletion mutant background (Δ<i>suxA</i>), in presence of 20 mM (triangles) or 100 µM (squares) sucrose. (C) Kinetics of <i>LacZ</i> expression in the wild-type background in the presence of 100 µM sucrose, fructose or glucose.</p

Figure 8

<p>[¹⁴C]sucrose transport into <i>Xanthomonas campestris</i> pv. <i>campestris</i> (<i>Xcc</i>). (A) Transport of [¹⁴C]sucrose over 120 minutes into the <i>Xcc</i> wild-type strain precultured in minimal medium without sugar (uninduced) or supplemented with 20 mM sucrose (induced). (B and C) Transport of [¹⁴C]sucrose over 8 minutes into the <i>Xcc</i> wild-type strain, the insertion mutants in <i>suxR</i> and in <i>suxC</i> (<i>suxR</i>::pVO and <i>suxC</i>::pVO, respectively), the deleted mutants in <i>suxA</i> and <i>suxB</i> (Δ<i>suxA</i> and Δ<i>suxB</i>, respectively) and their corresponding complemented strains [Δ<i>suxA</i> (pL-<i>suxA</i>) and Δ<i>suxB</i> (pL-<i>suxB</i>), respectively]. Cells were grown in minimal medium without sucrose. Transport was measured using 0.5 µM [¹⁴C]sucrose.</p

Candidate Fur-boxes identified in the promoter regions of <i>Xanthomonas campestris</i> pv. <i>campestris</i> Fur regulated TonB-dependent receptor (TBDR) genes, TBDR containing operons and the XCC3593 FecI sigma factor gene.

a<p>Gene identification (ID), name and putative function from <i>Xanthomonas campestris</i> pv. <i>campestris</i> strain ATCC33913 as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000224#pone.0000224-daSilva1" target="_blank">[14]</a>.</p>b<p>Matches to the Fur consensus sequence AATGAgAATcATTctCATT, determined in this study, appear in boldface.</p>c<p>Distance after start codon reannotation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000224#pone.0000224.s003" target="_blank">Table S1</a>).</p

Figure 11

<p>Genetic organization of putative <i>Xanthomonas campestris</i> pv. <i>campestris</i> CUT loci and specific induction of the TonB-dependent receptor gene by plant carbohydrates. Putative pectin (A), xylan (B) and maltodextrin (C) utilization CUT loci and specific induction of the corresponding TBDR gene in the presence of PGA, xylose and maltose, respectively. β-glucuronidase assays were performed in at least two independent experiments with TBDR insertion mutants cultivated in minimal medium supplemented with glutamate 20 mM (GT) or specific carbohydrates (PGA: polygalacturonic acid 0.125%; XYL: xylose 20 mM; MAL: maltose 20 mM). TBDR: TonB-dependent receptor; PME: pectin methyl esterase; PL: pectate lyase; CGTase: cyclomaltodextrin glucanotransferase.</p

Figure 3

<p>Palindromic consensus Fur-box sequence identified upstream of 9 <i>Xanthomonas campestris</i> pv. <i>campestris</i> Fur regulated TonB-dependent receptor genes. (A) Sequence logos generated by WebLogo (<a href="http://weblogo.berkeley.edu/" target="_blank">http://weblogo.berkeley.edu/</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000224#pone.0000224-Crooks1" target="_blank">[123]</a>) of the <i>Xcc</i> Fur-box motif as predicted by the MotifSampler program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000224#pone.0000224-Thijs1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000224#pone.0000224-Thijs2" target="_blank">[42]</a>. (B and C) Multiple alignement generated with Multalin program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000224#pone.0000224-Corpet1" target="_blank">[116]</a> with previously proposed Fur-box consensus sequences of <i>Bacillus subtilis </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000224#pone.0000224-Baichoo1" target="_blank">[46]</a>, <i>Shewanella oneidensis </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000224#pone.0000224-Wan1" target="_blank">[45]</a>, <i>X. campestris</i> pv. <i>campestris</i> and <i>Escherichia coli </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000224#pone.0000224-Escolar1" target="_blank">[44]</a> (B) and after a 3 bp shift of the <i>E. coli</i> sequence (C).</p

Relative expression ratios of <i>suxR, suxC, suxA</i> and <i>suxB</i> in the <i>suxR</i> insertion mutant (<i>suxR</i>::pVO) <i>vs</i> wild-type strain.

a<p>From real-time quantitative reverse-transcriptase polymerase chain reaction perfomed in at least three independent experiments. Cells were cultivated in minimal medium with or without sucrose. Calculation of relative expression includes normalization against the 16S rRNA endogenous control gene. SD: standard deviation.</p

Figure 9

<p>Concentration-dependent [¹⁴C]sucrose transport into wild-type <i>Xanthomonas campestris</i> pv. <i>campestris</i>. Cells were grown in minimal medium without sucrose and transport was measured for 15 sec at the indicated [¹⁴C]sucrose concentrations. The insert shows the Scatchard transformation of the binding data.</p

Figure 6

<p>Genetic organization of the <i>Xanthomonas campestris</i> pv. <i>campestris sux</i> locus. Location of mutations are indicated above the map: arrowheads indicate pVO155 insertions and deleted sequences are represented by horizontal dotted bars. Regions cloned in plasmids are indicated below the map: horizontal thick black bars indicate sequences used for plasmid constructions.</p

Figure 5

<p>Quantitative analysis of the interaction between <i>Xanthomonas campestris</i> pv. <i>campestris</i> (<i>Xcc</i>) and <i>Arabidopsis thaliana Sf</i>-2 plants. (A) Pathogenicity tests with the <i>Xcc</i> wild-type strain and the <i>XCC3358</i> insertion mutant (<i>XCC3358</i>::pVO). (B) Pathogenicity and complementation tests with the <i>Xcc</i> wild-type strain, the <i>XCC3356</i> and <i>XCC3357</i> insertion mutants <i>(XCC3356</i>::pVO and <i>XCC3357</i>::pVO, respectively), the <i>XCC3358</i> and <i>XCC3359</i> deleted mutants (<i>XCC3358</i>D1 and <i>XCC3359</i>D1, respectively) and their corresponding complemented strains [<i>XCC3358</i>D1 (pL-<i>XCC3358</i>) and <i>XCC3359</i>D1 (pL-<i>XCC3359</i>), respectively]. Disease symptoms were scored 5 to 8 days after inoculation. Each inoculated leaf was individually scored as: no symptom = 0; weak chlorosis surrounding the wound sites = 1; strong V-shaped chlorosis = 2; developing necrosis = 3; leaf death = 4. The represented average disease scores and the standard deviations were calculated from the values of four plants with four inoculated leaves per plant.</p

Figure 12

<p>Model of CUT loci functioning based on the <i>Xanthomonas campestris</i> pv. <i>campestris sux</i> locus. This scheme shows sucrose outer membrane transport <i>via</i> the SuxA TBDR or by passive diffusion through a putative porin. After crossing the inner membrane through the SuxC transporter, sucrose is proposed to interact with the SuxR repressor (thus allowing <i>sux</i> gene induction) and also to serve as a substrate for the SuxB amylosucrase. The large double headed arrow below the <i>sux</i> locus represents the balance between metabolic adaptation and virulence control putatively mediated by CUT loci.</p