CAT specific activities of <i>P_YG</i> promoter in <i>E</i>. <i>coli</i> NR698.

<p>^a Results are expressed in nanomoles of product formed per minute and per milligram of protein in cytoplasmic extracts. Data are means ± standard deviation obtained from a minimum of three independent extracts.</p><p>^b The <i>P</i>_<i>spank</i> promoter is constitutive due to low expression in the absence of induction by IPTG.</p><p>^C<i>term</i> corresponds to the T4 transcription terminator.</p><p>CAT specific activities of <i>P_YG</i> promoter in <i>E</i>. <i>coli</i> NR698.</p

CAT specific activities of <i>P</i>_<i>UG</i> promoter in <i>E</i>. <i>coli</i> NR698.

<p>^a Results are expressed in nanomoles of product formed per minute and per milligram of protein in S100 extracts. Induction was performed with vancomycin (0.25 μg/ml). Data are means ± standard deviation obtained from a minimum of three independent extracts.</p><p>^b The <i>P</i>_<i>spank</i> promoter is constitutive due to low expression in the absence of induction by IPTG.</p><p>^C<i>term</i> corresponds to the T4 transcription terminator.</p><p>CAT specific activities of <i>P</i>_<i>UG</i> promoter in <i>E</i>. <i>coli</i> NR698.</p

Binding sites of VanU_G to the <i>P</i>_<i>UG</i> regulatory promoter (A, B) and regulatory genes transcription (C, D).

<p>(A) Sequence of the <i>P</i>_<i>UG</i> region. The transcriptional start site (+1) is in boldface and the -35 and -10 sequences are boxed. The translational start site is in boldface and underlined and the ribosome binding site (RBS) is in boldface and in italics. Regions protected from DNase I cleavage by VanU_G are delineated by a bracket. The binding motif is composed of two 14-bp imperfect inverted repeats indicated in orange and purple and by arrows; the complementary bases are underlined. (B) DNase I footprinting analysis of the binding of VanU_G to <i>P</i>_<i>UG</i>. A 357-bp DNA fragment was amplified from the <i>P</i>_<i>UG</i> promoter region using a labeled reverse primer (VanG126) to radiolabel the template strand. Increasing amounts of VanU_G, indicated above each lane, were incubated with the DNA probe. The bracket indicates the region protected from DNase I cleavage by VanU_G and the co-ordinates of protection relative to the transcriptional start site are indicated on the left. M is the A+G Maxam and Gilbert sequencing reaction lane of the probe used as a size marker and the nucleotide positions are indicated at the right. Transcription of the regulatory genes by RT-qPCR in transconjugant BM4522 (C) and deletant derivatives relative to the same genes of BM4522 (D). The strains are indicated at the bottom. Results are presented in arbitrary units normalized to the <i>rpoB</i> transcripts in the same strain and in BM4522 under similar conditions. Each strain, not induced or induced by vancomycin, was tested in triplicate in two independent experiments. The bars represent the means and the error bars the standard deviations; nd, not detectable. NI, not induced. Vm, vancomycin.</p

Growth rate.

<p>^a Exponential growth rate measured in the absence of antibiotic or in the presence of vancomycin (1μg/ml) (Vm1); average of at least four independent experiments ± standard deviations.</p><p>^b Relative growth rate was calculated as the ratio of the growth rate of the strain induced by 1μg/ml of vancomycin versus the non induced strain.</p><p>Growth rate.</p

VanT_G racemase specific activity in membrane extracts from clinical isolate BM4518, transconjugant BM4522, and its deletant derivatives.

<p>Vancomycin (Vm) inducing concentrations (μg/ml) and MICs are indicated at the bottom. NI, not induced. The error bars represent the standard deviations from at least three independent experiments (eight for BM4723) and the values above the bars are the means of specific activity defined as the number of nanomoles of product formed at 37°C per minute per milligram of protein contained in the extracts.</p

Autophosphorylation of VanS_G (A), phosphotransfer from VanS_G-P to VanR_G (B), phosphorylation of VanR_G by acetyl [³²P] phosphate (C), hydrolysis of VanR_G-P by VanS_G (D), and phosphotransfer from VanS_G to VanU_G (E) or to VanU_G plus VanR_G (F).

<p>Quantitative analysis of phosphorylated VanR_G in panels C and D (G). (A) Purified VanS_G was incubated with [γ-³²P]-ATP for 1 h at room temperature to test autophosphorylation. (B) After autophosphorylation of VanS_G (time 0), purified VanR_G was added, samples were removed at the indicated times (in min), mixed with β-mercaptoethanol stop solution on ice and separated by SDS-PAGE (15%). Transfer of radioactivity to VanR_G was revealed by autoradiography. (C) Purified VanR_G was incubated with acetyl[³²P]phosphate for 1 h at room temperature (time0), excess acetyl[³²P]phosphate was removed by using a Sephadex G-50 Quick-Spin column, and phosphorylated VanR_G was incubated at room temperature either alone or (D) following the addition of purified VanS_G. Samples were removed at the indicated times (in min), mixed with β-mercaptoethanol-stop solution on ice, resolved by SDS-PAGE (15%), and subjected to autoradiography. After autophosphorylation of VanS_G (time 0), purified VanU_G was added alone (E) or with VanR_G (F), samples were removed at the indicated times (in min), mixed with β-mercaptoethanol stop solution on ice and separated by SDS-PAGE (12%). Transfer of radioactivity to VanR_G but not to VanU_G was revealed by autoradiography. (G) Hydrolysis in the absence (blue line, panel C) or in the presence (pink line, panel D) of VanS_G of purified VanR_G labeled with acetyl[³²P]phosphate was detected on a phosphor storage screen and percent quantified. Results are the means of four independent experiments and the bars indicate standard deviations.</p

Competition between VanU_G and VanR_G for binding to the <i>P_YG</i> resistance promoter.

<p>(A) DNase I footprinting analysis. A 233-bp DNA fragment was amplified from the <i>PYG</i> region using a labeled reverse primer (YG10) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005170#pgen.1005170.s006" target="_blank">S2 Table</a>) to radiolabel the template strand. Increasing amounts of VanR_G and two fixed amounts of VanU_G, indicated at the top, were incubated with the DNA probe. The bracket indicates the region protected from DNase I cleavage by VanU_G and/or VanR_G and the co-ordinates of protection relative to the transcriptional start site are indicated on the left. M is the A+G Maxam and Gilbert sequencing reaction lane of the probes used as a size marker and the nucleotide positions are indicated at the right. (B) Model for the binding competition between VanU_G and VanR_G-P in the absence or in the presence of various concentrations of vancomycin (Vm).</p

Schematic representation of the <i>vanG</i> operon.

<p>Open arrows represent coding sequences and indicate direction of transcription. The regulatory genes are in red, the resistance genes in blue and accessory genes in green. The additional regulatory gene, <i>vanUG</i>, is in yellow. The vertical bar in <i>vanYG</i> indicates a frameshift mutation leading to a truncated protein.</p