PCP induction of PA3720-<i>armR</i>.

<p>qRT-PCR results showing impact of PCP and/or a <i>nalC</i> mutation on PA3720 expression (as a measure of PA3720-<i>armR</i> expression). Expression of PA3720 is reported relative to the <i>rpsL</i> internal control for wild type <i>P. aeruginosa</i> K767 and its <i>nalC</i> derivative, K1454 exposed or not to PCP (0.75 mM; 1.5 hr). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032684#s3" target="_blank">Results</a> shown are the mean +/− standard error of one cDNA sample for each, processed in triplicate, and are representative of 2 independent experiments.</p

Lack of impact of PCP on MexR repressor binding to the <i>mexR-mexA</i> intergenic region.

<p>Mobility shift assay in which purified MexR-His (500 ng) was incubated with 40 ng of a 351-bp DNA fragment encompassing the <i>mexR-mexA</i> intergenic region and increasing amounts of PCP as indicated. Lane 1, DNA only control.</p

Mapping the PA3720-<i>armR</i> transcriptional start site.

<p>The <i>nalC</i>/PA3720-<i>armR</i> intergenic region highlighting the RACE-determined transcription start site for PA3720-<i>armR</i> (bolded and italicized), the predicted transcriptional initiation site (marked with an asterisk; assessed using neural network promoter prediction software provided by M. G. Reese at <a href="http://www.fruitfly.org/seq_tools/promoter.html" target="_blank">http://www.fruitfly.org/seq_tools/promoter.html</a>), the <i>nalC</i> and PA3720 start codons (arrows), and putative −10/−35 sites for <i>nalC</i> (overlined) and PA3720-<i>armR</i> (underlined) promoters. The end-points of PCR-generated PA3720-distal (I) and -proximal (II) fragments and oligonucleotides (III and IV) used in EMSAs with purified NalC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032684#pone-0032684-g002" target="_blank">Fig. 2B</a>) are identified by arrowheads above the sequence. The shaded sequence corresponds to an inverted repeat and possible NalC-binding site.</p

ArmR-/PA3720-independence of PCP induction of <i>mexAB-oprM</i>.

<p>Expression of <i>mexA</i> (as a measure of <i>mexAB-oprM</i>) was assessed in <i>P. aeruginosa</i> strains K767 (wild type; WT), K3145 (Δ<i>armR</i>), K3146 (ΔPA3720) and K3130 (ΔPA3720-<i>armR</i>) exposed or not to PCP (0.75 mM; 1.5 hr) using qRT-PCR. Expression was normalized to <i>rpsL</i> controls and is reported relative (fold change) to untreated <i>P. aeruginosa</i> K767. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032684#s3" target="_blank">Results</a> shown are the mean +/− standard error of one cDNA sample for each, processed in triplicate, and are representative of 2 independent experiments.</p

PCP modulation of NalC repressor binding to the PA3720-<i>armR</i> upstream region.

<p>A) Mobility shift assay in which 50 ng of a 209-bp DNA fragment encompassing the <i>nalC</i>-PA3720 intergenic region was incubated without (lane 1) or with 200 (lane 2), 300 (lane 3), 400 (lane 4), 500 (lane 5), 800 (lane 6), 1000 (lane 7), 2000 (lane 8) or 3000 (lane 9) ng of purified NalC-His. B) Mobility shift assay in which purified NalC-His (800 ng) was incubated with 50 ng of a 209-bp DNA fragment encompassing the <i>nalC</i>-PA3720 intergenic region and increasing amounts of PCP as indicated. Lane 1, DNA only control.</p

Bacterial strains and plasmids.

<p>Ap^r, ampicillin resistant; Km^r, kanamycin resistant; Tc^r, tetracycline resistant.</p

Bacterial strains and plasmids.

a<p>Tc^r, tetracycline resistance; Ap^r, ampicillin resistance.</p>b<p>The amino acid substitutions in the ArmZ products produced by the indicated <i>armZ</i> mutant strains are highlighted in parentheses.</p>c<p>The amino acid substitutions in the mutant ArmZ products encoded by the indicated plasmids are highlighted in parentheses. WT, wild type.</p

Influence of ArmZ on spectinomycin-inducible <i>mexXY</i> expression.

<p><i>mexX</i> expression was assessed in the indicated <i>P. aeruginosa</i> strains in the absence (−) or presence (+) of spectinomycin (SPC) using quantitative RT-PCR. The <i>armZ</i> and <i>mexZ</i> status (−, absent; +, present) of the strains is highlighted. Expression was normalized to <i>rpoD</i> and is reported relative (fold change) to the wild-type <i>P. aeruginosa</i> PAO1 strain K767 not exposed to spectinomycin. Values represent the mean ± SEM from at least three independent determinations, each performed in triplicate.</p

MexZ-ArmZ interaction.

<p>A MexZ-ArmZ interaction and the impact of <i>armZ</i> mutations this interaction was assessed using a bacterial 2-hybrid assay, in which the interaction leads to repression of <i>lacZ</i> expression and reduced β-galactosidase activity in the reporter <i>E. coli</i> strain SU202. (a) The β-galactosidase activity of <i>E. coli</i> SU202 harbouring pMS604 derivatives expressing wild type (WT) or no (−) MexZ together with DP804 derivatives expressing wild type (WT), mutant (amino acid substitutions highlighted) or no (−) ArmZ is indicated. The results shown are mean ± SEM from at least three independent determinations, each performed in triplicate. (b) Western immunoblot of whole cell extracts of <i>E. coli</i> SU202 harbouring pMS604 derivatives expressing wild type MexZ and pDP804 derivatives expressing wild type and mutant ArmZ developed with anti-LexA antibodies.</p

Location of mutations in ArmZ that compromise its interaction with MexZ.

<p>The 3-dimensional model of ArmZ was created by threading the ArmZ amino acid sequence onto the crystal structure of the PH1602-extein protein from <i>Pyrococcus horikoshii</i> (PDB code: 1UC2, chain A).</p