33 research outputs found
IscR-regulated <i>nfuA</i> expression and <i>nfuA</i> promoter analysis.
<p>(A) IscR-regulated <i>nfuA</i> expression. RNA samples were isolated from uninduced and 0.5 mM plumbagin (PB)-induced cultures of the indicated <i>P</i>. <i>aeruginosa</i> strains. qRT-PCR using primers BT2841 and BT2860 for monitoring <i>nfuA</i> expression and performed as described in the Materials and Methods. Relative expression (fold) is defined as the changes in the <i>nfuA</i> expression levels across multiple samples relative to the level of the uninduced culture of PAO1. The data are presented as the means ± SD from three independent experiments. (B) The primer extension assay was performed using <sup>32</sup>P-labeled primer BT3577 and RNA extracted from <i>P</i>. <i>aeruginosa</i> PAO1 and Δ<i>iscR</i> grown under uninduced (UN) and 0.25 mM PB-induced conditions. G, A, T, and C represent the DNA ladder sequence prepared using <sup>32</sup>P-labeled primer BT3577 and plasmid pP<sub><i>nfuA</i></sub> as the template. The arrowhead indicates the transcription start site (+1). The -10 and -35 elements are in italic type. The consensus sequence of the Type-I <i>E</i>. <i>coli</i> IscR-binding site is aligned above the corresponding underlined sequence, and the homologous bases are marked with asterisks. The mutated IscR-binding site on the <i>nfuA</i> promoter was aligned below the underlined sequence line. The putative ribosome-binding site (RBS) is indicated in bold type. (C) The electrophoretic mobility shift assay was performed using <sup>32</sup>P-labeled native or mutagenized <i>nfuA</i> promoter fragments and increasing concentrations of purified IscR. CC and HD represent an addition of 1 μg unlabeled <i>nfuA</i> promoter and 2.5 μg of heterologous DNA (pUC18 plasmid), respectively, to the binding reaction mixtures containing 3.0 μM IscR. F and B indicate free and bound probes, respectively.</p
Gel mobility shift assay.
<p>Purified protein preparation and protein-DNA binding assay were performed as described in the experimental procedures. The gel mobility shift assay was performed using the <sup>32</sup>P-labeled native (A) and mutagenic (B) <i>fprB</i> promoter fragment and increasing concentrations of purified IscR. UP and HD represent the addition of 1 μg unlabeled <i>iscR</i> promoter and 2.5 μg of heterologous DNA (pUC18 plasmid), respectively, to the binding reaction mixtures containing 3.0 μM IscR. F and B indicate free and bound probes, respectively. The data presented was a representative of three biologically independent experiments.</p
<i>P</i>. <i>aeruginosa</i> and Δ<i>nfuA</i> mutant plating efficiency and growth under aerobic and anaerobic conditions.
<p>The plating efficiency and growth of PAO1 and various mutant strains were determined. Bacteria were grown in LB for aerobic conditions and in LB plus 1% KNO<sub>3</sub> for anaerobic conditions. The plating efficiency was defined as the number of CFU under anaerobic conditions divided by the number of CFU under aerobic conditions. The asterisk indicates statistically significant differences (paired <i>t</i>-test, <i>p</i> < 0.05) compared with the aerobic condition. (A) The plating efficiency and colony morphology of PAO1 and <b>Δ</b><i>nfuA</i> mutant strains under aerobic and anaerobic conditions was determined. The PAO1, <b>Δ</b><i>nfuA</i>::NfuA strains expressing wild-type NfuA or mutated NfuA (NfuA<sub>C152S</sub>, NfuA<sub>C155S</sub>, and NfuA<sub>C43,47S</sub>) were spotted onto plates containing 1% KNO<sub>3</sub> and incubated under aerobic and anaerobic conditions. (B) Plating efficiency of the PAO1 (PAO1::Tn7T), <b>Δ</b><i>nfuA</i> mutant (<b>Δ</b><i>nfuA</i>::Tn7T) and various complemented strains as in (A), with the addition of <b>Δ</b><i>iscR</i>/pIscR and <b>Δ</b><i>iscR</i>::NfuA/pIscR strains performed under aerobic and anaerobic conditions. Growth of <i>P</i>. <i>aeruginosa</i> strains under aerobic (C) and anaerobic (D) conditions in broth supplemented with 1% KNO<sub>3</sub> incubated at 37°C with 180 rpm shaking was determined. The OD<sub>600nm</sub> was monitored at hourly intervals for 24 h. Typically representative results of five independent experiments are shown.</p
List of primers used in this study.
<p>List of primers used in this study.</p
Determination of the resistance levels against oxidative and metal stresses in <i>P</i>. <i>aeruginosa</i> strains.
<p>The resistance levels of PAO1::Tn7T, Δ<i>nfuA</i>::Tn7T and the Δ<i>nfuA</i>::NfuA mutant strains expressing transposed wild-type NfuA or mutated NfuA (NfuA<sub>C152S</sub>, NfuA<sub>C155S</sub>, and NfuA<sub>C43,47S</sub>) against substances were determined using plate sensitivity assays on plates containing oxidants (A) i.e., 250 μM Paraquat, 1 mM Plumbagin, 0.5 mM H<sub>2</sub>O<sub>2</sub>, 1.6 mM CuOOH, and 0.045% NaOCl, and metals (B) i.e., 1.2 mM 2,2’-dipyridyl, 4.2 mM CuCl<sub>2</sub>, 0.8 mM CdCl<sub>2</sub>, 0.5 mM CoCl<sub>2</sub>, and 5 mM MgCl<sub>2</sub>. Survival (%) was defined as the percentage of colony-forming units (CFU) on plates containing oxidants over the number of CFU on plates without oxidants. The data shown are the means ± SD from three independent experiments. The asterisk indicates statistical significance (paired <i>t</i>-test, <i>p</i> < 0.05) compared with PAO1 treated with the same condition.</p
Expression levels of <i>soxR</i>, <i>iscR</i> and their targeted genes in <i>P</i>. <i>aeruginosa</i> strains.
<p>Analysis of the expression levels of genes encoding [2Fe-2S]-containing transcriptional regulators, namely, SoxR and IscR, and their targeted genes, <i>soxR</i>, <i>PA2274</i>, <i>iscR</i> and <i>fdx2</i>, in <i>P</i>. <i>aeruginosa</i> wild type (PAO1::Tn7T), <b>Δ</b><i>nfuA</i> mutant (<b>Δ</b><i>nfuA</i>::Tn7T) and the complemented strain (<b>Δ</b><i>nfuA</i>::NfuA) grown in uninduced and 0.5 mM Paraquat-induced conditions. qRT-PCR was performed as described in the Materials and Methods, and the data are shown as the fold expression relative to the level of the uninduced PAO1 (PAO1::Tn7T).</p
Iron-sulfur cluster-containing protein activities.
<p>(A) Aconitase and succinate dehydrogenase (Sdh) activities in <i>P</i>. <i>aeruginosa</i> PAO1 and <i>fprB</i> mutant harboring empty vector (pBBR), pFprB, or pISC strains under normal growth condition were measured. Cell culture conditions, protein preparation and enzymatic activity assays were performed as described in the experimental procedures. The data shown are means and SD of triple biologically independent replications. The asterisk indicates a statistically significant difference (P < 0.01) compared with the PAO1/pBBR strain and pBBR referred as an empty vector control. (B) Real-time RT-PCR analysis of <i>anr</i>, <i>narG</i>, <i>soxR</i> and PA2274 in indicated <i>P</i>. <i>aeruginosa</i> strains cultivated under normal growth condition. RNA isolation and real-time RT-PCR were performed as described in the experimental procedures. The data shown are means and SD of three independent experiments. The asterisk indicates a statistically significant difference (P < 0.01) compared to those of the PAO1/pBBR strain.</p
Bacterial strains and plasmids used in this study.
<p>Bacterial strains and plasmids used in this study.</p
Multiple amino acid sequence alignment and gene organization of <i>P</i>. <i>aeruginosa fprB</i>.
<p>(A) Alignment of <i>P</i>. <i>aeruginosa</i> FprB with other characterized bacterial FprB enzymes (Ppu, <i>P</i>. <i>putida</i>; Avn, <i>A</i>. <i>vinelandii</i>; and Ecl, <i>E</i>. <i>coli</i> ATCC 8739) was performed using the CLUSTALW program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134374#pone.0134374.ref057" target="_blank">57</a>]. Black (YW) and light gray (RAYS) boxes indicate the subclass II signature amino acid and the FAD-binding domain, respectively. The asterisk, colon, and period symbols indicate identical residues, conserved substitutions, and semi-conserved substitutions, respectively. Number indicates percent identity of the aligned protein with that of <i>P</i>. <i>aeruginosa</i>. (B) Gene organization at the <i>fprB</i> locus among <i>Pseudomonas</i> spp. <i>fnr</i> and <i>fnr-2</i>, <i>fprB</i>-homologous genes; <i>katB</i> and <i>katE</i>, catalase genes; <i>mscL</i>, gene encoding large conductance mechanosensitive channel; <i>dctP</i>, gene encoding propable periplasmic C4-dicarboxylate binding-protein; <i>rsmC</i>, gene encoding propable 16S RNA G1207 methylase; <i>luxR</i>, LuxR family DNA-binding transcriptional regulator gene; <i>radA</i>, gene encoding propable DNA repair protein; non-labelled, gene with unknown annotation.</p
Expression profile and characterization of <i>fprB</i> promoter.
<p>(A) Real-time RT-PCR analysis of <i>fprB</i> expression. The PAO1 cultures were induced with 1 mM H<sub>2</sub>O<sub>2</sub>, 1 mM CHP, 1 mM tBH, 1 mM DM, 0.5 mM PB, 0.5 mM PQ, 0.5 mM MD, 1 mM DIPY, 5 mM NaCl or 5 mM KCl for 15 min prior to RNA preparation and real-time RT-PCR analysis as described in the experimental procedures. The data shown are the means and SD of three independent experiments. (B) Promoter characterization using primer extension assay. Reverse transcription was performed using <sup>32</sup>P-labeled BT3552 primer and RNA extracted from PAO1 cultured under uninduced (UN) conditions or 0.5 mM PB. The extension products were separated on an 8% acrylamide-7 M urea sequencing gel. G, A, T, C represent the DNA sequence ladder prepared using a sequencing kit (Epicentre) with <sup>32</sup>P-labeled primer and the putative <i>fprB</i> promoter fragment as template. The arrowhead points to the transcription start site (+1). The putative -10 and -35 elements are underlined. The consensus sequence of the <i>E</i>. <i>coli</i> IscR binding site is aligned above the sequence line in corresponding letters, and the homologous bases are marked by asterisks. The mutagenized bases (from GCC to TTT) in the putative IscR binding motif are shown below the sequence line.</p