A DNA segment encoding the anticodon stem/loop of tRNA determines the specific recombination of integrative-conjugative elements in <i>Acidithiobacillus</i> species

<p>Horizontal gene transfer is crucial for the adaptation of microorganisms to environmental cues. The acidophilic, bioleaching bacterium <i>Acidithiobacillus ferrooxidans</i> encodes an integrative-conjugative genetic element (ICE<i>Afe1</i>) inserted in the gene encoding a tRNA^Ala. This genetic element is actively excised from the chromosome upon induction of DNA damage. A similar genetic element (ICE<i>Aca_TY.2</i>) is also found in an equivalent position in the genome of <i>Acidithiobacillus caldus</i>. The local genomic context of both mobile genetic elements is highly syntenous and the cognate integrases are well conserved. By means of site directed mutagenesis, target site deletions and <i>in vivo</i> integrations assays in the heterologous model <i>Escherichia coli</i>, we assessed the target sequence requirements for site-specific recombination to be catalyzed by these integrases. We determined that each enzyme recognizes a specific small DNA segment encoding the anticodon stem/loop of the tRNA as target site and that specific positions in these regions are well conserved in the target <i>attB</i> sites of orthologous integrases. Also, we demonstrate that the local genetic context of the target sequence is not relevant for the integration to take place. These findings shed new light on the mechanism of site-specific integration of integrative-conjugative elements in members of <i>Acidithiobacillus</i> genus.</p

Bioinformatic characterization of predicted Trx.

<p>Amino acid sequences from <i>Leptospirillum</i> “5 way CG” were obtained from NCBI data base.</p>*<p>Encoding genes detected by PCR method in <i>L. ferriphilum</i> DSM14647.</p>**<p>pI/MW was calculated for the precursor protein.</p

Glutathione reductase activity.

<p>The protein crude extract from <i>L. ferriphilum</i> (gray) were obtained after 1 hour of exposure to 0, 150, 250, or 260 mM Fe³⁺. <i>E. coli</i> (black) and <i>H. pylori</i> (light gray) were used as positive and negative controls, respectively. Bars represent µmol of oxidized NADPH by µgram of protein per min during 15 min of incubation (initial velocity). The average was calculated from three independent trials (lines on top of bars represent values range).</p

Time-course curve of Trx and TR relative activity.

<p>Activities of TR (square) and Trx (triangle) were measured every 10 minutes until 60 minutes after exposure to 260 mM Fe³⁺. This data represents the mean of 3 independent trials (bars indicate values range). All data are normalized by their respective controls.</p

Thiol/Disulfide System Plays a Crucial Role in Redox Protection in the Acidophilic Iron-Oxidizing Bacterium <em>Leptospirillum ferriphilum</em>

<div><p>Thiol/disulfide systems are involved in the maintenance of the redox status of proteins and other molecules that contain thiol/disulfide groups. <em>Leptospirillum ferriphilum</em> DSM14647, an acidophilic bacterium that uses Fe²⁺ as electron donor, and withstands very high concentrations of iron and other redox active metals, is a good model to study how acidophiles preserve the thiol/disulfide balance. We studied the composition of thiol/disulfide systems and their role in the oxidative stress response in this extremophile bacterium. Bioinformatic analysis using genomic data and enzymatic assays using protein extracts from cells grown under oxidative stress revealed that the major thiol/disulfide system from <em>L. ferriphilum</em> are a cytoplasmic thioredoxin system (composed by thioredoxins Trx and thioredoxin reductase TR), periplasmic thiol oxidation system (DsbA/DsbB) and a <em>c</em>-type cytochrome maturation system (DsbD/DsbE). Upon exposure of <em>L. ferriphilum</em> to reactive oxygen species (ROS)-generating compounds, transcriptional activation of the genes encoding Trxs and the TR enzyme, which results in an increase of the corresponding activity, was observed. Altogether these data suggest that the thioredoxin-based thiol/disulfide system plays an important role in redox protection of <em>L. ferriphilum</em> favoring the survival of this microorganism under extreme environmental oxidative conditions.</p> </div

Comparison of thioredoxin activity between <i>L. ferriphilum</i> and neutrophilic bacteria.

<p>The reduction of the insulin alfa-chain was monitored at 650 nm for 25 minutes in <i>E. coli</i> (triangle), <i>B. subtilis</i> (circle) and <i>L. ferriphilum</i> (square) after 4 mM diamide exposure during 60 minutes (close figures). Controls are shown with open figures. The negative control (mixture without protein) did not reduced insulin at 30 min. This data represents 2 independent trials.</p

Thioredoxin activity.

<p>The reduction of the insulin alfa-chain was monitored at 650 nm in bacteria treated with 260 mM Fe³⁺, 4 mM diamide, or 1 mM H₂O₂, at 30 (A) or 60 minutes (B) of stress exposure. The reaction was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044576#s2" target="_blank">materials and methods</a> after 15 min of incubation. Activity in the control reaction corresponds to 100%. Data represents the average of two independent trials (lines on top of bars indicate values range). The negative control (mixture without protein) did not reduced insulin alfa-chain at 15 min.</p

Relative expression of <i>trx</i> genes in oxidative stressed <i>L. ferriphilum</i>.

<p>Bacteria were treated with 260 mM Fe³⁺ (gray) or 4 mM diamide (Black) for 20 or 50 min. Cells grown under standard growth conditions were used as negative control (white). Data was normalized by 16S rRNA. Data represents the average of 3 independent trials (bars indicate values range).</p

Primers used in this work.

<p>Primers used in this work.</p

DNA sequence logos of Fur-binding sites derived from several bioinformatic and experimental prediction strategies

<p><b>Copyright information:</b></p><p>Taken from "Bioinformatic prediction and experimental verification of Fur-regulated genes in the extreme acidophile "</p><p></p><p>Nucleic Acids Research 2007;35(7):2153-2166.</p><p>Published online 13 Mar 2007</p><p>PMCID:PMC1874648.</p><p>© 2007 The Author(s)</p> () Fur box consensus sequence (), () Heterologous training set logo (66 Fur boxes, Supplementary Data S1A), () information-theory-based logo (90 Fur boxes, Supplementary Data S1B), () HMM-based logo (79 Fur boxes, Supplementary Data S1D), () Fur box logo derived for EMSA-validated genes (9 Fur boxes), () consensus Fur-binding sequence. Boxed and blue letters represent bases that are protected by Fur DNA binding in (,,)