<i>In vitro</i> RNase assay of ICE<i>Afe</i>1 toxins.

<p>1.6 µg of MS2 RNA was incubated with (+) or without (−) the purified toxins in 10 mM Tris-HCl (pH 7.8) in the absence of divalent ions (A) or with 10 mM MgCl₂ (B) or MnCl₂ (C). The reactions were incubated at 37°C for 15 (A and C) or 30 minutes (B). 12 mM EDTA was added to some reactions as a control (lanes 6-10).</p

Plasmids used.

<p>Plasmids used.</p

Plasmid maintenance test. <i>E. coli</i> BL21(DE3) was double transformed with the plasmids indicated.

a<p>percentage of chloramphenicol-resistant bacteria (resistance gene encoded on pACYCDuet-1) when cultured on ampicillin-containing media (resistance gene encoded on pETDuet-1) for the days indicated. The data are expressed as the means of three independent cultures ± standard deviation.</p><p>Plasmid maintenance test. <i>E. coli</i> BL21(DE3) was double transformed with the plasmids indicated.</p

Oligonucleotides used.

<p>Oligonucleotides used.</p

Putative type II TA systems encoded on <i>A. ferrooxidans</i>^§.

§<p>Locus, accession gi and hits in CDD are according to the NCBI.</p>a<p>according to the classification by Leplae et al., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112226#pone.0112226-Leplae1" target="_blank">[19]</a>; NI: not identified.</p>b<p>Chr: chromosomal TA II; when a TA II is encoded in a MGE, the name of the element is indicated; in the case of ICE<i>Afe</i>2, the name is followed by the number of <i>A. ferrooxidans</i> ATCC strain where the TA is present.</p><p>Putative type II TA systems encoded on <i>A. ferrooxidans</i>^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112226#nt101" target="_blank">§</a>}.</p

Comparison of the relative genomic locations of <i>A. ferrooxidans</i> TA systems.

<p>Using BLASTP, TA from each <i>A. ferrooxidans</i> genome were paired according to protein similarity. TA encoded in MGEs are shown in red (ICE<i>Afe</i>1), pink (ICE<i>Afe</i>2) and blue (Genomic island, GI). In black are shown TA in which the gene that must encode the toxin are pseudo genes. Black lines link TA that have 94-100% amino acid identity between the two strains. The blue line links a TA that has 49% (antitoxin) and 52% (toxin) amino acid identity with its counterpart in the other strain. Numbers of the TA are according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112226#pone-0112226-t002" target="_blank">Table 2</a>.</p

Effect of ICE<i>Afe</i>1 TA systems expression in <i>E. coli</i> growth.

<p>Cellular growth of <i>E. coli</i> BL21(DE3)pLysS cells harboring plasmids containing toxin (T, blue curves), antitoxin (A, red curves) or both (TA, green curves) genes of TA 26 (A), TA 27 (B) and TA 28 (C) was monitored by measuring the OD₆₀₀. Cells containing the empty vector (gray curves) were used as a control. The arrows indicate the moment when 1 mM IPTG was added to each culture. 3 hours after the induction 10-fold serial dilutions of each culture were spotted on LB plates without IPTG (panels below each graph). The means and standard deviation of three different experiments are plotted.</p

Phylogenetic relationship between TA toxins of <i>A. ferrooxidans</i> ATCC 23270.

<p>Circular unrooted dendogram built using Neighbor-Joining method. Scale shows the evolutionary distance in number of base substitutions per site. Toxins described by Leplae et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112226#pone.0112226-Leplae1" target="_blank">[19]</a> belonging to RelE/ParE (red full-filled circle), CcdB/MazF (blue full-filled triangles) and VapC (green full-filled squared) super-families were introduced in the analysis as reference. Toxin classifications performed according the homologs with lower evolutionary distance (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112226#pone.0112226.s007" target="_blank">Table S1</a>) are show in open symbols. The sequences whose homologs with lower evolutionary distance correspond to a non previously classified toxin are show in open rhomboid. The accession numbers of the sequences used in the analysis are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112226#pone.0112226.s010" target="_blank">Supporting information S3</a>.</p

Functionality of tRNAs encoded in a mobile genetic element from an acidophilic bacterium

<p>The genome of the acidophilic, bioleaching bacterium <i>Acidithiobacillus ferrooxidans</i>, strain ATCC 23270, contains 95 predicted tRNA genes. Thirty-six of these genes (all 20 species) are clustered within an actively excising integrative-conjugative element (ICEA<i>fe</i>1). We speculated that these tRNA genes might have a role in adapting the bacterial tRNA pool to the codon usage of ICEA<i>fe</i>1 genes. To answer this question, we performed theoretical calculations of the global tRNA adaptation index to the entire <i>A. ferrooxidans</i> genome with and without the ICEA<i>fe</i>1 encoded tRNA genes. Based on these calculations, we observed that tRNAs encoded in ICEA<i>fe</i>1 negatively contribute to adapt the tRNA pool to the codon use in <i>A. ferrooxidans.</i> Although some of the tRNAs encoded in ICE<i>Afe</i>1 are functional in aminoacylation or protein synthesis, we found that they are expressed at low levels. These findings, along with the identification of a tRNA-like RNA encoded in the same cluster, led us to speculate that tRNA genes encoded in the mobile genetic element ICEA<i>fe</i>1 might have acquired mutations that would result in either inactivation or the acquisition of new functions.</p