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. 2014 Nov 10;9(11):e112226.
doi: 10.1371/journal.pone.0112226. eCollection 2014.

Toxin-antitoxin systems in the mobile genome of Acidithiobacillus ferrooxidans

Paula Bustamante  1 , Mario Tello  2 , Omar Orellana  1
Affiliations

Toxin-antitoxin systems in the mobile genome of Acidithiobacillus ferrooxidans

Paula Bustamante et al. PLoS One. .

Abstract

Toxin-antitoxin (TA) systems are genetic modules composed of a pair of genes encoding a stable toxin and an unstable antitoxin that inhibits toxin activity. They are widespread among plasmids and chromosomes of bacteria and archaea. TA systems are known to be involved in the stabilization of plasmids but there is no consensus about the function of chromosomal TA systems. To shed light on the role of chromosomally encoded TA systems we analyzed the distribution and functionality of type II TA systems in the chromosome of two strains from Acidithiobacillus ferrooxidans (ATCC 23270 and 53993), a Gram-negative, acidophilic, environmental bacterium that participates in the bioleaching of minerals. As in other environmental microorganisms, A. ferrooxidans has a high content of TA systems (28-29) and in twenty of them the toxin is a putative ribonuclease. According to the genetic context, some of these systems are encoded near or within mobile genetic elements. Although most TA systems are shared by both strains, four of them, which are encoded in the active mobile element ICEAfe1, are exclusive to the type strain ATCC 23270. We demonstrated that two TA systems from ICEAfe1 are functional in E. coli cells, since the toxins inhibit growth and the antitoxins counteract the effect of their cognate toxins. All the toxins from ICEAfe1, including a novel toxin, are RNases with different ion requirements. The data indicate that some of the chromosomally encoded TA systems are actually part of the A. ferrooxidans mobile genome and we propose that could be involved in the maintenance of these integrated mobile genetic elements.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Comparison of the relative genomic locations of A. ferrooxidans TA systems.
Using BLASTP, TA from each A. ferrooxidans genome were paired according to protein similarity. TA encoded in MGEs are shown in red (ICEAfe1), pink (ICEAfe2) 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 Table 2.
Figure 2
Figure 2. Phylogenetic relationship between TA toxins of A. ferrooxidans ATCC 23270.
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 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 (Table S1) 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 Supporting information S3.
Figure 3
Figure 3. Effect of ICEAfe1 TA systems expression in E. coli growth.
Cellular growth of E. coli 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.
Figure 4
Figure 4. In vitro RNase assay of ICEAfe1 toxins.
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).
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Grants and funding

This work was supported by grants from Fondecyt Chile 1110203 to OO (http://www.conicyt.cl/fondecyt/) and Proyecto Bicentenario PDA20 (www.conicyt.cl) and Proyecto FIA PYT20120056 to MT (www.fia.cl). PB was the recipient of a graduate studies fellowship and supporting fellowship AT-24100112 from Conicyt (www.conicyt.cl), Chile. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.