Mutagenesis and functional characterization of the RNA and protein components of the toxIN abortive infection and toxin-antitoxin locus of Erwinia

Abstract

Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multiple adaptive resistance mechanisms. These mechanisms include the abortive infection systems, which promote "altruistic suicide" of an infected cell, protecting the clonal population. A cryptic plasmid of Erwinia carotovora subsp. atroseptica, pECA1039, has been shown to encode an abortive infection system. This highly effective system is active across multiple genera of gram-negative bacteria and against a spectrum of phages. Designated ToxIN, this two-component abortive infection system acts as a toxin-antitoxin module. ToxIN is the first member of a new type III class of protein-RNA toxin-antitoxin modules, of which there are multiple homologues cross-genera. We characterized in more detail the abortive infection phenotype of ToxIN using a suite of Erwinia phages and performed mutagenesis of the ToxI and ToxN components. We determined the minimal ToxI RNA sequence in the native operon that is both necessary and sufficient for abortive infection and to counteract the toxicity of ToxN. Furthermore, site-directed mutagenesis of ToxN revealed key conserved amino acids in this defining member of the new group of toxic proteins. The mechanism of phage activation of the ToxIN system was investigated and was shown to have no effect on the levels of the ToxN protein. Finally, evidence of negative autoregulation of the toxIN operon, a common feature of toxin-antitoxin systems, is presented. This work on the components of the ToxIN system suggests that there is very tight toxin regulation prior to suicide activation by incoming phage.

FIG. 1.

Role of ToxI repeats in phage resistance. (A) EZ::Tn transposon mutants of pECA1039 were assessed in E. carotovora subsp. atroseptica SCRI 1043 for resistance to φA2. The toxIN locus is not drawn to scale. Promoter elements and the transcriptional start are indicated by rectangles and a bent arrow, respectively. Each open arrow in toxI represents a DNA repeat, and the first imperfect repeat is indicated by a black arrow. The toxI transcriptional terminator is indicated by facing gray arrows, and the toxN ORF is indicated by a large open arrow. Transposon insertions are indicated by triangles, and dashed lines link these insertions with phage resistance data for them. The following plasmids were used (from left to right): no plasmid, pECA1039-Km12 (control insertion), pECA1039-Km22, pECA1039-Km13, pECA1039-Km8, pECA1039-Km6, pECA1039-Km5, pECA1039-Km9, pECA1039-Km15, and pECA1039-Km23 (toxN insertion). (B) φA2 phage resistance of E. carotovora subsp. atroseptica SCRI 1043 carrying plasmids with different numbers of internal toxI repeat deletions. The plasmids used were pTA47 (control) (toxI [5.5], toxN-FS), pTA46 (toxI [5.5], toxN), pTA69 (toxI [4.5], toxN), pTA63 (toxI [3.5], toxN), and pTA75 (toxI [2.5], toxN).

FIG. 2.

Conservation of amino acids in ToxN homologues (ClustalW2 alignment) and predicted secondary structure. The shading indicates relative conservation of residues among homologues, and a black background indicates a fully conserved residue. The residues chosen for mutagenesis were Y10, K20, F35, G37, Y47, S53, S64, K87, K116, G142, C148, and E154, and their positions are indicated by asterisks. Predicted alpha-helices and beta-strands are indicated by arrows and cylinders, respectively, and are linked by random coils indicated by solid lines. Where applicable, the host plasmid is listed with the strain. Abbreviations: Eca pECA1039, E. carotovora subsp. atroseptica SCRI 1039; Tcarboxy Nor1, Thermosinus carboxydivorans Nor1; Cbotulinum Eklund, Clostridium botulinum C strain Eklund; Bweihen, Bacillus weihenstephanensis KBAB4; Rtorques 27756, Ruminococcus torques ATCC 27756; Bthuring, Bacillus thuringiensis serovar kurstaki; Vharveyi BAA-1116, Vibrio harveyi ATCC BAA-1116; Llact, Lactococcus lactis; Hia, Haemophilus influenzae biotype aegyptius.

FIG. 3.

Impact of site-directed mutagenesis on ToxN phage resistance, stability, and toxicity. (A) EOP of φA2 with native (open bars) and FLAG-tagged (filled bars) SDM ToxN mutants in E. carotovora subsp. atroseptica SCRI 1043. (B) Western blot analysis of the mutant ToxN-FLAG proteins in E. carotovora subsp. atroseptica SCRI 1043. Blots representative of two biological repeats with identical results are shown. (C) Toxicities of wild-type and SDM ToxN mutants in E. coli DH5α. All proteins were FLAG tagged, unless indicated otherwise. In panels A and the C the data are the means ± standard deviations of three biological replicates. Plasmid designations are shown in Table 1. WT, wild type.

FIG. 4.

Analysis of ToxN levels during phage infection and ToxIN autoregulation. (A) Western blot for ToxN-FLAG levels during infection of E. carotovora subsp. atroseptica SCRI 1043 by wild-type and “escape” phages. Pre, before infection. (B) β-Galactosidase (β-gal) assay for a P_toxIN-lacZ low-copy-number promoter-probe plasmid (pTA104) in E. coli in the presence (+) and in the absence (−) of ToxI overexpression (pTA76 and pTA100, respectively) and in the presence and in the absence of ToxN overexpression (pTA49 and pTA50, respectively). (C) β-Galactosidase activity of a P_toxIN-lacZ promoter fusion plasmid (pTA104) with no expression (−) of ToxI or ToxN compared to the activity of a P_toxIN-toxI-lacZ operon fusion construct (pTA119) that expresses ToxI (+) but not ToxN (−). (D) β-Galactosidase activity of a P_toxIN-toxI-lacZ operon fusion construct (pTA106) that expresses ToxI (+) but not ToxN (−) compared to the activity of a P_toxIN-toxIN-lacZ operon fusion construct (pTA120) that expresses ToxI (+) and ToxN (+). (E) Schematic diagrams of P_toxIN-lacZ constructs used in the experiments whose results are shown in panels B, C, and D. Promoter elements of P_toxIN and the transcriptional start are indicated by rectangles and a bent arrow, respectively. Each open arrow in toxI represents a DNA repeat, and the first imperfect repeat is indicated by a black arrow. The toxI transcriptional terminator is indicated by facing gray arrows, and the toxN and lacZ ORFs are indicated by large open arrows. In panels B and C data are the means and standard deviations of three biological replicates.