The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair

Abstract

Various mechanisms exist that enable bacteria to resist bacteriophage infection. Resistance strategies include the abortive infection (Abi) systems, which promote cell death and limit phage replication within a bacterial population. A highly effective 2-gene Abi system from the phytopathogen Erwinia carotovora subspecies atroseptica, designated ToxIN, is described. The ToxIN Abi system also functions as a toxin-antitoxin (TA) pair, with ToxN inhibiting bacterial growth and the tandemly repeated ToxI RNA antitoxin counteracting the toxicity. TA modules are currently divided into 2 classes, protein and RNA antisense. We provide evidence that ToxIN defines an entirely new TA class that functions via a novel protein-RNA mechanism, with analogous systems present in diverse bacteria. Despite the debated role of TA systems, we demonstrate that ToxIN provides viral resistance in a range of bacterial genera against multiple phages. This is the first demonstration of a novel mechanistic class of TA systems and of an Abi system functioning in different bacterial genera, both with implications for the dynamics of phage-bacterial interactions.

Fig. 1.

toxIN on plasmid pECA1039 encodes a phage-resistance system. (A) Linear map of pECA1039 and subsequent constructs, together with (B) EOP data of each construct versus φA2 and φM1. Here * denotes a frameshift mutation, and ▼ denotes a transposon insertion site.

Fig. 2.

ToxN is growth-inhibiting. (A) Growth (OD₆₀₀) and (B) viable counts of E. coli DH5α were measured after induction of the toxN gene (pTA49) or a toxN-FS control (pTA50); for details, see Materials and Methods. (C) Serial dilutions of exponentially grown cultures of E. coli DH5α with ToxN-FS (pTA50) or ToxN (pTA49) plated on LBA, Ap, and L-ara (inducing conditions).

Fig. 3.

Organization of the toxIN locus. (A) Schematic representation of the toxIN locus. The transcription start (+1), toxI tandem repeats (black arrows), rho-independent terminator (gray arrows), toxN gene (white arrow), and promoter -35 and -10 elements are indicated. The hypothetical toxI ORF also is shown. (B) toxIN promoter lacZ transcriptional fusions in E. coli DH5α using plasmids (from top to bottom) pTA104, pTA105, pTA106, and pTA119.

Fig. 4.

The toxIN locus encodes a bacteriostatic TA system. (A) Protection of E. coli DH5α from ToxN inhibition by transcription of toxI. Protection assays were conducted as described in Materials and Methods, and the strains shown are E. coli DH5α, pTA49, pTA100 (toxN, vector) and E. coli DH5α, pTA49, pTA76 (toxN, P_tac-toxI). The symbols “+” and “−” refer to induction or repression of toxN (L-ara and glu) and toxI (+/− IPTG). Empty vector induction is indicated by 0. (B) Serial dilutions of E. coli DH5α, pTA49, pTA76 on LBA plates with Ap, Sp and, glu (control), glu and IPTG (ToxN), L-ara (ToxN), and L-ara and IPTG (ToxN+ToxI). (C) ToxI is less stable than ToxN. E. coli DH5α, pTA49, pTA76 was grown expressing both ToxI and ToxN, as described in Materials and Methods, and plated under conditions resulting in continued expression or repression of either ToxI or ToxN or both (+ and −). (D) ToxN is bacteriostatic. ToxN was induced in E. coli DH5α, pTA49, pTA76 for different times, and viable counts were determined on LBA Ap, Sp, and glu plates without (ToxN) or with (ToxN+ToxI) IPTG. Time (hr) refers to hours after ToxN induction.

Fig. 5.

ToxIN is an RNA-protein TA system. (A) Protection of E. coli DH5α from ToxN inhibition (pTA49) by expression of toxI deletions composed of 5.5 (pTA76), 4.5 (pTA78), 3.5 (pTA79), 2.5 (pTA80), 1.5 (pTA81), 0.5 (pTA93), a WT single 36-nt repeat (pTA103), a nonsense 36-nt repeat mutant (pTA122), or a silent 36-nt repeat mutant (pTA107). (B) Sequences of the 36-nt inserts in pTA103 (WT), pTA122 (nonsense), and pTA107 (silent) and the putative short peptides that they may encode. A putative ribosome binding site is underscored, the possible start codon is in italic type, and the single repeat is in bold type. (C) Expression of the ToxI RNA results in the stable production of ToxN. E. coli DH5α, pTA76, pTRB1 and E. coli DH5α, pTA76, pTA49 were grown, and samples were probed with a polyclonal anti-FLAG antibody as described in Materials and Methods. (D) Protection of E. coli DH5α from ToxN_Bt inhibition by transcription of toxI_Bt. Protection assays were performed as described in Materials and Methods; the strain shown here is E. coli DH5α, pTA117, pTA115 (toxN_Bt, P_tac-toxI_Bt). The symbols “+” and “−” refer to induction or repression of toxN (L-ara and glu) and toxI (+/− IPTG).