Diversity of bacterial type II toxin-antitoxin systems: a comprehensive search and functional analysis of novel families

Abstract

Type II toxin-antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as 'addiction', and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the 'guilt by association' principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes.

Figure 1.

Association by guilt bioinformatics approach. Detection of ‘associated by guilt’ (AG) sequences (A). ‘Similar’ sequences (purple box) to the 48 ‘original’ sequences (white box) were detected by PSI-BLAST searches using the indicated parameters (gray boxes) and a database composed of 2181 genome sequences (blue cylinder). First round: AG toxins and antitoxins denoted as AG1 (orange box) were identified by paring of the ‘similar’ sequences using the indicated parameters (gray box). Number and type of sequences are indicated (T for toxin, A for antitoxin and U for unassigned). Second round: AG1 sequences were used as query for a second round of detection using the same parameters as in the initial steps to identify AG-like sequences (orange box). AG2 sequences were identified by pairing the AG-like sequences. Number and type of sequences are indicated. Pair definition (B). An additional round of pair definition was performed using sequences detected in (A) using the indicated parameters (gray box). Protein families (C). Protein families (green box) were generated by grouping all the proteins present in the dataset using the Markov clustering algorithm (MCL) with the indicated parameters (gray boxes). Names of ‘original’ or ‘similar’ sequences were propagated to the AG proteins within the same protein family. These sequences were then defined as ‘associated by guilt and annotated’ (AGA).

Figure 2.

The ecoA1-ecoT1_EDL933 and the nspA5-nspT5_PC7120 pairs constitute TA systems. The DJ624Δara strain containing the pBAD33 and pLac-staby plasmids (1), the pBAD33-ecoT1_EDL933 and pLac-staby plasmids (2), the pBAD33-ecoT1_EDL933 and pLac-staby-ecoA1_EDL933 plasmids (3), the pBAD33-nspT5_PC7120 and pLac-staby plasmids (4) or the pBAD33-nspT5_PC7120 and pLac-staby- nspA5_PC7120 plasmids (5) were grown in log phase in M9 medium supplemented with glucose (1%) and appropriate antibiotics. Serial dilutions (as indicated) were spotted on LB plates containing arabinose (1%) and IPTG (1 mM). Plates were incubated overnight at 37°C.

Figure 3.

Overexpression of the CcrT4_CB15 and AtuT1_C58 toxins induce the SOS system in E. coli. The DJ624λsfiA::lacZ strain containing the pBAD33 (1), pBAD-parE_RK2 (2), pBAD-ccrT4_CB15 (3) or pBAD-atuT1_c58 (4) plasmids were grown in M9 medium. After induction of toxin expression by arabinose addition, samples were taken to perform β-galactosidase assays as described in ‘Materials and Methods’ section.

Figure 4.

Overexpression of the novel toxins inhibits translation in E. coli. The DJ624Δara strain containing the pBAD33 (1), pBAD-parE_RK2 (2), pBAD-yoeB (3), pBAD-ecoT1_EDL933 (4), pBAD-mavT1_K10 (5), pBAD-spyT1₁₀₂₇₀ (6), pBAD-spyT2₁₀₂₇₀ (7), pBAD-bceT1_E33L (8), pBAD-smeT1₁₀₂₁ (9), pBAD-spyT1_M1 (10), pBAD-bceT5_E33L (11), pBAD-nspT1_PC7120 (12), pBAD-nspT2_PC7120 (13), pBAD-spyT4₁₀₂₇₀ (14), pBAD-spyT5₁₀₂₇₀ (15), pBAD-spyT3₁₀₂₇₀ (16), pBAD-spyT1₉₄₂₉ (17) and pBAD-lmoT1_EGD-e (18) were grown in M9 medium. After induction of toxin expression by arabinose addition, cultures were labeled with ³⁵S-methionine. Translation rate was measured as described in ‘Materials and Methods’ section.

Figure 5.

Associations of toxin super-families. The 12 toxin super-families are indicated above the pie chart. Each section of the pie chart represents the relative abundance of antitoxin sequences belonging to a given super-family associated with toxin sequences. Each antitoxin super-family is represented by a specific color. In (A), toxin super-families that are associated with multiple antitoxin super-families (>4). In (B), toxin super-families that are associated with three different antitoxin super-families. In (C), toxin super-families that are associated with AG sequences and another antitoxin super-family. Associations occurring at less than 1% were not considered for clarity.

Figure 6.

Associations of antitoxin super-families. The 20 antitoxin super-families are indicated above the pie chart. Each section of the pie chart represents the relative abundance of toxin sequences belonging to a given super-family associated with antitoxin sequences. Each toxin super-family is represented by a specific color. In (A), antitoxin super-families that are associated with multiple toxin super-families (five or more). In (B), antitoxin super-families that are associated with three or four different toxin super-families. In (C), antitoxin super-families that are associated with AG sequences and another toxin super-family. In (D), antitoxin super-families that are associated with a specific toxin super-family or to AG sequences. Associations occurring at <1% were not considered for clarity.

Figure 7.

Phyletic distribution of toxin and antitoxin super-families. Sequences of toxin super-families (A) and antitoxin super-families (B) are detected in the bacterial phyla indicated at the left of the figure in different proportions: not detected (white), 0.1–20% (gray), 20.1–60% (dark gray), above 60.1% (black).

Figure 8.

No correlation between the total number of CDS and the number of predicted antitoxin and toxin sequences. Only ‘original’ and ‘similar’ antitoxin and toxin sequences were considered.