Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems

Abstract

In their initial stages of discovery, prokaryotic toxin-antitoxin (TA) systems were confined to bacterial plasmids where they function to mediate the maintenance and stability of usually low- to medium-copy number plasmids through the post-segregational killing of any plasmid-free daughter cells that developed. Their eventual discovery as nearly ubiquitous and repetitive elements in bacterial chromosomes led to a wealth of knowledge and scientific debate as to their diversity and functionality in the prokaryotic lifestyle. Currently categorized into six different types designated types I-VI, type II TA systems are the best characterized. These generally comprised of two genes encoding a proteic toxin and its corresponding proteic antitoxin, respectively. Under normal growth conditions, the stable toxin is prevented from exerting its lethal effect through tight binding with the less stable antitoxin partner, forming a non-lethal TA protein complex. Besides binding with its cognate toxin, the antitoxin also plays a role in regulating the expression of the type II TA operon by binding to the operator site, thereby repressing transcription from the TA promoter. In most cases, full repression is observed in the presence of the TA complex as binding of the toxin enhances the DNA binding capability of the antitoxin. TA systems have been implicated in a gamut of prokaryotic cellular functions such as being mediators of programmed cell death as well as persistence or dormancy, biofilm formation, as defensive weapons against bacteriophage infections and as virulence factors in pathogenic bacteria. It is thus apparent that these antitoxins, as DNA-binding proteins, play an essential role in modulating the prokaryotic lifestyle whilst at the same time preventing the lethal action of the toxins under normal growth conditions, i.e., keeping the proverbial wolves at bay. In this review, we will cover the diversity and characteristics of various type II TA antitoxins. We shall also look into some interesting deviations from the canonical type II TA systems such as tripartite TA systems where the regulatory role is played by a third party protein and not the antitoxin, and a unique TA system encoding a single protein with both toxin as well as antitoxin domains.

Keywords: DNA-binding motifs; autoregulation; conditional cooperativity; toxin-antitoxin; transcriptional repressor proteins.

Figure 1

Three-dimensional structures showing the most frequent DNA-binding domains found in prokaryotic type II antitoxin proteins. The most frequent DNA-binding domains found in type II antitoxins include: (A) the HTH-motif (of which the smallest structural motif is shown) that has two α-helices (red) connected by a small loop (green); (B) the RHH folding motif, in which the minimal structure (as the one depicted corresponding to the CopG transcriptional repressor) is generated by two antiparallel β-strands (arrows, with arrowheads pointing to the C-terminal part of the protomer) that generate a ribbon; each strand comes from one of two protein monomers and they are involved both in dimer formation and in specific interactions with the DNA bases in the antitoxin DNA target (adapted from Gomis-Rüth et al., 1998), and (C) the SpoVT/Abr DNA binding motif in which the dimeric molecules are constructed by three- and four-stranded antiparallel β-sheets (upper part of the molecule) that are tightly packed, generating the DNA-binding domain. Loops keeping the outer part of the molecules are indicated by light green and red colors.

Figure 2

The E. coli-encoded MazEF TA system. (A) Genetic organization of the E. coli mazEF operon, and the regulatory elements on the mazEF promoter. Black arrows denote the transcriptional start sites of promoters P2 and P3. The stop codon of RelA and the start codon of MazE are underlined in brown. The FIS binding site is indicated with a blue line. Alternating palindromic regions “c-a” or “a-b” are indicated with red arrows. Adapted and modified from Marianovsky et al. (2001). (B) Structure of the MazE¹⁻⁵⁰ antitoxin homodimer-DNA complex (PDB accession: 2MRU). The MazE¹⁻⁵⁰ homodimer is indicated in blue and purple with the operator DNA indicated in orange. The N- and C-termini of the two MazE¹⁻⁵⁰ units are as labeled. The key amino acid residues of MazE that are involved in binding to the major groove of the double-stranded “a” operator DNA, i.e., Trp-9, Asn-11, and Arg-16 (Zorzini et al., 2015), are shown for one of the MazE monomers (blue).

Figure 3

Stoichiometries of Kis-Kid complexes and their binding affinities to parD DNA. When the Kis antitoxin is in excess, or in equal amounts as Kid toxin, various Kis-Kid complexes are formed (e.g., [kid₂-Kis₂]_n or [kid₂-Kis₂-kid₂-Kis₂]_n etc.). The most abundant one, the Kid₂-Kis₂-Kid₂-Kis₂ octamer complex, binds strongly to the two half-sites of the parD DNA regions I and II with two Kis dimers, and thus strongly represses the transcription of parD operon. When the Kid toxin exceeds Kis antitoxin, the Kid₂-Kis₂-Kid₂ hexamer is the most abundant. The Kid₂-Kis₂-Kid₂ hexamer has weak affinity toward parD DNA as it can only bind to the two half-sites of regions I and II using one dimer. Adapted and modified from Diago-Navarro et al. (2010).

Figure 4

The YefM-YoeB TA systems from E. coli and S. pneumoniae. (A) Tertiary structure of the E. coli YefM₂ YoeB heterotrimeric complex (PDB accession: 2A6Q). The YefM homodimer is indicated in yellow and green with the monomeric unit containing the disordered C-termini in yellow whilst the other unit with the ordered C-termini that binds to the YoeB monomer (shown in red) is depicted in green. The N- and C-termini of the two YefM units are indicated in their respective colors. (B) Sequence and organization of the upstream regulatory region of the E. coli yefM-yoeB locus. The −10 and −35 regions of the promoter are shown in blue boxes, the core hexameric 5′-TGTACA-3′ sequence is indicated in green bold letters, and the long (L) and short (S) palindromic sequences are denoted by inverted green arrows. A red asterisk denotes the transcription start site. (C) Sequence and organization of the upstream regulatory region of the S. pneumoniae yefM-yoeB_Spn locus. The −10 and −35 regions of the two promoters P_yefM1 and P_yefM2 are shown in blue boxes. The imperfect palindrome sequence, PS, that is the operator site for P_yefM2 is indicated by inverted purple arrows whereas the hexameric 5′-TGTACA-3′ sequence is underlined. Sequences that are part of the BOX element are depicted in orange letters. Red asterisks denote the transcription start sites from P_yefM1 and P_yefM2.

Figure 5

Structure of the E. coli MqsA-DNA complex. Tertiary structure of the E. coli-encoded MqsA dimer in complex with its operator DNA (PDB accession: 3O9X). The monomers of the MqsA dimer are colored either in green or in blue with their N- and C-termini indicated in their respective colors; zinc ions are shown as gray spheres; the mqsRA operator DNA is depicted in orange. For clarity, the MqsA amino acid residues that are crucial for interaction with operator DNA (Phe-22, Arg-23, Lys-58, and Arg-61) are shown for only one of the monomers as are the cysteine residues (Cys-3, Cys-6, Cys-37, and Cys-40) involved in coordination with the zinc ion (Brown et al., 2011).

Figure 6

Genetic organization of tripartite type II TA systems. The tripartite TA systems depicted here are the pasABC TA system of plasmid pTF-FC2 from A. ferroxidans (Smith and Rawlings, 1997), the ω-ε-ζ TA system of plasmid pSM19035 from S. pyogenes (Cegłowski et al., 1993), the paaR2-paaA2-parE2 system from E. coli O157:H7 (Hallez et al., 2010), and the spoIIS TA system from B. cereus (Melničáková et al., 2015). The antitoxin gene is depicted as blue arrows, the toxin gene as red arrows, the regulatory or third component gene as yellow arrows while gray arrows are for surrounding non-TA genes. Black line arrows indicate the relevant promoters for each TA system with the weaker P_ε promoter (in comparison to the P_ω promoter) shown as a dotted arrow. Note that the diagram is not drawn to scale.