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Toxin-antitoxin systems play key roles in bacterial adaptation, including protection from antibiotic assault and infection by bacteriophages. The type IV toxin-antitoxin system AbiE encodes a DUF1814 nucleotidyltransferase-like toxin, and a two-domain antitoxin. In Streptococcus agalactiae, the antitoxin AbiEi negatively autoregulates abiE expression through positively co-operative binding to inverted repeats within the promoter. The human pathogen Mycobacterium tuberculosis encodes four DUF1814 putative toxins, two of which have antitoxins homologous to AbiEi. One such M. tuberculosis antitoxin, named Rv2827c, is required for growth and whilst the structure has previously been solved, the mode of regulation is unknown. To complete the gaps in our understanding, we first solved the structure of S. agalactiae AbiEi to 1.83 Å resolution for comparison with M. tuberculosis Rv2827c. AbiEi contains an N-terminal DNA binding domain and C-terminal antitoxicity domain, with bilateral faces of opposing charge. The overall AbiEi fold is similar to Rv2827c, though smaller, and with a 65° difference in C-terminal domain orientation. We further demonstrate that, like AbiEi, Rv2827c can autoregulate toxin-antitoxin operon expression. In contrast with AbiEi, the Prv2827c promoter contains two sets of inverted repeats, which bind Rv2827c with differing affinities depending on the sequence consensus. Surprisingly, Rv2827c bound with negative co-operativity to the full Prv2827c promoter, demonstrating an unexpectedly complex form of transcriptional regulation.

Graphical abstract, Highlights • The crystal structure of the anti-CRISPR repressor Aca2 has been solved to 1.34 Å. • Aca2 contains a new dimerization domain for HTH transcriptional regulators. • Aca2-like regulators are found encoded in diverse biological contexts., Bacteria use adaptive CRISPR-Cas immune mechanisms to protect from invasion by bacteriophages and other mobile genetic elements. In response, bacteriophages and mobile genetic elements have co-evolved anti-CRISPR proteins to inhibit the bacterial defense. We and others have previously shown that anti-CRISPR associated (Aca) proteins can regulate this anti-CRISPR counter-attack. Here, we report the first structure of an Aca protein, the Aca2 DNA-binding transcriptional autorepressor from Pectobacterium carotovorum bacteriophage ZF40, determined to 1.34 Å. Aca2 presents a conserved N-terminal helix-turn-helix DNA-binding domain and a previously uncharacterized C-terminal dimerization domain. Dimerization positions the Aca2 recognition helices for insertion into the major grooves of target DNA, supporting its role in regulating anti-CRISPRs. Furthermore, database comparisons identified uncharacterized Aca2 structural homologs in pathogenic bacteria, suggesting that Aca2 represents the first characterized member of a more widespread family of transcriptional regulators.