Toxin-antitoxin systems consist of toxin and antitoxin gene pairs. Toxins affect cell metabolism, antitoxins regulate and neutralize toxin in normal growing conditions. Their most widely accepted function is stabilisation of gene elements, other proposed functions include altruistic cell death, role in persister cell formation and protection from bacteriophages. Toxin-antitoxin module higBA2 from Vibrio cholerae is characterised by regulation with negative cooperativity caused by HigA2 antitoxin’s N-terminal intrinsically disordered domain’s ability to exclusively bind to either toxin HigB2 (blocking toxin’s endonuclease activity) or contribute to antitoxins affinity for operator region (increasing repression of the higBA2 module). In order to analyse higBA2 module’s regulation, we have produced vector constructs in E. coli in which RFP expression is regulated by higBA2 module’s operator, toxin HigB2 and antitoxin HigA2 are expressed under constitutive promoters with different activities and are consequently present in different molar ratios. By measuring the fluorescence, we confirmed antitoxin HigA2 is a strong repressor of higBA2 operator, its N-terminal intrinsically disordered domain cooperates in antitoxin’s binding to DNA, without it antitoxin HigA2 binds to higBA2 operator region more weakly. At an overabundance of antitoxin HigA2 the repression of higBA2 module is stronger, overabundance of toxin HigB2, contrary to our hypothesis, does not lower higBA2 module’s repression, but rather increases it. At low antitoxin concentrations enhanced activity of higBA2 module was observed. The presence of wild type higBA2 module in E. coli did not affect persister cell formation, the module is therefore not a suitable target for the development of drugs combating V. cholerae presistence.
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