Biochemistry and biology of the Vibrio cholerae parde toxin-antitoxin systems.

摘要

Toxin-antitoxin (TA) systems were originally identified in plasmids where they ensure plasmid maintenance by killing plasmid-free daughter cells. Recently, numerous chromosomal loci exhibiting homology to plasmid-borne TA loci have been identified in diverse bacterial species. However, the biochemical activities and biological roles of chromosome-encoded TA loci are poorly understood. Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, has 13 TA loci, all located in the superintegron on chromosome II. These TA loci include 3 parDE loci that exhibit modest similarity to the plasmid RK2 parDE locus. My thesis work is focused on the activities of these 3 parDE loci and their potential biological role(s). I found that the ParE2 toxin targets a fragment of the A subunit of DNA gyrase (GyrA59), stalls the gyrase DNA-cleavage-union cycle, and causes DNA damage. Unlike other known gyrase inhibitors, ParE2 action requires ATP hydrolysis, and ParE2 only inhibits gyrase-mediated supercoiling and does not block gyrase-mediated relaxation of DNA. Thus, these observations suggest that a particular conformation of the gyrase complex formed during the supercoiling cycle is required for ParE2 to gain access to its binding site(s). Together, my findings reveal that ParE2 works differently than other known gyrase inhibitors.;I examined the activities of the three V. cholerae parDE loci in vivo and found that each of them encodes a functional TA system. Transcription of parDE1 (which has both coding and promoter sequences identical to those of parDE3) and parDE2 are independently regulated by their cognate ParD antitoxins. Furthermore, the ParD proteins only neutralize the toxicity of their cognate ParEs. Expression of ParE1 or ParE2 inV. cholerae led to pronounced DNA damage and stimulated the V. cholerae SOS response. Presumably as a consequence of this cellular response to DNA damage, the ParE toxins blocked cell division. Finally, I found that the 3 ParE toxins act in a redundant fashion to degrade chromosome I in V. cholerae cells that have lost chromosome II. Taken together, my findings suggest that the parDE loci aid in maintenance of the integrity of the V. cholerae superintegron and in ensuring the inheritance of chromosome II.