The Yersinia pestis chromosome encodes active addiction toxins

Toxin-antitoxin (TA) loci consist of two genes in an operon, encoding a stable toxin and an unstable antitoxin. The expression of toxin leads to cell growth arrest and sometimes bacterial death, while the antitoxin prevents the cytotoxic activity of the toxin. In this study, we show that the chromosome of Yersinia pestis, the causative agent of plague, carries 10 putative TA modules and two solitary antitoxins that belong to five different TA families (HigBA, HicAB, RelEB, Phd/Doc, and MqsRA). Two of these toxin genes (higB2 and hicA1) could not be cloned in Escherichia coli unless they were coexpressed with their cognate antitoxin gene, indicating that they are highly toxic for this species. One of these toxin genes (higB2) could, however, be cloned directly and expressed in Y. pestis, where it was highly toxic, while the other one (hicA1) could not, probably because of its extreme toxicity. All eight other toxin genes were successfully cloned into the expression vector pBAD-TOPO. For five of them (higB1, higB3, higB5, hicA2, and tox), no toxic activity was detected in either E. coli or Y. pestis despite their overexpression. The three remaining toxin genes (relE1, higB4, and doc) were toxic for E. coli, and this toxic activity was abolished when the cognate antitoxin was coexpressed, showing that these three TA modules are functional in E. coli. Curiously, only one of these three toxins (RelE1) was active in Y. pestis. Cross-interaction between modules of the same family was observed but occurred only when the antitoxins were almost identical. Therefore, our study demonstrates that of the 10 predicted TA modules encoded by the Y. pestis chromosome, at least 5 are functional in E. coli and/or in Y. pestis. This is the first demonstration of active addiction toxins produced by the plague agent.