Abstract
The largest family of bacterial toxin–antitoxin (TA) modules is formed by the
vapBC operons, and these are grouped together by virtue of their toxin components belonging to the PilT N-terminal domain family of proteins that are thought to function as ribonucleases. We have identified a single
vapBC operon in the genome of
Mycobacterium smegmatis and herein report the molecular and biochemical characterisation of this TA module. In
M. smegmatis, the
vapBC genes are transcribed as a leaderless mRNA that is constitutively synthesised throughout the growth cycle. The
vapBC operon is autoregulated by the VapBC protein complex as demonstrated by a threefold increase in
vapBC expression (
promoter-vapB-lacZ) in a Δ
vapBC mutant. Electrophoretic mobility shift assays using purified VapBC protein complex show that the complex binds to inverted repeat DNA sequences in the
vapBC promoter region that overlap the −
35 and −
10 promoter elements, thus explaining the autoregulation and the low-level constitutive expression of this operon in
M. smegmatis. Neither a Δ
vapBC nor a Δ
vapB mutant strain exhibited any phenotypic deviation to that of the isogenic wild-type parent strain under normal laboratory growth conditions, but conditional overexpression of VapC in
M. smegmatis inhibited growth by a bacteriostatic mechanism and this phenotype is exacerbated in a Δ
vapBC mutant. This effect is mediated through VapC-dependent inhibition of translation, not inhibition of DNA replication or transcription. The growth inhibitory effect of VapC was neutralised when co-expressed with its cognate antitoxin VapB. Western blot analysis revealed the overproduction of VapC under inducing conditions and that the VapC protein is not produced in the Δ
vapB mutant despite the presence of mRNA transcript. Taken together, these data demonstrate that VapBC from
M. smegmatis has all the hallmarks of a TA module with the capacity to cause growth inhibition by regulating translation.