The vapBC Operon from Mycobacterium smegmatis Is
An Autoregulated Toxin–Antitoxin Module That Controls
Growth via Inhibition of Translation
Jennifer Robson
1
, Joanna L. McKenzie
2
, Ray Cursons
2
,
Gregory M. Cook
1
and Vickery L. Arcus
2
⁎
1
Department of Microbiology
and Immunology, Otago School
of Medical Sciences, University
of Otago, P.O. Box 56, Dunedin
9054, New Zealand
2
Department of Biological
Sciences, University of Waikato,
Private Bag 3105, Hamilton
3240, New Zealand
Received 4 February 2009;
received in revised form
4 May 2009;
accepted 7 May 2009
Available online
13 May 2009
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)inaΔvapBC
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 ΔvapBC nor a ΔvapB
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 ΔvapBC
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 ΔvapB 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.
© 2009 Elsevier Ltd. All rights reserved.
Edited by J. Karn
Keywords: toxin–antitoxin; translation; RNase; mycobacteria; growth
regulation
Introduction
Bacterial toxin–antitoxin (TA) modules are gener-
ally bicistronic operons whose protein products form
a tight heterodimeric protein complex.
1–4
One
member of the protein complex is toxic, and its
cognate antitoxin acts as an efficient inhibitor. These
TA operons are abundant and can be found in nearly
every sequenced bacterial and archaeal genome.
1,5
The toxic components of these operons can be
divided into seven families,
3
and in the case of four
⁎Corresponding author. E-mail address:
varcus@waikato.ac.nz.
Abbreviations used: TA, toxin–antitoxin; PIN domain,
PilT N-terminal domain; RT, reverse transcriptase; IR,
inverted repeat; PvapB-lacZ, promoter-vapB-lacZ; DLS,
dynamic light scattering; EMSA, electrophoretic mobility
shift assay; DIG, digoxigenin-11-ddUTP; LF, left flank; RF,
right flank.
doi:10.1016/j.jmb.2009.05.006 J. Mol. Biol. (2009) 390, 353–367
Available online at www.sciencedirect.com
0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.