DOI: 10.1128/mbio.01592-26 ISSN: 2150-7511
Paradigm shift for
cry
gene expression in
Bacillus thuringiensis
Emilie Verplaetse, Alicia Nevers, Leyla Slamti, Didier Lereclus ABSTRACT
In most
Bacillus thuringiensi
s strains, the
cry
genes are transcribed by RNA polymerases containing sporulation-sigma factors E or K, leading to the formation of an insecticidal crystal within the mother cell along spore development. The
kurstaki
HD1 strain, a parent of commercial strains, also releases the insecticidal proteins Cry1I and Vip3A in the extracellular medium.
vip3A
expression is activated by the transcriptional regulator VipR at the onset of the stationary phase. Here, we expanded the VipR regulon in strain HD1 by identifying the VipR-binding box upstream from the
cry2Aa
,
cry2Ab
, and
cry1Ia
genes, and conducting transcription assays. Unexpectedly, a VipR box was located in the promoter of a putative N-acetylmuramoyl-
l
-alanine amidase (
ami
) gene upstream from
cry1Ac
in strain
kurstaki
HD73, closely related to the HD1 but devoid of
vipR
. Introduction of
vipR
in this strain led to the expression of the
ami-cry1Ac
operon, resulting in an early and increased production of Cry1Ac. We demonstrated that Cry1Ac was also produced in a VipR-dependent manner in an HD73 ∆
spo0A
mutant. Similarly, an HD1 ∆
spo0A
strain produces all the insecticidal proteins encoded in its genome, including
cry2Ab
, previously considered unexpressed. A genomic analysis also revealed the presence of putative VipR-binding sequences in lepidopteran-active strains, upstream from
cry
genes such as
cry1E
,
cry1F
,
cry9D
, and
cry9E
. Overall, our results break the dogma on the regulation of
cry1A
and
cry2A
genes and provide evidence of sporulation-independent Cry toxin production in biopesticidal Bt strains.
IMPORTANCE
Bacillus thuringiensis
is a remarkably efficient entomopathogen due to its ability to produce various insecticidal proteins, such as Cry or Vip. This property has made it a highly effective biopesticide used worldwide. Our work modifies the paradigm of
cry1
and
cry2
genes being regulated solely by sporulation-specific sigma factors and thus exclusively expressed during this process. Indeed, we demonstrated that the VipR regulator controls the transcription of
vip3Aa
,
cry2Aa
,
cry2Ab
,
cry1Ia
, and the
ami-cry1A
operons encoded by a strain closely related to that of commercial biopesticides and specifically turns on their expression from the onset of the stationary phase, leading to the production of insecticidal crystals independently of sporulation. By providing new knowledge on the regulation of insecticidal protein genes, these findings bring new insight for the genetic improvement of Bt strains used as commercial biopesticides.