Affiliation:
1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
2. Department of Biology, Northeastern University, Boston, Massachusetts, USA
Abstract
ABSTRACT
In this study, a sporulation-specific gene (tentatively named
cwlC
) involved in mother cell lysis in
Bacillus thuringiensis
was characterized. The encoded CwlC protein consists of an N-terminal
N
-acetylmuramoyl-
l
-alanine amidase (Mur
N
Ac-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified from
Escherichia coli
were able to directly bind to and digest the
B. thuringiensis
cell wall. The CwlC point mutations at the two conserved glutamic acid residues (Glu-24 and Glu-140) shown to be critical for the catalytic activity in homologous amidases resulted in a complete loss of cell wall lytic activity, suggesting that CwlC is an
N
-acetylmuramoyl-
l
-alanine amidase. Results of transcriptional analyses indicated that
cwlC
is transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σ
K
). Deletion of
cwlC
completely blocked mother cell lysis during sporulation without impacting the sporulation frequency, Cry1Ac protein production, and insecticidal activity. Taken together, our data suggest that CwlC is an essential cell wall hydrolase for
B. thuringiensis
mother cell lysis during sporulation. Engineered
B. thuringiensis
strains targeting
cwlC
, which allows the crystal inclusion to remain encapsulated in the mother cell at the end of sporulation, may have the potential to become more effective biological control agents in agricultural applications since the crystal inclusion remains encapsulated in the mother cell at the end of sporulation.
IMPORTANCE
Mother cell lysis has been well studied in
Bacillus subtilis
, which involves three distinct yet functionally complementary cell wall hydrolases. In this study, a novel cell wall hydrolase, CwlC, was investigated and found to be essential for mother cell lysis in
Bacillus thuringiensis
. CwlC of
B. thuringiensis
only shows 9 and 21% sequence identity with known
B. subtilis
mother cell hydrolases CwlB and CwlC, respectively, suggesting that mechanisms of mother cell lysis may differ between
B. subtilis
and
B. thuringiensis
. The
cwlC
gene deletion completely blocked the release of spores and crystals from the mother cell without affecting insecticidal activity. This may provide a new effective strategy for crystal encapsulation against UV light inactivation.
Funder
The National Key Research and Development Program of China
National Natural Science Foundation of China
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
21 articles.
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