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governments have restricted the usage of chemi-
cal insecticides due to their negative environmen-
tal effects. As a result, the use of biopesticides, as
a component of an integrated pest management
(IPM), has been gaining worldwide acceptance.
Bacillus thuringiensis (Bt) has been used as a
biopesticide. Its advantages are specific toxic-
ity against target insects, non-polluting residues
and safety to non-target organisms. Bt produces
crystals, composed of protein/s known as insecti-
cidal crystal protein/s (ICPs) during sporulation,
which are selectively toxic to different species
of several invertebrate phyla. In addition, some
Bt strains secrete vegetative insecticidal proteins
(VIPs) during vegetative growth that are toxic to
many lepidopteran pests, causing lysis of midgut
epithelial cells and gut paralysis. Some Bt strains
also secrete proteases, chitinases, and other viru-
lence factors conferring insecticidal activity.
Genetic improvement of Bt strains for the
development of novel biopesticides entails in-
creasing their potency against target insects,
broadening the insecticidal spectra for specific
crop applications, improving persistence on
plants and optimizing fermentation production
and the most important of all meeting the resis-
tance development needs. This review focuses
on the developments of genetic manipulation for
improving Bt strains.
Genetic Manipulation of BT
Transduction
The first genetic exchange system available in
Bt was generalized transduction mediated by the
Bacillus cereus Frankland and Frankland phages
CP-51 or CP-54. This system allows the trans-
fer of chromosomal markers. Since then, various
phage-based systems for mapping chromosomal
genes have been developed for several groups.
The generalized transducing phage CP-55, ob-
tained by Thorne’s laboratory, was able to realize
inter-varietal transduction between Bt subspecies
galleriae and israelensis. TP-13, a broad host-
range temperate phage, which infected 18 of 19
tested Bt subspecies, is useful in scanning large
segments of chromosome. TP-18, a narrower
host-range phage, which infected 9 of 21 Bt sub-
species and had a head size seven times smaller
than that of TP-13, was effective for ordering
markers that were too closely linked. CP-51
could also be used to transduce plasmid mole-
cules such as pBC16 and pC194 into strains of B.
cereus, B. anthracis and Bt, at frequencies as high
as 10−5. Transduction has allowed the transfer of
recombinant plasmids carrying crystal protein
genes between Bt strains at detectable frequen-
cies. This is of particular value for biotechnologi-
cal applications because introduction of cloned
crystal protein genes into various Bt subspecies,
including both Cry− and Cry+ strains can expand
the insecticidal spectrum of each strain, allowing
native strains to have better insecticidal activity
against more insect species. Transduction is an
efficient means of gene transfer yielding high
levels of gene expression, but not all strains are
amenable to phage transduction. Furthermore,
the stability of the introduced plasmids depends
mainly on the host.Conjugation
The second important advance in genetic ex-
change is the discovery of a conjugation process
where the donor and the recipient strains are
grown together for a few generations in nutrient
broth, and the mixture is plated onto an appro-
priate medium that is selective for the recipient
cells. The transcipient cells are scored by analyz-
ing the plasmid profiles of the resulting recipient
colonies by electrophoretic analysis. Using the
cell-mating method, both large and small plas-
mids were found to transfer equally well between
strains Bt, B. cereus and B. anthracis. Subse-
quently, there were many reports on the use of
this conjugation-like process for transferring spe-
cific plasmids in Bt. A recombinant plasmid from
B. subtilis (containing a cloned Bt toxin gene)
transferred readily into Bt subsp. kurstaki, israel-
ensis and sotto. This mating system can also be
used for Bt and other gram-positive species. Plas-
mid pC194 from Bt strain 0016 and pBC16 from
B. cereus could be transferred to Bt subsp. israel-
ensis. Plasmid pBC16 transfers from B. subtilis
to B. anthracis, B. cereus, B. licheniformis, B.
megaterium, B. pumilus, B. subtilis, and Bt using
the conjugation-like process. In soil microcosm,
the conjugation-like process has been poorly