Constructed Bacillus thuringiensis strains producing...

Details Constructed Bacillus thuringiensis strains producing... Constructed Bacillus thuringiensis strains producing...

1999-08-12

2002-11-19

Bugaisky, Gabriele (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S320100, C435S252300, C435S252500

Reexamination Certificate

active

06482636

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to constructed bacterial strains that produce mosquito-toxic proteins. Compared with the highly mosquito-toxic strain
B.t. israelensis
, the constructed strains have improved characteristics, such as reduced spore number or increased variety of mosquito-toxic proteins. The invention also relates to a method of constructing improved mosquito-toxic strains of bacteria wherein the mosquito-toxin plasmid of
B.t. israelensis
is tagged with an antibiotic-resistance gene, thus permitting the detection of transfer of the mosquito-toxin
B.t. israelensis
plasmid from a donor bacteria strain into a recipient bacteria strain.
BACKGROUND OF THE INVENTION
Bacillus thuringiensis
(
B.t
.) is a gram-positive bacterium that produces proteinaceous crystalline inclusions during sporulation. These
B.t
. crystal proteins are often highly toxic to specific insects. Insecticidal activity has been identified for crystal proteins from various
B.t
. strains against insect larvae from the insect orders Lepidoptera (caterpillars), Diptera (mosquitos, flies) and Coleoptera (beetles).
Individual
B.t
. crystal proteins, also called delta-endotoxins or parasporal crystals or toxin proteins, can differ extensively in their structure and insecticidal activity. These insecticidal proteins are encoded by genes typically located on large plasmids, greater than 30 megadaltons (mDa) in size, that are found in
B.t
. strains. A number of these
B.t
. toxin genes have been cloned and the insecticidal crystal protein products characterized for their specific insecticidal properties. Hofte et al. provides a review of cloned
B.t
. toxin genes and crystal proteins (
Microbiol. Rev
., 1989, 53, 242-255).
The insecticidal properties of
B.t
. have long been recognized and
B.t
. strains have been incorporated into commercial biological insecticide products for over forty years. Commercial
B.t
. insecticide formulations typically contain dried
B.t
. fermentation cultures whose crystal protein is toxic to various insect species.
Traditional commercial
B.t
. bioinsecticide products are derived from “wild-type”
B.t
. strains, i.e., purified cultures of
B.t
. strains isolated from natural sources. Newer commercial
B.t
. bioinsecticide products are based on genetically altered
B.t
. strains, such as the transconjugant
B.t
. strains described in U.S. Pat. No. 5,080,897, issued Jan. 14, 1992, and U.S. Pat. No. 4,935,353, issued Jun. 19, 1990.
Various
B.t
. strains have been classified based on the reaction of the
B.t
. flagella with antibodies. A
B.t
. strain whose flagella react with a unique antibody is classified as a unique serovar, and over thirty different
B.t
. serovars or subspecies have been described (DeBarjac and Frachon,
Entomophaga
, 1990, 35, 233-240). Each
B.t
. subspecies often produces unique types of insecticidal crystal proteins. For example,
B.t
. subspecies
kurstaki
produces crystal proteins of approximatley 130 kilodaltons (kD) and 70 kD in size that are toxic to caterpillars, whereas
B.t
. subspecies
tenebrionis
produces a crystal protein of 72 kD which is toxic to beetles.
B.t
. subspecies
israelensis
(
B.t. israelensis
) is a mosquito-toxic bacterium that produces at least four crystal proteins designated Cry4A, Cry4B, Cry4D and CytA that have been shown to be toxic to mosquito larvae. The
B.t
. crystal toxin gene designations have recently been revised (Crickmore et al.,
Microbiol. Molec. Biol. Rev
., 1998, 62, 807-813).
B.t. israelensis
contains several native plasmids of approximate sizes 3.3, 4.2, 4.9, 10.6, 68, 75, 105 and 135 mDa (Gonzalez and Carlton,
Plasmid
, 1984, 11, 28-38). The genes for the Cry4A, Cry4B, Cry4D and CytA proteins are all carried on a single large plasmid, the mosquito-toxin plasmid, of approximately 75 mDa in
B.t. israelensis
(Gonzalez and
Carlton, Plasmid
, 1984, 11, 28-38). Gene cloning experiments have shown that recombinant non-
B.t. israelensis
bacteria containing cloned
B.t. israelensis
mosquito-toxin genes are generally less toxic to Aedes aegypti mosquitos than wild-type
B.t. israelensis
(Sekar and Carlton,
Gene
, 1985, 33, 151-158; Donovan et al.,
J. Bacteriol
., 1988, 170, 4732-4738; Trisrisook et al.,
Appl. Envir. Microbiol
., 1990, 56, 1710-1716; Bar et al.,
J. Invert. Pathology
, 1991, 57, 149-158; Angsuthanasombat et al.,
FEMS Microbiol. Letts
., 1992, 94, 63-68; Wu et al.,
Molec. Microbiol
., 1994, 13, 965-972).
One explanation for the reduced mosquito toxicity seen with recombinant bacteria containing cloned
B.t. israelensis
mosquito-toxin-genes compared with wild-type
B.t. israelensis
is that all four
B.t. israelensis
crystal protein genes (i.e., cry4A, cry4B, cry4D and cytA) must be present in the same cell for maximum mosquito toxicity. Other factors of
B.t. israelensis
also contribute to its mosquito toxicity. For example,
B.t. israelensis
produces specific sugar residues that are attached to the
B.t. israelensis
crystal proteins (Pfannenstiel et al.,
J. Bacteriol
., 1987, 169, 796-801) and these sugar residues make a significant contribution to the mosquito toxicity of
B.t. israelensis
(Muthukumar and Nickerson,
App. Environ. Microbiol
., 1987, 53, 2650-2655).
It has been found that certain strains of
B.t
. naturally transfer or conjugate their native plasmids to other strains of
B.t
. (Gonzalez and Carlton in “Genetic Exchange,” U. N. Streips, S. H. Goodgal, W. R. Guild and G. A. Wilson, Eds., 1982, p. 85-95, Marcel Dekker, Inc., New York). However, despite the ability of
B.t
. to naturally conjugate plasmids from one strain to another, the transfer of the 75 mDa mosquito-toxin plasmid from
B.t. israelensis
to a non-
B.t. israelensis
strain of
B.t
. has not been reported. Gonzalez and Carlton (
Plasmid
, 1984, 11, 28-38) have reported the lack of ability to transfer
B.t. israelensis
mosquito-toxin plasmid from
B.t. israelensis
to a non-israelensis strain of
B.t
. However, it is possible to transfer the mosquito-toxin plasmid from a donor
B.t. israelensis
strain to a recipient
B.t. israelensis
strain which had previously lost its 75 mDa mosquito-toxin plasmid.
Jensen et al. (
Current Microbiol
., 1996, 33, 228-236) have described an aggregation-mediated conjugation system of
B.t. israelensis
wherein a non-toxin plasmid of approximately 130 mDa, designated plasmid pXO16, was tagged with an antibiotic-resistance marker and the antibiotic-tagged, non-toxin plasmid was transferred into various recipient host cells. It should be emphasized that the antibiotic-tagged plasmid pXO16 described by Jensen et al. is known to transfer readily to non-
B.t. israelensis
strains. It is especially emphasized that the
B.t. israelensis
plasmid pXO16 does not carry mosquito-toxin genes. Although the non-toxin 130 mDa plasmid pXO16 of
B.t. israelensis
conjugates readily to non-
B.t. israelensis
strains, the mosquito-toxin 75 mDa plasmid of
B.t. israelensis
has not been found to conjugate into non-
B.t. israelensis
strains as shown by Gonzalez and Carlton (
Plasmid
, 1984, 11, 28-38).
SUMMARY OF THE INVENTION
The present invention relates to the 75 mDa mosquito-toxin plasmid of
B.t. israelensis
which is tagged with an antibiotic-resistance gene. The antibiotic-tagged plasmid is useful in detection of the rare event in which the mosquito-toxin plasmid transfers from a donor strain of
B.t
. into a recipient strain of
B.t
., the recipient strain being a non-
B.t. israelensis
strain.
The present invention also relates to constructed mosquito-toxic strains of
B.t
. which are non-
B.t. israelensis
strains and which contain the 75 mDa mosquito-toxin plasmid of
B.t. israelensis
. The mosquito-toxic strains are constructed by natural conjugation in which the antibiotic-tagged mosquito-toxin plasmid of
B.t. israelensis
is transferred from a donor strain into a non-israelensis recipient strain of
B.t
. It is within the scope of this invention to use other means, such as electroporation, to introduce the antibiotic-tagged, 75 mDa
B.t. i

Affiliated with

Also associated with

Rating

Constructed Bacillus thuringiensis strains producing... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Constructed Bacillus thuringiensis strains producing..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Constructed Bacillus thuringiensis strains producing... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2993411