Insecticidal bacteria, and methods for making and using them

Details Insecticidal bacteria, and methods for making and using them Insecticidal bacteria, and methods for making and using them

2000-08-14

2004-04-13

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S252300, C435S071300, C435S471000, C435S252310, C435S252500, C536S024100, C536S023700

Reexamination Certificate

active

06720167

ABSTRACT:

CROSS-REFERENCES TO RELATED APPLICATIONS
Not applicable.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
Despite advances in medical science and new drugs, malaria, filariasis, dengue and the viral encephalilitides remain important diseases of humans, with an estimated 2 billion people worldwide living in areas where these are endemic (
The World Health Report—
1999, World Health Organization, Geneva, Switzerland (1999)). The causative agents of these diseases are transmitted by mosquitoes, and therefore disease control methods have relied heavily on broad spectrum chemical insecticides to reduce mosquito populations. However, chemical insecticide usage is being phased out in many countries due the development of insecticide resistance in mosquito populations. Furthermore, many governments restrict use of these chemicals because of concerns over their effects on the environment, especially on non-target beneficial insects, and vertebrates through contamination of food and water supplies.
As a result of these problems, the World Health Organization is facilitating the replacement of chemical with bacterial-based insecticides through the development of standards for their registration and use (Guideline specifications for bacterial larvicides for public health use, WHO Document WHO/CDS/CPC/WHOPES/99.2, World Health Organization, Geneva, Switzerland (1999)). Products based on bacteria have been designed to control mosquito larvae, and the two most widely used are Vectobac® and Teknar®, both of which are based on
Bacillus thuringiensis
subsp.
israelensis
. In addition, Vectolex®, a new product based on
B. sphaericus
has come to market recently for control of the mosquito vectors of filariasis and viral diseases. These products have achieved moderate commercial success, but their high cost and lower efficacy compared to many chemical pesticides prevents them from being used more extensively in many developing countries. Moreover, concerns have been raised about their long term utility due to resistance, which has already been reported to
B. sphaericus
in field populations of Culex mosquitoes in India, Brazil, and France (Sinègre, et al. First field occurrence of Culex pipiens resistance to
Bacillus sphaericus
in southern France, VII European Meeting, Society for Vector Ecology, 5-8 September Barcelona, Spain (1994); Rao et al.,
J. Am. Mosq. Control Assoc
. 11:1-5 (1995); Silva-Filha et al.,
J. Econ. Entomot
88: 525-530 (1995)).
The insecticidal properties of these bacteria are due primarily to insecticidal proteins produced during sporulation. In
Bacillus thuringiensis
subsp.
israelensis
(Bti), the key proteins are Cyt1A (27 kDa), Cry11A (72 kDa), Cry4A (128 kDa) and Cry4B (134 kDa), whereas
B. sphaericus
(Bs) produces 41- and 52-kDa proteins that serve, respectively, as the toxin and binding domains of a single binary toxin (Federici et al. in
Bacterial Control of Mosquitoes and Blackflies
, eds.: de Barjac & Sutherland, D. J, 11-44 (Rutgers University Press, New Brunswick, N.J.) (1990); Baumann et al.,
Microbiol. Rev
. 55:425-436 (1991)).
Biochemical and toxicological differences in the Bti and Bs toxins suggested that it might be possible to construct an improved bacterium by combining their toxins into a single bacterium. Numerous attempts using this approach have been made over the past decade to create a recombinant bacterium with the desired toxicity. Several groups, for example, have introduced Bti toxin genes in Bs. For example, Bar et al.,
J. Invertebrate Pathol
. 149-158 (1991 cloned Bti endotoxin genes into Bs 2362, but found that the biological activity of the recombinant organism was lower than that of Bti. Poncet et al.,
FEMS Microbiol Lett
. 117:91-96 (1994) cloned the cry4B and cry11A genes of Bti into Bs 2297, and Poncet et al.,
Appl Environ, Microbiol
. 63:4413-4420 (1997) introduced the cry11A gene into the same strain by homologous recombination. Thoéry et al.,
Appl. Environ. Microbiol
. 64:3910-3916 (1998) introduced a Bt cyt1Ab1 gene into Bs, but reported that the level of expression of the cyt1Ab1 gene was probable too low to have any significant effect on toxicity. Servant et al., Appl Environ Microbiol. 65:3021-3026 (1999) introduced Cry11A and Cry11Ba Bt toxins in Bs 2297 by in vivo recombination, and showed that the host range could thereby be increased. Bourgouin et al.,
Appl. Environ Microbiol
. 56:340-344 (1990) introduced Bs toxin into Bti, but found no synergistic or additive effect between the toxins against mosquito larvae. Attempts to combine the advantages of Bs and Bt in other manners have also apparently not proven commercially useful. Simply growing cultures of Bs and Bt and then combining the two organisms, for example, is not effective because the spores of the two organisms are considered to form too large a proportion of the resulting mix in proportion to the weight of the toxins to provide adequate toxicity.
Commercial development of new biopesticides is costly, in part because of EPA regulations requiring extensive testing, and margins are low relative to, for example, pharmaceutical agents. It does not appear that any of the recombinant organisms reported in the past decade have shown sufficient improvement over current commercial Bti or Bs strains to warrant development for commercial use in mosquito control.
SUMMARY OF THE INVENTION
The invention provides nucleotide sequences, expression vectors, host cells and methods for achieving the high level expression of Bs binary toxin, particularly in cells of Bacillus species and especially in Bs and in Bt cells.
In particular, the invention provides nucleic acid sequences comprising, in the following order, A nucleic acid sequence comprising, in the following order, a
B. thuringiensis
promoter selected from the group consisting of a BtI promoter, a BtII promoter, and a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a
B. sphaericus
binary toxin. In some embodiments, the bacterial STAB-SD sequence is selected from the group consisting of GAAAGGAGG (SEQ ID NO:1), GAAGGGGGG (SEQ ID NO:2), GAGGGGGGG (SEQ ID NO:3), GAAAGGGGG (SEQ ID NO:4), GAAAGGAGG (SEQ ID NO:5), and GAAAGGGGT (SEQ ID NO:6). The
B. thuringiensis
promoter is a cry promoter, and in particular can be a cry1 promoter.
Further, the
B. thuringiensis
promoter can be cry1Aa1, cry1Aa2, cry1Aa3, cry1Aa4, cry1Aa5, cry1Aa6, cry1Ba1, cry1Ba2, cry1Ca1, cry1Ca2, cry1Ca3, cry1Ca4, cry1Ca5, cry1Ca6, cry1Ca7 cry1Fa1, cry1Fa2, cyt1Aa1, cyt1Aa2, cyt1Aa3, or cyt1Aa4. In some preferred embodiments, the
B. thuringiensis
promoter is a cyt1Aa1 promoter. The nucleic acid can have both a BtI promoter and a BtII promoter, and the two promoters can be overlapping.
The invention further provides expression vectors comprising the nucleic acid described above, and host cells comprising the expression vectors. The host cells can further comprise a 20 kD protein encoded by the Bti cry11A operon. In preferred embodiments, the host cell is a
B. thuringiensis
cell or a
B. sphaericus
cell.
The invention further provides a nucleic acid sequence comprising, in the following order, a
B. thuringiensis
promoter which binds a sigma factor A protein, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a
B. sphaericus
binary toxin.
The invention also relates to a method of enhancing production of
B. sphaericus
binary toxin in a host bacterial cell, said method comprising: transforming the host cell with a gene comprising, in the following order, a
B. thuringiensis
promoter selected from the group consisting of a BtI promoter, a BtII promoter, and a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a
B. sphaericus
binary toxin; and expressing said gene in the host cell; whereby expression of said g

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