WO1995015383A2 - Agents de lutte biologique - Google Patents

Agents de lutte biologique Download PDF

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Publication number
WO1995015383A2
WO1995015383A2 PCT/GB1994/002628 GB9402628W WO9515383A2 WO 1995015383 A2 WO1995015383 A2 WO 1995015383A2 GB 9402628 W GB9402628 W GB 9402628W WO 9515383 A2 WO9515383 A2 WO 9515383A2
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asn
sphaericus
leu
mosquitocidal
bacterium
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PCT/GB1994/002628
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WO1995015383A3 (fr
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Thirumaran Thanabalu
Alan George Porter
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National University Of Singapore
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Priority to GB9503697A priority Critical patent/GB2289049A/en
Priority to AU11144/95A priority patent/AU1114495A/en
Publication of WO1995015383A2 publication Critical patent/WO1995015383A2/fr
Publication of WO1995015383A3 publication Critical patent/WO1995015383A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins

Definitions

  • the present invention relates to bacteria capable of exhibiting toxicity to mosquito larvae and the use of such bacteria as biological control agents to kill mosquito larvae.
  • C Culex
  • Aedes Ae.
  • Anopheles Anopheles
  • high toxicity strains of B.sphaericus and B.thurin ⁇ iensis subsp. israelensis which have good toxicity for representative species of the Culex genus and/or Aedes genus.
  • neither known high toxicity strains of B.sphaericus nor toxic strains of B.thurin ⁇ iensis subsp. israelensis are satisfactorily toxic against a wide spectrum of Culex, Aedes and Anopheles mosquitoes.
  • no toxic strain of a Bacillus species is known to be highly effective against Anopheles mosquito larvae.
  • No insecticidal bacterium has been tested in the field and shown to be more effective against Anopheles mosquitoes than against Culex or Aedes mosquitoes.
  • B.sphaericus is an aerobic, gram positive, spore forming bacterium which is widespread in soil and aquatic environments.
  • the strains of B.sphaericus have been divided into 5 groups (groups I to V) based on DNA homology, and group II is further subdivided into groups IIA and IIB (Krych et al. Int. J. Syst. Bacteriol. (1980)10.,476-484) .
  • group IIA and IIB Korean-chain bacterium
  • the vast majority of the toxic strains of B.sphaericus have been found to be in DNA homology group IIA.
  • the toxic strains have been further subdivided into the high toxicity strains (e.g. 2362, 2297 and IAB59) and the low toxicity strains (e.g.
  • the coding sequence of the mtx gene corresponding to the full length protein starts at nucleotide (nt) 1207 and ends at nt 3817. All nucleotide and amino acid numbering as used hereinafter corresponds to that of Figure 7.
  • the putative ribosome binding site is located between nt 1188 and nt 1198.
  • the putative promoter sequences -10 TATAAC (nt 1122 to nt 1127) and -35 TTCACA (nt 1092 to nt 1097) show homology to the consensus sequences for the ⁇ 55 vegetative promoter of B.subtilis (-10 TATAAA and -35 TTGACA) .
  • a G+C rich inverted repeat sequence capable of forming a hairpin loop structure with a free energy of -27.6 kcal is found between nt 3905 and nt 3940. This repeat sequence is followed by a T-rich sequence and thus has the characteristics of a transcriptional termination signal.
  • the mtx gene has been found to be widely distributed in both low toxicity and high toxicity strains of B.sphaericus tested.
  • the corresponding protein of 870 amino acids has no significant homology to the mosquitocidal toxins of B.thurin ⁇ iensis subsp. israelensis or to the binary toxin of B.sphaericus. Regions have, however, been found to have significant amino acid sequence homology with the catalytic subunits of ADP- ribosyltransferase toxins (Thanabalu et al. J. Bacteriol. (1991) 172(9), 2776-2785).
  • Amino acids 95 to 148 resemble sequences important for toxicity in the SI subunit of pertussis toxin and the A subunits of cholera toxin and E.coli heat labile toxin, which exert their action by ADP-ribosylation of cellular proteins.
  • the amino acid sequence Glu-X-X-X-X-Trp at amino acid positions 219 to 224 is also a characteristic of known ADP-ribosylating proteins. Such a sequence is present in exotoxin A of P.aeru ⁇ inosa and the A subunit of diphtheria toxin.
  • the high toxicity strains of B.sphaericus show maximal toxicity at the sporulation phase of growth.
  • the toxicity of B.sphaericus SSII-1 is not sporulation dependent, although very much lower.
  • the relatively low mosquitocidal toxicity of B.sphaericus SSII-1 is not due to low toxicity of the mtx gene toxin per se. This is clear from the previously reported finding that the 97kDa mtx toxin has an LC50 value of 15 ng/ml against C. ⁇ uin ⁇ uefasciatus larvae (Thanabalu et al. J. Bacteriol. (1992) 174, 5051-5056) .
  • B.sphaericus SSII-1 This is the same order of toxicity as exhibited by the binary mosquitocidal toxin from B.sphaericus group IIA strains. Low toxin stability has previously been suggested as a factor influencing observed expression of the mtx gene in bacterial hosts. It is known that the toxicity of B.sphaericus SSII-1 is sensitive to heat and can also be decreased by refrigeration, a freeze- thaw cycle and two methods of cell disruption (Myers and Yousten Infect. Immun. (1978) 19(3), 1047-1053, Myers et al. Can. J. Microbiol. (1979) 15, 1227-1231) .
  • the inventors for the present application have now found that high levels of expression of mosquitocidal toxins encoded by the B.sphaericus mtx gene may be obtained by transferring the mtx gene into normally non-toxic B.sphaericus strains of DNA homology groups III and IV, thereby providing recombinant B.sphaericus bacteria having good toxicity for mosquitoes of the Culex, Aedes. and Anopheles genera during both the vegetative and sporulation phases.
  • B.sphaericus strains of DNA homology groups III and IV lack extracelluar proteases that degrade the 97 kDa mtx toxin. Additionally, cell extracts of such bacteria have now been shown to have little or no intracellular protease activity capable of degrading the 97 kDa mtx toxin.
  • B.sphaericus strains of other groups do exhibit extracellular protease activity, and in some cases significant intracellular protease activity, against the same toxin.
  • B.sphaericus 2362 (a high toxicity group IIA B.sphaericus strain) has previously been reported to produce at least two extracellular proteases (Broadwell and Baumann Appl. Environ. Microbiol. (1986) 52., 758-764) .
  • the present invention provides a bacterium which contains a DNA coding sequence for a mosquitocidal toxin encoded by the B.sphaericus mtx gene or a modified mosquitocidal form thereof and which is capable of expressing the said toxin or modified mosquitocidal form thereof, said bacterium substantially lacking extracellular or intracellular proteases capable of degrading the said toxin or modified mosquitocidal form thereof.
  • a bacterium of the present invention may have a DNA coding sequence for the complete lOOkDa mtx toxin or a modified mosquitocidal form thereof.
  • a bacterium of the present invention may have a gene for the lOOkDa mtx toxin minus the putative signal peptide, ie. the 97kDa mtx toxin, or a modified mosquitocidal form thereof.
  • a modified mosquitocidal form of a mosquitocidal toxin as encoded by the B.sphaericus mtx gene may have one or more amino acid substitutions, deletions or insertions and/or an extension at either or each end so that ability to confer mosquitocidal toxicity on bacterial host cells is retained. There may be a degree of homology of at least 80%, for example at least 90% to 95%, preferably at least 98% or 99%, between the modified mosquitocidal form of the toxin and the natural toxin. Where a modified mosquitocidal form of a mtx gene encoded toxin incorporates amino acid substitutions, these may preferably be conservative substitutions, although other types of substitution may be incorporated which do not destroy toxin function.
  • amino acid changes either substitutions, deletions or insertions, may be present which result in retention of toxicity.
  • one or more amino acid changes may be present which preserve the physicochemical character of the toxin, ie. in terms of charge density, hydrophilicity/hydrophobicity and configuration, and hence preserve mosquitocidal toxicity.
  • a hydrophobic amino acid may be replaced by an alternative hydrophobic amino acid, e.g. Trp(w) may be substituted by Phe(F) or vice versa.
  • Ala(A) may be replaced by Val (V) or vice versa; Val (V) by Leu(L), Lys(K) by Arg(R), Asp(D) by Glu(E) or vice versa.
  • Val (V) by Leu(L), Lys(K) by Arg(R), Asp(D) by Glu(E) or vice versa.
  • a bacterium of the present invention may, for example, have a gene for a variant of the lOOkDa mtx toxin in which the natural signal sequence of this protein has been replaced by an heterologous signal sequence.
  • Derivatives of the lOOkDa mtx toxin which retain mosquitocidal toxicity when expressed in a bacterial host may be readily determined by appropriate assay for such toxicity (see Examples 2 and 3) .
  • modified mosquitocidal forms of a mtx gene encoded toxin include toxic variants having an N-terminal or C-terminal extension. Such an extension may be a few amino acids, e.g. 1 to 5 amino acids, but far longer extensions are possible, e.g.
  • a mtx gene encoded toxin may, for example, be fused at the N-terminus to a non mtx-gene encoded polypeptide as part of a fusion protein in which the mtx gene encoded toxin retains mosquitocidal toxicity.
  • fusion proteins are described, for example, in Thanabalu et al. J. Bacteriol (1992) 174, 5051-5056.
  • a bacterium of the present invention may preferably encode the complete 100 kDa B.sphaericus mtx toxin or a modified mosquitocidal form of this protein which has an N-terminal signal sequence.
  • protease deficiency of a bacterium for the purpose of the present invention may be determined by assay of protease activity in bacterial culture supernatants and bacterial cell extracts against an appropriate purified toxin protein using SDS-PAGE electrophoresis.
  • a bacterium of the present invention produces the 100 kDa B.sphaericus mtx toxin, it will be understood to equate with substantial lack of protease activity either in culture supernatant of the bacterium or cell extracts against the 97 kDa mtx toxin.
  • a bacterium of the present invention is preferably derived from a wild-type bacterium capable of surviving in an aquatic environment which has appropriate deficiency of protease activity compared with natural hosts of the mtx gene such as B.sphaericus SSII-1. As indicated above, this criterion is met by B.sphaericus strains of DNA homology groups III and IV.
  • the present invention provides a recombinant bacterium derived from a B.sphaericus strain selected from B.sphaericus strains of DNA homology groups III and IV which contains a DNA coding sequence for a mosquitocidal toxin encoded by the B.sphaericus mtx gene or a modified mosquitocidal form thereof and which is capable of expressing the said toxin or modified mosquitocidal form thereof so as to exhibit mosquitocidal toxicity.
  • B.sphaericus strains of DNA homology groups III and IV have now been found to have sensitivity to chloramphenicol. This renders them favourable for transformation to obtain bacteria according to the present invention using a chloramphenicol antibiotic resistance gene as a marker for selection of successful transformants.
  • B.sphaericus strain 1693 of DNA homology group IV and B.sphaericus strain 4525 of DNA homology III have been found to have particularly good sensitivity to chloramphenicol.
  • B.sphaericus strain 1693 this has been found to enable particularly convenient transformation to a bacterium of the present invention.
  • the present invention provides a recombinant B.sphaericus bacterium as defined above which is derived from a B.sphaericus strain selected from B.sphaericus strains 1693 and 4525, most preferably B.sphaericus 1693.
  • a bacterium of the present invention may contain an mtx gene obtainable from a natural host for this gene such as B.sphaericus SSII-1.
  • variants of this gene may be employed.
  • the DNA coding sequence for the 100 kDa mtx protein may be substituted by an alternative sequence coding for the same protein, i.e. a degenerate variant sequence, or by a sequence coding for a modified mosquitocidal form of the lOOkDa mtx toxin.
  • the DNA coding sequence may be under the control of a promoter other than the mtx gene promoter.
  • This may be a heterologous promoter which is sporulation dependent or allows expression of the 100 kDa mtx toxin or a modified mosquitocidal form thereof throughout both vegetative and sporulation phases of the bacterial host.
  • the coding sequence for the toxin of interest may be operably linked to a promoter from a bacterial gene other than the mtx gene, e.g. the SpoVG promoter (Molecular Cloning and Gene Regulation in Bacilli (1982) ed. A.T. Ganesan, p.
  • the mtx gene promoter has resemblance to a B.subtilis vegetative promoter and has previously been categorised as a vegetative promoter, it has now been established that it can direct good expression of the mtx gene coding sequence when present in a sporulating bacterium of the present invention during either the vegetative phase or the sporulation phase. Indeed, in recombinant B.sphaericus 1693 carrying the mtx gene high expression of the mtx gene coding sequence has been unexpectedly observed in sporulated cells.
  • mtx gene promoter in a bacterium of the present invention to direct expression of a mtx gene encoded toxin or a modified mosquitocidal form thereof is highly favourable from the point of view of obtaining a good time course of expression throughout vegetative and sporulation phases with optimum expression in sporulated cells.
  • the present invention provides a bacterium according to the invention in which the DNA coding sequence for the mtx gene encoded toxin or a modified mosquitocidal form thereof is operably-linked to the B.sphaericus mtx gene promoter or a functional variant of this promoter.
  • a bacterium may advantageously contain the complete B.sphaericus mtx gene.
  • the invention provides a bacterium according to the invention wherein the DNA coding sequence of the B.sphaericus mtx gene, or a degenerate variant thereof, is operably-linked to a heterologous promoter.
  • the present invention provides a method of preparing a bacterium of the present invention which comprises transforming an appropriate bacterium with a DNA containing a gene coding for a mosquitocidal toxin encoded by the B.sphaericus mtx gene or a modified mosquitocidal form thereof such that the said toxin or modified mosquitocidal form thereof can be expressed by the transformed bacterium and is capable of conferring on the bacterium mosquitocidal toxicity.
  • Transformation in such a method may be achieved in conventional manner.
  • an expression vector carrying an appropriate gene e.g. a gene for the 100 kDa B.sphaericus mtx protein or an appropriate modified mosquitocidal form thereof, may be introduced into a chosen bacterial host, e.g. by electroporation.
  • An expression vector for use in a transformation method of the present invention will contain an appropriate origin of replication sequence for replication of the vector and the appropriate sequences for expression of the coding sequence for the mtx gene encoded toxin or modified mosquitocidal form thereof. These sequences will include a transcription promoter operably- linked to the mosquitocidal toxin coding sequence and a transcription termination site.
  • Such an expression vector may conveniently include a natural coding sequence for the 100 kDa B.sphaericus mtx protein, most conveniently the complete mtx gene as present, for example, in the DNA of
  • an expression vector for use in a method of preparing a recombinant bacterium of the present invention will include a marker gene, e.g. an antibiotic resistance gene, which enables transformed bacterial cells to be readily selected.
  • PHV1431 is a 10.865 kb vector previously designed as a shuttle vector for E.coli and B.subtilis. This vector is a hybrid of PBR322 and pAMBl, the origin of replication being derived from pAMBl.
  • PHV1431 can also be used to transform B.sphaericus strains belonging to DNA homology group IV using the chloramphenicol resistance gene for selection of transformants. Electroporation is, for example, a suitable means for introducing the vector into the bacterial cells.
  • a gene having a coding sequence for a mtx gene encoded toxin or a modified mosquitocidal form thereof may be conveniently introduced at the EcoRV restriction site of the PHV1431 vector so resulting in insertional inactivation of the tetracycline marker gene and retention of the chloramphenicol marker gene, although an alternative restriction site may be used, e.g. BamHI, SphI, Sail or Nrul (see Figure 4) .
  • a suitable expression vector for obtaining a recombinant B.sphaericus bacterium of the present invention belonging to DNA homology group IV may, for example, be particularly conveniently obtained by inserting into the EcoRV site of PHV1431 an appropriate DNA fragment containing the natural mtx gene.
  • Recombinant bacteria of the present invention which are a B.sphaericus strain of DNA homology group IV and have been transformed by an expression vector derived from PHV1431 carrying a gene for a mtx gene encoded toxin or a modified mosquitocidal form thereof inserted at the EcoRV site represent a particularly preferred embodiment of the present invention.
  • recombinant bacteria derived from B.sphaericus strain 1693 e.g. recombinant B.sphaericus 1693 transformed by PHV1431 containing the mtx gene.
  • transformation in a method of the present invention for preparing a recombinant bacterium may be carried out so that homologous recombination occurs resulting in a gene for a mosquitocidal toxin encoded by the B.sphaericus mtx gene or a modified mosquitocidal form thereof being integrated into the chromosomal DNA of the parent bacterium.
  • the DNA carrying the sequence to be introduced into the parent bacterium may be in the form of a plasmid or a linear fragment.
  • bacteria of the present invention provide an important addition to the armoury against mosquito larvae. It has previously been appreciated that the mtx gene confers on B.sphaericus SSII-1 low toxicity for representative species of the Aedes and Culex mosquito genera. The unexpected discovery by the inventors for the present application that the mtx gene can confer on alternative bacterial hosts good toxicity for Anopheles mosquitoes, in addition to good toxicity against both Aedes and Culex mosquitoes, has particular importance in enabling provision of new bioinsecticides to combat Anopheles mosquitoes, which transmit a number of disease parasites including malarial parasites.
  • the present invention provides use of a mosquitocidal toxin encoded by the B.sphaericus mtx gene or a modified mosquitocidal form thereof as a biological control agent for mosquitoes, e.g. Culex. Aedes, Mansonia and Haemagogus mosquitoes, most especially Anopheles mosquitoes.
  • the 97 kDa mtx toxin has been shown to exhibit, for example, particularly favourable toxicity against Anopheles albimanus.
  • a bacterium of the present invention may be used directly as a biological control agent.
  • a bacterium of the present invention may be formulated in a composition.
  • the present invention provides an insecticidal composition
  • a bacterium of the present invention may be present as vegetative cells and/or sporulated cells depending upon the promoter chosen for expression of the mtx gene encoded toxin or modified mosquitocidal form thereof.
  • a composition comprises, for example, a bacterium according to the present invention in which the DNA coding sequence for the mtx gene encoded toxin or modified mosquitocidal form thereof is operably-linked to the mtx gene promoter, e.g.
  • a bacterium may be present as vegetative cells or sporulated cells.
  • a bacterium may be employed principally or totally in the sporulation phase.
  • a composition of the present invention is suitable for application to a water surface.
  • the invention also provides a method of controlling mosquitoes which comprises applying at a recognised or potential feeding locus for mosquito larvae an effective amount of a bacterium of the present invention, preferably in the form of a composition.
  • the selected locus for such a method may be a wetland or an alternative site providing a suitable water surface to support mosquito larvae feeding and breeding. Bacteria so applied which leave the water surface region are not available for ingestion by mosquito larvae which feed only at the water surface, e.g. Anopheles larvae.
  • compositions of the present invention are preferably formulated so as to delay settlement of the toxin-producing bacteria.
  • a composition of the present invention may, for example, be in the form of a powder.
  • compositions of the present invention are, however, floating formulations, e.g. slow release floating pellets. Methods of preparing such floating formulations have previously been described for mosquitocidal toxin producing bacteria (Lacey et al. Mosquito News (1984) 44. 26-32, Aly et al. J.Am Mosquito Control Assoc. (1987) 3., 583-588; Cheung et al. Appl. Environ. Microbiol. (1985) 50, 984-988; Margalit et al. Appl. Microbiol. Biotechnol. (1984) 19, 382-383) . To reduce loss of cell viability due to U.V. radiation, floating formulations of the present invention will preferably include a U.V. screening agent.
  • compositions of the present invention may be formulated so as to exhibit in a conventional laboratory test for mosquito larvae toxicity (see, for example, Example 3) an LC50 of at least approx 10 1 - 10 3 cells per ml, preferably at least approx 10 2 - 10 3 cells per ml.
  • a composition of the present invention may, for example, be formulated so as to exert effective control of mosquito larvae in the field at a dose in the range 0.05 - 0.5 kg/hectare, preferably 0.1 - 0.2 kg/hectare.
  • Figure 1 shows the restriction map of the 4,133 bp Sau3AI-PstI DNA fragment sequenced by Thanabalu et al. containing the mtx gene as present in B.sphaericus SSII-1 (J.Bacteriol. (1991) 173 (9), 2776-2785).
  • the shaded boxes represent the mtx gene and the 3' end of another open reading frame (ORF) . Arrows show gene orientation.
  • the numbers represent nucleotide numbers beginning at the first nucleotide of the Sau3AI site.
  • the letters below the shaded boxes indicate the determined restriction enzyme sites:
  • Figure 2a Lane 1:9062 (gpI),Lane 2:10208 (gpl) , Lane 3:14577 (gpl), Lane 4:SSII-1 (gpIIA) , Lane 5:2173 (gpIIA) , Lane 6:2315 (gpIIA) , Lane 7:2362 (gpIIA) , Lane 8:7054 (gpIIB) , Lane 9:7055 (gpIIB) .
  • Figure 2b Lane 1:9062 (gpI),Lane 2:10208 (gpl) , Lane 3:14577 (gpl), Lane 4:SSII-1 (gpIIA) , Lane 5:2173 (gpIIA) , Lane 6:2315 (gpIIA) , Lane 7:2362 (gpIIA) , Lane 8:7054 (gpIIB) , Lane 9:7055 (gpIIB) .
  • Figure 2b Lane 1:9062
  • Lane 3 in the same Figure corresponds to 3.subtilis DB104.
  • Figures 3 (a) , (b) and (c) show the results of SDS-PAGE analysis of intracellular protease activity against the 97 kDa mtx protein in cell extracts from various B.sphaericus strains and B.subtilis DB104.
  • the gel lanes correspond to B.sphaericus strains as follows: Figure 3a.
  • Figure 3b Lane 1:12300 (gpIIB) , Lane 2:PI (gpIII) , Lane 3:592 (gpIII) , Lane 4:4525 (gpIII), Lane 5:400 (gpIV) , Lane 6:1693 (gpIV) , Lane 7:13805 (gpIV) , Lane 8:1198 (gpV) , Lane 9:1199 (gpV) .
  • Lane 3c Lane 2:13502 (gpIIA) .
  • Lane 1 in Figure 3c corresponds to untreated 97 kDa toxin. Lane 3 in the same Figure corresponds to B.subtilis DB104.
  • Figure 4 is a restriction map of plasmid
  • PHV1431. The unique restriction sites are marked.
  • Cm chloramphenicol resistance marker.
  • Tet tetracycline resistance marker.
  • Amp ampicillin resistance marker.
  • Figure 5 is a restriction map of plasmid pXP33 obtainable by inserting the 3.8kb PstI fragment of the DNA shown in Figure 1 into the PstI site in pUC18.
  • the production of the 100 kDa mtx protein can also be driven by the lac Z promoter (LP) .
  • Figure 6 shows the restriction map of plasmid pC35. This is obtainable by inserting the 3.25 kb Clal fragment from pXP33, after the cohesive ends have been converted to blunt ends, into the EcoRV site of PHV1431. X denotes the EcoRV/Clal sites.
  • Figure 7 shows the nucleotide sequence of the DNA fragment illustrated in Figure 1.
  • the deduced amino acid sequence is shown below the nucleotide sequence.
  • Lines above the nucleotide sequence denote inverted repeat, single underlining indicates putative promoter sequences and double underlining indicates the putative ribosome- binding site.
  • B.sphaericus 2362 was previously reported to produce at least two extracellular proteases maximally at 28 hours in NYSM (Broadwell and Baumann, Appl. Environ. Microbiol. (1986) J52., 758-764).
  • B.sphaericus strains from each of the 6 different DNA homology groups (I, IIA,IIB,III,IV and V) were grown at 30oC in 6 ml of NYSM.
  • B.subtilis DB104 a non- toxic mutant B.subtilis strain deficient in 2 of at least 5 extracellular proteases (Kawamura and Doi, J. Bacteriol. (1984) , 160, 442 444) , was also grown under the same conditions. After 28 hours of incubation, 1 ml of cell suspension was removed from each culture, the cells were harvested and the protein concentration of the culture supernatant was determined using the BIO-RAD protein assay kit.
  • the 97 kDa mtx protein (the 100 kDa B.sphaericus protein minus the putative signal peptide) was purified as described in Thanabalu et al. J. Bacteriol.
  • the 97 kDa mtx B.sphaericus toxin protein (the 100 kDa mtx protein minus the putative signal peptide) was purified as described in Thanabalu et al. J. Bacteriol. 174 (15), 5051-5056.
  • the purified protein was used to assay for toxicity against first or second instar larvae of C.quin ⁇ uefasciatus.
  • An.albimanus and Ae.aeqvpti in accordance with a previously published assay for assessment of mosquitocidal toxicity (Matsumura, F. (1975) Toxicity of Insecticides, p20-22, Plenum Publishing Corp., New York) .
  • the 97 kDa mtx protein has a broad spectrum of toxicity. It is effective against representative species of Aedes, Culex and Anopheles mosquitoes. The toxicity of this protein against Anopheles mosquitoes, e.g.
  • An.albimanus. is significantly higher than against either Culex mosquitoes, e.g. C. ⁇ uinquefasciatus. or Aedes mosquitoes, e.g. Ae.ae ⁇ vpti.
  • B.sphaericus strains (obtainable from Dr. A. Yousten, Microbiology Section, Virginia Polytechnic Institute, State University, Blacksburg, Virginia, 24061, USA) were tested for their natural resistance to chloramphenicol by inoculating one drop of overnight cultures of the various strains in L- broth containing various concentrations of chloramphenicol.
  • B.sphaericus 1693 (gpIV) and B.sphaericus 4525 (gpIII) were found to be the most sensitive to chloramphenicol in the 2 groups.
  • B.sphaericus 1693 and 4525 cells were prepared for electroporation based on the method of Taylor, L.D. and W.F.Burke (FEMS Microbiol. Lett. (1990) _5£. 125-128) 2 ml of an overnight culture of bacterial cells in L-broth was inoculated into 40 ml of L-broth and incubated at 37oC for 90 mins with shaking (175 rpm) . The cells were harvested by centrifugation and washed 3 times with 20 ml of 10% glycerol and finally resuspended in 1 ml of 10% glycerol. The cell suspension was aliquoted in 100 ⁇ l lots and stored
  • the frozen cells were thawed at room temperature just before transformation. 1 ⁇ l (1 ⁇ g DNA) of plasmid DNA was mixed with the cells and the cells/DNA mixture placed on ice for 5 mins. The cells/DNA mixture was then transferred to a cold 0.2 cm electroporation cuvette (Biorad) . The cells were given a single pulse using the BIO-RAD Gene Pulser (2.5 kV, 25 ⁇ F) with the pulse controller (200 ⁇ ) . The cells were resuspended in 1 ml of L-broth and allowed to regenerate at 37°C for 90 mins. The cells were plated onto L-agar plates containing chloramphenicol (4 ⁇ g/ml) and incubated at 30°C overnight. While B.sphaericus 1693 was transformed by pHV1431 (10 3 transformants/ ⁇ g) , no transformants were observed with B.sphaericus 4525 even after 3 days.
  • the plasmid pXP33 (see Figure 5) was prepared by inserting the 3.8kb PstI fragment of the DNA shown in Figure 1 into the PstI site in pUC18 as previously described by Thanabalu et al. in J.Bacteriol. (1991) 171(9), 2776-2185.
  • pXP33 was digested with Clal and Neil (to destroy the vector sequences) and the restriction fragments were resolved in a 0.7% agarose gel by electrophoresis.
  • the cohesive ends produced by Clal were made blunt-ended with DNA polymerase I Klenow fragment and the fragment was ligated into pHV1431 (see Figure 4) at the EcoRV site.
  • This plasmid has not previously been used in B.sphaericus. but the plasmid pAMBI, from which the origin of replication is derived, has been introduced into B.sphaericus strain 1593 of group IIA by conjugation (Ozech and Burke FEMS Microbiol. Lett. (1984) 25, 91-95). Recombinants were selected by screening for insertional inactivation of tetracycline resistance. After restriction analysis, pC35 ( Figure 6) was chosen for transformation into B.sphaericus strain 1693.
  • B.sphaericus 1693 The larvicidal activity of recombinant B.sphaericus 1693 was assayed on first or second instar larvae of laboratory-reared Culex quinquefasciatus and Aedes ae ⁇ vpti. Comparative assays were carried out with B.sphaericus 2362, which produces the binary mosquitocidal toxin, and B.sphaericus SSII-1.
  • the cells were grown in L-Broth for 24 hrs at 30°C.
  • the sporulated cells were grown in NYSM medium at 30oc for 48 hrs.
  • the medium was supplemented with chloramphenicol at 4 ⁇ g/ l when growing B.sphaericus 1693 harbouring plasmid.
  • Cells from 6 ml culture were harvested by centrifugation, washed once with 0.85% saline and resuspended in 1 ml of 0.85% saline.
  • the OD 550 was measured and the cell suspension was diluted to a OD 5S0 of 0.5.
  • C. ⁇ uin ⁇ uefasciatus is comparable to the toxicity of sporulated cells of B.sphaericus strains such as 2362, which produce the B.sphaericus binary toxin. These strains develop high toxicity during sporulation, having a much lower level of toxicity in the vegetative phase.
  • B.sphaericus 1693 expressing the 100 kDa B.sphaericus mtx protein would be significantly (about 7 fold) more potent against An.albimanus than known B.sphaericus strains producing the B.sphaericus binary toxin (e.g. 2362) assuming that floating type formulations are used to override differences in feeding habits between C. ⁇ uin ⁇ uefasciatus and A .albimanus.
  • B.sphaericus 1693.pC35 thus represents a mosquitocidal bacterium with significant toxicity to representatives of all three major mosquito genera, Aedes, Anopheles and Culex. in both the vegetative and sporulation phases.
  • MOLECULE TYPE DNA (genomic)
  • GAA AAA ACG ATA ATC GAA TCG A ⁇ ATC CAC C ⁇ GCC CAT AAA ⁇ A GGT 864 Glu Lys Thr He He Glu Ser He He His Leu Ala His Lys Leu Gly 275 280 285
  • GGT GAA A ⁇ G ⁇ AGA ATA TGG A ⁇ AAC CCT AAT T ⁇ A ⁇ AAT CCA TCA 1905 Gly Glu He Val Arg He Trp He Asn Pro Asn Phe He Asn Pro Ser 220 225 230

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Abstract

L'invention concerne une bactérie qui contient une séquence d'ADN, codant une toxine tuant les moustiques et codée par le gène mtx de B.sphaericus, ou codant une forme de cette toxine tueuse de moustiques modifiée, et qui peut exprimer cette toxine ou sa forme tueuse modifiée, cette bactérie manquant pour l'essentiel des protéases extracellulaires ou intracellulaires pouvant dégrader ladite toxine ou sa forme tueuse modifiée. Cette bactérie peut contenir par exemple le gène mtx complet tel qu'il est présent dans B.sphaericus SSII-1 et peut s'obtenir par transformation appropriée d'une souche B.sphaericus à homologie d'ADN de groupe III ou IV.
PCT/GB1994/002628 1993-11-30 1994-11-30 Agents de lutte biologique WO1995015383A2 (fr)

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GB9503697A GB2289049A (en) 1993-11-30 1994-11-30 Biological control agents
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GB939324529A GB9324529D0 (en) 1993-11-30 1993-11-30 Biological control agents
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010083A1 (fr) * 1994-09-28 1996-04-04 Novartis Ag Nouvelles proteines et souches pesticides
CN106632624A (zh) * 2016-09-21 2017-05-10 中国农业科学院植物保护研究所 一种杀虫蛋白及其核酸、制备方法和应用
CN112342159A (zh) * 2020-11-09 2021-02-09 海南师范大学 一种芽孢杆菌新菌株hsy204及其杀虫基因和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989010976A1 (fr) * 1988-05-05 1989-11-16 The Public Health Research Institute Of The City O Bacteries gram-positives deficientes en proteases et leur utilisation comme organismes hotes pour la production de produits recombinants

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989010976A1 (fr) * 1988-05-05 1989-11-16 The Public Health Research Institute Of The City O Bacteries gram-positives deficientes en proteases et leur utilisation comme organismes hotes pour la production de produits recombinants

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J BACTERIOL 173 (9). 1991. 2776-2785, THANABALU, T. ET AL. 'CLONING SEQUENCING AND EXPRESSION OF A GENE ENCODING A 100-KILODALTON MOSQUITOCIDAL TOXIN FROM BACILLUS - SPHAERICUS SSII-1.' cited in the application *
J BACTERIOL 174 (15). 1992. 5051-5056, THANABALU, T. ET AL. 'PROTEOLYTIC PROCESSING OF THE MOSQUITOCIDAL TOXIN FROM BACILLUS - SPHAERICUS SSII-1.' cited in the application *
JOURNAL OF BIOTECHNOLOGY, vol. 7, no. 1, January 1988 AMSTERDAM NL, pages 71-82, SOUZA, A. ET AL. 'Cloning and expression in Escherichia coli of two DNA fragments from Bacillus sphaericus encoding mosquito-larvicidal activity' *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849870A (en) * 1993-03-25 1998-12-15 Novartis Finance Corporation Pesticidal proteins and strains
WO1996010083A1 (fr) * 1994-09-28 1996-04-04 Novartis Ag Nouvelles proteines et souches pesticides
EP1754789A2 (fr) * 1994-09-28 2007-02-21 Syngeta Participations AG Souches de bacillus et protéines pesticides
EP1754789A3 (fr) * 1994-09-28 2009-11-11 Syngeta Participations AG Souches de bacillus et protéines pesticides
CN106632624A (zh) * 2016-09-21 2017-05-10 中国农业科学院植物保护研究所 一种杀虫蛋白及其核酸、制备方法和应用
CN106632624B (zh) * 2016-09-21 2021-01-05 中国农业科学院植物保护研究所 一种杀虫蛋白及其核酸、制备方法和应用
CN112342159A (zh) * 2020-11-09 2021-02-09 海南师范大学 一种芽孢杆菌新菌株hsy204及其杀虫基因和应用
CN112342159B (zh) * 2020-11-09 2022-11-22 海南师范大学 一种芽孢杆菌新菌株hsy204及其杀虫基因和应用

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WO1995015383A3 (fr) 1995-07-13
AU1114495A (en) 1995-06-19
GB9503697D0 (en) 1995-07-26
GB9324529D0 (en) 1994-01-19

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