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PubMed1995

ssp genes and spore osmotolerance in Bacillus thuringiensis israelensis and Bacillus sphaericus.

It was shown previously that spores and vegetative cells of Bacillus sphaericus (Bf) and Bacillus thuringiensis israelensis (Bti) are very sensitive to osmotic variations. Since spore osmotolerance has been associated with their SASP (small acid soluble spore proteins) content coded by ssp genes, hybridization assays were performed with sspE and sspA genes from B. subtilis as probes and showed that Bti and Bf strains could lack an sspE-like gene. The B. subtilis sspE gene was then introduced into Bti 4Q2 strain; spores were obtained and showed a 65 to 650 times higher level of osmotolerance to NaCl, without affecting other important properties: hypoosmotic resistance in vegetative cells, spore UV resistance, and larvicidal activity against diptera larvae.

Animals↗

PubMed2001

Cyt1Ab1 and Cyt2Ba1 from Bacillus thuringiensis subsp. medellin and B. thuringiensis subsp. israelensis Synergize Bacillus sphaericus against Aedes aegypti and resistant Culex quinquefasciatus (Diptera: Culicidae).

The interaction of two cytolytic toxins, Cyt1Ab from Bacillus thuringiensis subsp. medellin and Cyt2Ba from Bacillus thuringiensis subsp. israelensis, with Bacillus sphaericus was evaluated against susceptible and resistant Culex quinquefasciatus and the nonsensitive species Aedes aegypti. Mixtures of B. sphaericus with either cytolytic toxin were synergistic, and B. sphaericus resistance in C. quinquefasciatus was suppressed from >17,000- to 2-fold with a 3:1 mixture of B. sphaericus and Cyt1Ab. This trait may prove useful for combating insecticide resistance and for improving the activity of microbial insecticides.

Aedes↗

PubMed1993

A complete physical map of a Bacillus thuringiensis chromosome.

Bacillus thuringiensis is the source of the most widely used biological pesticide, through its production of insecticidal toxins. The toxin genes are often localized on plasmids. We have constructed a physical map of a Bacillus thuringiensis chromosome by aligning 16 fragments obtained by digestion with the restriction enzyme NotI. The fragments ranged from 15 to 1,350 kb. The size of the chromosome was 5.4 Mb. The NotI DNA fingerprint patterns of 12 different B. thuringiensis strains showed marked variation. The cryIA-type toxin gene was present on the chromosome in four strains, was extrachromosomal in four strains, and was both chromosomal and extrachromosomal in two strains. A Tn4430 transposon probe hybridized to 5 of the 10 cryIA-positive chromosomal fragments, while cryIA and the transposon often hybridized to different extrachromosomal bands. Ten of the strains were hemolytic when grown on agar plates containing human erythrocytes. Nine of the strains were positive when assayed for the presence of Bacillus cereus enterotoxin. We conclude that B. thuringiensis is very closely related to B. cereus and that the distinction between B. cereus and B. thuringiensis should be reconsidered.

Bacillus thuringiensis↗

PubMed1996

[Construction of shuttle vector containing delta-endotoxin gene of Bacillus thuringiensis].

The Bacillus thuringiensis toxin gene CryIA(c) was inserted into the shuttle vector pBE-2 to construct pAMY for expressing the B.t. gene in both Gram-negative and -positive bacterial systems. pAMY was introduced into wild type Bacillus cereus, B.brevis and B.subtilis by electroporation. Transformants containing delta-endotoxin gene produced proteins reacted with B.t. crystal protein antibody. Upon biological toxicity tests, the transformants gave a mortality of 100% against Ostrinia furnacilis, 58.8% against Heliothis armigera and 100% against Heliothis assulta. The ability of promoting plant growth of the original strains is retained.

Bacillus cereus↗

PubMed1994

Tn5401 disruption of the spo0F gene, identified by direct chromosomal sequencing, results in CryIIIA overproduction in Bacillus thuringiensis.

The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation mutants derived from a B. thuringiensis transposon insertion library. A spo0F defect in B. thuringiensis, which was suppressed by multicopy hknA or kinA, resulted in the overproduction of the CryIIIA insecticidal crystal protein.

Amino Acid Sequence↗

PubMed1996

Genetic analysis of cryIIIA gene expression in Bacillus thuringiensis.

The Bacillus thuringiensis (Bt) cryIIIA gene is regulated by a different mechanism from that of most of the other cry genes. Its expression begins during late-exponential growth and not during sporulation as for the other classes of cry genes. Moreover, in Bacillus subtilis, cryIIIA expression is independent of the major sporulation-specific sigma factors and is increased in a spoOA genetic background. We used lacZ fusions and primer-extension analysis to follow the time-course of cryIIIA transcription in Bt wild-type and in various Spo- genetic backgrounds (spoOA, sigE and sigK). cryIIIA was activated from the end of vegetative growth to stage II of sporulation (t3) in the wild-type strain. Thereafter, transcription from the same promoter continued, at a decreasing rate, until the end of stage III. In the spoOA mutant strain, the same promoter was activated for at least 15 h during the stationary phase. cryIIIA activation in the sigK genetic background was similar to that in the wild-type but was extended in a sigma E mutant strain. Thus cryIIIA expression in Bt is not directly dependent on the major sporulation-specific sigma factors. Furthermore, an event linked with the thE-dependent period of sporulation ends cryIIIA activation, although transcription of this gene does not switch off before the end of stage III.

Bacillus thuringiensis↗

PubMed1998

Modulation of Cry IV A toxin protein expression by glucose in Bacillus thuringiensis israelensis.

Bacillus thuringiensis subsp. israelensis (Bti) produces Cry IV A protoxin protein as part of the insecticidal crystal toxin during sporulation. This study was conducted with the objective of identifying environmental signals which regulate toxin synthesis by Bti. Glucose was found to repress Cry IV A toxin induction at the mRNA level. The repressive effect of glucose was dependent on a phosphorylation step since protein kinase inhibitor calphostin c relieved the 130-kD protoxin synthesis at both the mRNA and protein level. Phosphorylation of HPr, the phosphocarrier protein of the phosphotransferase system, occurred during glucose repression of Cry IV A toxin synthesis in Bti cells was seen by Western blotting with anti-phosphoserine antibody and rabbit anti-HPr serum. Phosphorylation of HPr in vivo as well as in the in vitro assay was inhibited by calphostin c, a specific inhibitor of serine/threonine kinase. Calphostin c had no effect on sporulation efficiency of Bti cells.

Animals↗

PubMed1994

Location of a lepidopteran specificity region in insecticidal crystal protein CryIIA from Bacillus thuringiensis.

The Bacillus thuringiensis insecticidal crystal protein CryIIA has both high mosquito activity and gypsy moth activity; in contrast CryIIB, which is 87% homologous, displays no mosquito activity and has a three-fold lower gypsy moth activity. The regions responsible for specificity against gypsy moth (Lymantria dispar) and mosquito (Aedes aegypti) larvae were located by introducing MluI and XhoI sites into homologous positions within the putative domain II of both cryIIA and cryIIB genes, which divided almost equally the respective second domains into three regions. Taking advantage of naturally occurring NheI and NarI sites that border the putative domain II, a set of seven chimeric proteins were produced by exchanging all combinations of those regions between CryIIA and CryIIB. Analysis of the toxicity of these chimeric proteins demonstrated that the lepidopteran and dipteran specificity regions of CryIIA were not colinear. While the specificity region of CryIIA against mosquito larvae involved region 1 and probably also region 2, the specificity region of CryIIA against gypsy moth larvae was located within region 2.

Aedes↗

PubMed1995

Diarrhoeal enterotoxin production by strains of Bacillus thuringiensis isolated from commercial Bacillus thuringiensis-based insecticides.

Strains of Bacillus cereus and B. thuringiensis were tested by the Tecra VIA kit for the ability to produce a diarrhoeal enterotoxin. The strains of B. thuringiensis were isolated from commercial B. thuringiensis-based insecticides (Bactimos, DiPel, Florbac, FC, Foray 48B, Novodor FC, Turex, VecTobac, XenTari). The production of diarrhoeal enterotoxin varied by a factor of more than 100 among the different strains tested. B. cereus (F4433/73) produced the highest amount of enterotoxin and the B. thuringiensis strain isolated from DiPel the lowest. The products were tested for their content of diarrhoeal enterotoxin and all products, except MVP which does not contain viable B. thuringiensis spores, contained diarrhoeal enterotoxins. The results indicates an potential risk for gastroenteritis outbreak caused by B. thuringiensis.

Bacillus thuringiensis↗

PubMed2001

Lack of cross-resistance to Cry19A from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to cry toxins from Bacillus thuringiensis subsp. israelensis.

Culex quinquefasciatus mosquitoes with high levels of resistance to single or multiple toxins from Bacillus thuringiensis subsp. israelensis were tested for cross-resistance to the Bacillus thuringiensis subsp. jegathesan polypeptide Cry19A. No cross-resistance was detected in mosquitoes that had been selected with the Cry11A, Cry4A and Cry4B, or Cry4A, Cry4B, Cry11A, and CytA toxins. A low but statistically significant level of cross-resistance, three to fourfold, was detected in the colony selected with Cry4A, Cry4B, and Cry11A. This cross-resistance was similar to that previously detected with B. thuringiensis subsp. jegathesan in the same colony. These data help explain the toxicity of B. thuringiensis subsp. jegathesan against the resistant colonies and indicate that the Cry19A polypeptide might be useful in managing resistance and/or as a component of synthetic combinations of mosquitocidal toxins.

Animals↗

PubMed1998

Variable cross-resistance to Cry11B from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to single or multiple toxins of Bacillus thuringiensis subsp. israelensis.

A novel mosquitocidal bacterium, Bacillus thuringiensis subsp. jegathesan, and one of its toxins, Cry11B, in a recombinant B. thuringiensis strain were evaluated for cross-resistance with strains of the mosquito Culex quinquefasciatus that are resistant to single and multiple toxins of Bacillus thuringiensis subsp. israelensis. The levels of cross-resistance (resistance ratios [RR]) at concentrations which caused 95% mortality (LC95) between B. thuringiensis subsp. jegathesan and the different B. thuringiensis subsp. israelensis-resistant mosquito strains were low, ranging from 2.3 to 5.1. However, the levels of cross-resistance to Cry11B were much higher and were directly related to the complexity of the B. thuringiensis subsp. israelensis Cry toxin mixtures used to select the resistant mosquito strains. The LC95 RR obtained with the mosquito strains were as follows: 53.1 against Cq4D, which was resistant to Cry11A; 80.7 against Cq4AB, which was resistant to Cry4A plus Cry4B; and 347 against Cq4ABD, which was resistant to Cry4A plus Cry4B plus Cry11A. Combining Cyt1A with Cry11B at a 1:3 ratio had little effect on suppressing Cry11A resistance in Cq4D but resulted in synergism factors of 4.8 and 11.2 against strains Cq4AB and Cq4ABD, respectively; this procedure eliminated cross-resistance in the former mosquito strain and reduced it markedly in the latter strain. The high levels of activity of B. thuringiensis subsp. jegathesan and B. thuringiensis subsp. israelensis, both of which contain a complex mixture of Cry and Cyt proteins, against Cry4- and Cry11-resistant mosquitoes suggest that novel bacterial strains with multiple Cry and Cyt proteins may be useful in managing resistance to bacterial insecticides in mosquito populations.

Animals↗

PubMed2004

Laboratory and simulated field evaluation of a new recombinant of Bacillus thuringiensis ssp. israelensis and Bacillus sphaericus against Culex mosquito larvae (Diptera: Culicidae).

In the laboratory, three microbial mosquito larvicidal products consisting of Bacillus thuringiensis ssp. israelensis de Barjac (Bti), Bacillus sphaericus (Neide) (Bsph) (strain 2362), and the University of California Riverside (UCR) recombinant (producing toxins of both Bacillus sphaericus and Bacillus thuringiensis ssp. israelensis) were bioassayed against larvae of Culex quinequefasciatus Say (susceptible and resistant to Bsph 2362), and Aedes aegypti (L.). Bti proved highly effective against Cx. Quinequefasciatus susceptible and resistant strains, with LC50 values of 0.009 and 0.011 ppm and LC90 values of 0.057 and 0.026 ppm for Bsph-susceptible and -resistant strains, respectively. Bti was also highly active against Ae. eagypti with LC50 and LC90 values of 0.014 and 0.055 ppm, respectively. The UCR recombinant was equally active against both Bsph-susceptible and -resistant strains of Cx. Quinquefasciatus; LC50 values were 0.005 and 0.009 and LC90 values were 0.030 and 0.043 ppm, respectively. Bti and the UCR recombinant essentially showed similar activity against Bsph-susceptible and -resistant strains. UCR recombinant showed high toxicity against Ae. eagypti with LC50 and LC90 values of 0.023 and 0.064 ppm, respectively. Bsph was highly active against susceptible strain of Cx. quinequefasciatus with LC50 and LC9o values of 0.006 and 0.024 ppm, respectively. Bsph exhibited little toxicity against Ae. eagypti larvae and also no toxicity to Bsph resistance. In the field, we evaluated four experimental corn grit formulations of Bti (VBC 60021), Bsph (VBC 60022), UCR recombinants VBC 60023 (7.89%), and VBC 60024 (1.87%) in simulated field (microcosms) against Bsph-susceptible Culex mosquitoes. Bti and low-concentrate UCR recombinant showed similar initial activity as well as persistence. Both materials provided high-to-moderate level of control for 2-7 d posttreatment at low treatment rates. At low dosages, residual activity of Bti and UCR recombinant lasted for <7 d. Bsph and high-concentrate UCR recombinant (VBC 60023), however, were more effective against natural populations of Cullex and achieved longer control (7-21 d) than the other two materials.

Animals↗

PubMed2003

A plasmid encoding a combination of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus confers toxicity against a broad range of mosquito larvae when expressed in Gram-negative bacteria.

A recombinant plasmid harboring cry4A, cry4B and cry11A from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus has been constructed. The three cry genes were placed under the control of the cry4B promoter whereas the binary toxin gene was controlled by its native promoter. The expression of toxins in Escherichia coli harboring the resulting plasmid, p4BDA-5142, was investigated. Cry4B expression was highest compared to other toxins. Although the level of toxin expression was low compared with E. coli expressing single toxins, the recombinant E. coli strain harboring p4BDA-5142 exhibited broad range mosquito-larvicidal activity against all Aedes, Culex and Anopheles larvae. This work has shown that the development of the recombinant plasmid can be used to broaden the host range spectrum of the appropriate bacterial host for mosquito control.

Aedes↗

PubMed2003

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes.

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.

Amino Acid Sequence↗

PubMed2006

Occurrence of natural Bacillus thuringiensis contaminants and residues of Bacillus thuringiensis-based insecticides on fresh fruits and vegetables.

A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic.

Bacillus thuringiensis↗

PubMed2000

Biological control of Bactocera oleae (Diptera: Tephritidae) using a Greek Bacillus thuringiensis isolate.

Bacillus thuringiensis, isolate 114A, was used in toxicity experiments against the wild population ofthe olive pest Bactroceria oleae (Gmelin). In laboratory experiments, spores and crystals of the B.t. were delivered to the insects with the food. Longevity, oviposition period, number of eggs produced, and percent hatch were recorded. Olive fruits from the oviposition test were dipped into a suspension containing spores and crystals of B. thuringiensis 114A after the eggs were deposited. In field experiments, four to six sprayings per year of B.t. 114A isolate were applied for three successive years. It was found that, in addition to the longevity of B. oleae, the oviposition period, number of eggs and percent egg hatch decreased. Also, the percentage of pupation and emergence was reduced when olive fruits with eggs in their mesocarp were dipped in the solution of spores and crystals. Field applications with the toxins of 114A isolate of B. thuringiensis have resulted in significant protection of the olive production.

Animals↗

PubMed2006

Siderophores of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis.

Three Bacillus anthracis Sterne strains (USAMRIID, 7702, and 34F2) and Bacillus cereus ATCC 14579 excrete two catecholate siderophores, petrobactin (which contains 3,4-dihydroxybenzoyl moieties) and bacillibactin (which contains 2,3-dihydroxybenzoyl moieties). However, the insecticidal organism Bacillus thuringiensis ATCC 33679 makes only bacillibactin. Analyses of siderophore production by previously isolated [Cendrowski et al., Mol. Microbiol. 52 (2004) 407-417] B. anthracis mutant strains revealed that the B. anthracis bacACEBF operon codes for bacillibactin production and the asbAB gene region is required for petrobactin assembly. The two catecholate moieties also were synthesized by separate routes. PCR amplification identified both asbA and asbB genes in the petrobactin producing strains whereas B. thuringiensis ATCC 33679 retained only asbA. Petrobactin synthesis is not limited to the cluster of B. anthracis strains within the B. cereus sensu lato group (in which B. cereus, B. anthracis, and B. thuringiensis are classified), although petrobactin might be prevalent in strains with pathogenic potential for vertebrates.

Bacillus anthracis↗

PubMed2004

Synergy between toxins of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus.

Synergistic interactions among the multiple endotoxins of Bacillus thuringiensis subsp. israelensis de Barjac play an important role in its high toxicity to mosquito larvae and the absence of insecticide resistance in populations treated with this bacterium. A lack of toxin complexity and synergism are the apparent causes of resistance to Bacillus sphaericus Neide in particular Culex field populations. To identify endotoxin combinations of the two Bacillus species that might improve insecticidal activity and manage mosquito resistance to B. sphaericus, we tested their toxins alone and in combination. Most combinations of B. sphaericus and B. t. subsp. israelensis toxins were synergistic and enhanced toxicity relative to B. sphaericus, particularly against Culex quinquefasciatus Say larvae resistant to B. sphaericus and Aedes aegypti (L.), a species poorly susceptible to B. sphaericus. Toxicity also improved against susceptible Cx. quinquefasciatus. For example, when the CytlAa toxin from B. t. subsp. israelensis was added to Bin and Cry toxins, or when native B. t. subsp. israelensis was combined with B. sphaericus, synergism values as high as 883-fold were observed and combinations were 4-59,000-fold more active than B. sphaericus. These data, and previous studies using cytolytic toxins, validate proposed strategies for improving bacterial larvicides by combining B. sphaericus with B. t. subsp. israelensis or by engineering recombinant bacteria that express endotoxins from both strains. These combinations increase both endotoxin complexity and synergistic interactions and thereby enhance activity and help avoid insecticide resistance.

Aedes↗