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PubMed1983

[Interspecific recombinants of Bacillus thuringiensis x Bacillus cereus].

The possibility of interspecies recombination was shown by using protoplast fusion method. The Bacillus thuringiensis var. galleriae strain 48S Thi Nic Gua Rifr Strr and 56R Gua Rifr, and also Bac. cereus carrying the plasmid pBC16 responsible for resistance to tetracycline (150 mcg/ml) were used. Recombinants were selected on the medium containing rifampicin and tetracycline. They were shown to combine the properties of both parents. The majority of recombinants were resistant to phages Tg4 and Td15 and represented the mean level of sensitivity to phages Tg12, Tg13 and Td14. Examination of the plasmid profiles of recombinants revealed that their resistance to tetracycline was due to the plasmid with mobility analogous to pBC16. It was concluded that the protoplast fusion method can be used to obtain recombinants between relatively remote species of microorganisms.

Bacillus cereus↗
PubMed2003

Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides differentiation using a PCR-RE technique.

A method was developed to differentiate between Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis using the polymerase chain reaction combined with a restriction endonuclease (PCR-RE) technique. This fast and simple protocol, applied to pure culture strains, was developed using the gyrB DNA sequence, as previously proposed by other authors. Strains from international collections were used to optimize the method which was then applied to the identification of strains isolated from food samples. Amplifications were specific for the B. cereus group. Only Staphylococcus aureus gave the same size PCR product, but it was easily differentiated from strains in the B. cereus group by using restriction analysis, based on digestion with the RsaI, Sau3AI and EcoRI endonucleases. Specific amplifications and good differentiations were obtained using pure strains, suggesting the possibility of using the method described to identify the B. cereus group directly in food samples.

Bacillus↗
PubMed1995

Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus.

DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are closely related to cryI genes. Two cryV-type genes, cryV1 and cryV465, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465 protein was toxic only to Plutella xylostella.

Amino Acid Sequence↗
PubMed1987

Cytolytic activity and immunological similarity of the Bacillus thuringiensis subsp. israelensis and Bacillus thuringiensis subsp. morrisoni isolate PG-14 toxins.

The parasporal bodies of the mosquitocidal isolates of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 were compared with regard to their hemolytic and cytolytic activities and the immunological relatedness of the 28- and 65-kilodalton (kDa) proteins that occur in both subspecies. The alkali-solubilized parasporal bodies of B. thuringiensis subsp. israelensis caused 50% lysis of human erythrocytes at 1.14 micrograms/ml, whereas those of B. thuringiensis subsp. morrisoni caused similar lysis at 1.84 micrograms/ml. Preincubation of solubilized parasporal bodies with dioleolyl phosphatidylcholine significantly inhibited the hemolytic activity of both supspecies. In cytolytic assays against Aedes albopictus cells, the toxin concentrations causing 50% lysis for B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni were 1.87 and 11.98 micrograms/ml, respectively. Polyclonal antibodies raised separately against the 25-kDa protein (a tryptic digest of the 28-kDa protein) or the 65-kDa protein of B. thuringiensis subsp. israelensis cross-reacted, respectively, with the 28- and the 65-kDa proteins of B. thuringiensis subsp. morrisoni. However, neither of these antibodies cross-reacted with the 135-kDa protein of either subspecies. These results indicate that the mosquitocidal and hemolytic properties of B. thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni isolate PG-14 are probably due to the biologically related proteins that are present in the parasporal bodies of both subspecies. The lower hemolytic activity of the B. thuringiensis subsp. morrisoni may be due to the presence of lower levels of the 28-kDa protein in that subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)

Aedes↗
PubMed2006

Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis.

Members of the Bacillus cereus group are ubiquitously present in the environment and can adapt to a wide range of environmental fluctuations. In bacteria, these adaptive responses are generally mediated by two-component signal transduction systems (TCSs), which consist of a histidine kinase (HK) and its cognate response regulator (RR). With the use of in silico techniques, a complete set of HKs and RRs was recovered from eight completely sequenced B. cereus group genomes. By applying a bidirectional best-hits method combined with gene neighbourhood analysis, a footprint of these proteins was made. Around 40 HK-RR gene pairs were detected in each member of the B. cereus group. In addition, each member contained many HK and RR genes not encoded in pairs ("orphans"). Classification of HKs and RRs based on their enzymic domains together with the analysis of two neighbour-joining trees of these domains revealed putative interaction partners for most of the "orphans". Putative biological functions, including involvement in virulence and host-microbe interactions, were predicted for the B. cereus group HKs and RRs by comparing them with those of B. subtilis and other micro-organisms. Remarkably, B. anthracis appeared to lack specific HKs and RRs and was found to contain many truncated, putatively non-functional, HK and RR genes. It is hypothesized that specialization of B. anthracis as a pathogen could have reduced the range of environmental stimuli to which it is exposed. This may have rendered some of its TCSs obsolete, ultimately resulting in the deletion of some HK and RR genes.

Adaptation, Physiological↗
PubMed2005

Synthesis of additional endotoxins in Bacillus thuringiensis subsp. morrisoni PG-14 and Bacillus thuringiensis subsp. jegathesan significantly improves their mosquitocidal efficacy.

A fundamental principal of resistance management is that the more complex and potent a toxin mixture, the slower resistance will develop to the mixture in an insect population. Thus, to develop more complex and potent mosquitocidal bacteria, we genetically engineered Bacillus thuringiensis subsp. morrisoni PG-14 and Bacillus thuringiensis subsp. jegathesan, to synthesize, respectively, the binary (Bin) toxin of Bacillus sphaericus or a combination of Bin and the CytlA protein of Bacillus thuringiensis subsp. israelensis. Engineering these two larvicidal bacteria in general significantly improved their efficacy against fourth instars in comparison with their wild-type parental strains. For B. thuringiensis subsp. morrisoni PG-14, which naturally synthesizes Cyt1A, synthesis of Bin improved efficacy nine-fold (LC50 from 4.5 to 0.5 ng/ml) against Culex quinquefasciatus Say, although no improvement was observed (LC50 of 2 ng/ml for both strains) against Aedes aegypti L. For B. thuringiensis subsp. jegathesan, cosynthesis of Bin plus Cyt1A in combination with its normal complement of endotoxins improved efficacy 17-fold (LC50 from 34 to 2 ng/ml) against Cx. quinquefasciatus and 3.2-fold (LC50 from 68 to 21 ng/ml) against Ae. aegypti. Addition of Bin alone to B. thuringiensis subsp. jegathesan did not improve toxicity (LC50 from 68 to 65 ng/ml) against Ae. aegypti, indicating that CytlA synergized the activity of the endotoxins in this strain against Ae. aegypti. These results demonstrate that mosquitocidal efficacy of these strains and likely their resistance management properties can be improved significantly by increasing their toxin complexity and the amount of toxin they synthesize.

Animals↗
PubMed2004

MICs of selected antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a range of clinical and environmental sources as determined by the Etest.

This paper presents Etest determinations of MICs of selected antimicrobial agents for 76 isolates of Bacillus anthracis chosen for their diverse histories and 67, 12, and 4 cultures, respectively, of its close relatives B. cereus, B. thuringiensis, and B. mycoides derived from a range of clinical and environmental sources. NCCLS breakpoints are now available for B. anthracis and ciprofloxacin, penicillin, and tetracycline; based on these breakpoints, the B. anthracis isolates were all fully susceptible to ciprofloxacin and tetracycline, and all except four cultures, three of which had a known history of penicillin resistance and were thought to originate from the same original parent, were susceptible to penicillin. Based on NCCLS interpretive standards for gram-positive and/or aerobic bacteria, all cultures were susceptible to amoxicillin-clavulanic acid and gentamicin and 99% (one with intermediate sensitivity) of cultures were susceptible to vancomycin. No group trends were apparent among the different categories of B. cereus (isolates from food poisoning incidents and nongastrointestinal infections and food and environmental specimens not associated with illness). Differences between B. anthracis and the other species were as expected for amoxicillin and penicillin, with all B. anthracis cultures, apart from the four referred to above, being susceptible versus high proportions of resistant isolates for the other three species. Four of the B. cereus and one of the B. thuringiensis cultures were resistant to tetracycline and a further six B. cereus and one B. thuringiensis cultures fell into the intermediate category. There was a slightly higher resistance to azithromycin among the B. anthracis strains than for the other species. The proportion of B. anthracis strains fully susceptible to erythromycin was also substantially lower than for the other species, although just a single B. cereus strain was fully resistant. The Etest compared favorably with agar dilution in a subsidiary test set up to test the readings, and it compared with other published studies utilizing a variety of test methods.

Anti-Bacterial Agents↗
PubMed1983

[Experiments with Bacillus thuringiensis protoplasts. II. Study of the potential interspecies fusion of bacterial protoplasts: Bacillus thuringiensis and Bacillus megaterium].

In the present study the possibility of obtaining interspecific bacterial hybrids by polyethylene glycol-assisted fusion of protoplasts from Bacillus thuringiensis and Bac. megaterium has been examined. Electron microscopic and genetic data allow to confirm with great probability that cytological fusion takes place. However, genetic analysis revealed that neither of methods applied for protoplast fusion gave stable recombinants. Apparently, it is due to the lack of recombination or the death of recombinants that follows the functioning of the cell correction system. The mechanism of protoplast fusion and its most important steps are also studied in the present work.

Bacillus megaterium↗
PubMed1994

Integrated management of waste tire mosquitoes utilizing Mesocyclops longisetus (Copepoda: Cyclopidae), Bacillus thuringiensis var. israelensis, Bacillus sphaericus, and methoprene.

This study evaluated the compatibility and efficacy of using a predatory copepod, Mesocyclops longisetus in concert with 3 "biorational" compounds for mosquito control in waste tires. The toxicity of Bacillus thuringiensis var. israelensis (B.t.i), Bacillus sphaericus, and methoprene to Mesocyclops longisetus was assessed in the laboratory using concentrations 10 times the maximum labeled or suggested rate and based on a water depth of 7.6 cm. Microbials were tested using mature copepods exposed for durations of 24, 48, and 72 h. Methoprene bioassays consisted of individually exposing newly hatched copepods (i.e., nauplius larvae) and monitoring their development to maturity. The toxicity tests indicated B.t.i., B. sphaericus, and methoprene were not deleterious to copepods at concentrations exceeding those expected in the field. Copepods exposed to methoprene matured normally, and when mated, 50% developed egg sacs. A 5-month field test, integrating the copepod and B.t.i., B. sphaericus, and methoprene provided better mosquito reduction together than either copepods or control agents alone. When copepods were combined with B.t.i. or methoprene, overall reduction of 3rd- and 4th-instar larvae during the 5-month interval was equal to or greater than 90%. Bacillus thuringiensis var. israelensis alone temporarily produced a high degree of larval reduction (up to 100%), however reapplications were necessary to maintain that level of control. Of all the treatments, B. sphaericus alone produced the lowest degree of mosquito suppression due to lack of toxicity to Aedes albopictus, the predominant species during the study. It is recommended that mosquito control managers consider integrating M. longisetus and B.t.i. or methoprene against mosquitoes in waste tires.

Aedes↗
PubMed1997

[Extrachromosomal DNA spectrum of Bacillus thuringiensis Berliner and Bacillus sphaericus Meyer et Neide].

Analysis of the pattern of extrachromosomal DNA in different cultures of Bacillus thuringiensis and Bacillus sphaericus demonstrated a higher content of extrachromosomal DNA in B. thuringiensis than in B. sphaericus. The quantity and approximate molecular weights of the plasmids were determined. The assumption that the plasmid DNA content in B. thuringiensis strains is higher than in the other representatives of the genus Bacillus was confirmed.

Bacillus↗
PubMed1995

A new serovar of Bacillus thuringiensis possessing 28a28c flagellar antigenic structure: Bacillus thuringiensis serovar jegathesan, selectively toxic against mosquito larvae.

A novel Bacillus thuringiensis strain highly toxic to mosquitoes was isolated from soil samples in Malaysia. This strain was shown to display a new subfraction of the H-28 flagellar antigen determining a new serovar H28a28c, which was designated serovar jegathesan. Bioassays indicated that Culex quinquefasciatus larvae are the most susceptible to this new isolate, whereas toxicity to Anopheles maculatus and Aedes aegypti larvae was 10 times lower. The potency of this new serotype is also comparable to most of the Malaysian B. thuringiensis H-14 isolates.

Aedes↗
PubMed1994

Insecticidal activity of the CryIIA protein from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki expressed in Escherichia coli and Bacillus thuringiensis and in a leaf-colonizing strain of Bacillus cereus.

A 4.0-kb BamHI-HindIII fragment encoding the cryIIA operon from the NRD-12 isolate of Bacillus thuringiensis subsp. kurstaki was cloned into Escherichia coli. The nucleotide sequence of the 2.2-kb AccI-HindIII fragment containing the NRD-12 cryIIA gene was identical to the HD-1 and HD-263 cryIIA gene sequences. Expression of cryIIA and subsequent purification of CryIIA inclusion bodies resulted in a protein with insecticidal activity against Heliothis virescens, Trichoplusia ni, and Culex quinquefasciatus but not Spodoptera exigua. The 4.0-kb BamII-HindIII fragment encoding the cryIIA operon was inserted into the B. thuringiensis-E. coli shuttle vector pHT3101 (pMAU1). pMAU1 was used to transform an acrystalliferous HD-1 strain of B. thuringiensis subsp. kurstaki and a leaf-colonizing strain of B. cereus (BT-8) by using electroporation. Spore-crystal mixtures from both transformed strains were toxic to H. virescens and T. ni but not Helicoverpa zea or S. exigua.

Amino Acid Sequence↗
PubMed2005

Non-larvicidal effects of Bacillus thuringiensis israelensis and Bacillus sphaericus on oviposition and adult mortality of Culex quinquefasciatus Say (Diptera: Culicidae).

Two microbial mosquito larvicides, Bacillus thuringiensis ssp. israelensis (Bti) and Bacillus sphaericus (Bsph), have been shown to be highly effective in controlling mosquito larvae and have been used in larvicidal programs for many years. In exploring other modes of action of these agents, we studied the ovipositional response of Bsph susceptible and resistant Culex quinquefasciatus to aqueous suspensions of Bti and Bsph water dispersible granules (WDG). We quantified the level of mortality of adult mosquitoes caused by exposure to Bti and Bsph suspensions during oviposition. Significantly lower numbers of egg rafts were laid and collected from the treatments than the control regimen. There was an inverse relationship between Bacillus product concentrations and oviposition. As the concentration of Bti or Bsph increased from 0.0 to 2.0 mg/L, treated waters received progressively fewer egg rafts. In addition to the negative effects of Bacillus on oviposition, both male and female adult mosquitoes suffered high mortality on landing and imbibing on Bti and Bsph suspensions, the extent of mortality directly proportional to concentration. These two microbial agents used solely as mosquito larvicides thus have the additional benefits of reducing mosquito oviposition and killing adult mosquitoes, especially gravid females that come in contact with the treated water either for oviposition or drinking. Reducing the number of gravid females may also result in reduced transmission rates of pathogens. The combined effects of reduced oviposition and adult mortality could result in higher control potential of these microbial agents.

Animals↗
PubMed1992

Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis.

The Bacillus thuringiensis cryIIA delta-endotoxin gene is found as the third-gene in a three-gene operon, with a sporulation-dependent promoter lying upstream of the first gene, orf1. We show here that the polypeptide product of the middle gene (orf2) is required for efficient expression of the toxin gene. In the absence of a functional ORF2 polypeptide the toxin does not form the crystalline inclusions characteristic of other known Bacillus thuringiensis toxins. We discuss the importance of this finding with respect to the possible role of chaperonins in the crystallization of these proteins.

Amino Acid Sequence↗