Distribution of restriction endonucleases among some entomopathogenic strains of Bacillus sphaericus

The restriction enzyme Bsp TI, an isoschizomer of Hae III (recognition site GGCC), has been detected in eight strains of serotype 5a5b and two serotype 3 strains of the entomopathogenic bacterium Bacillus sphaericus . Strains from other serotypes contained the enzymes Bsp TII and Bsp TIII, which digested pBR322 DNA into similar banding patterns after agarose gel electrophoresis but differed in their susceptibility to methylation of the substrate. Strains from serotypes 9, 25 and 26a26b were lacking in restriction enzyme activity. There was little correlation between phage typing and restriction enzyme activity, suggesting that restriction and modification are not responsible for phage specificity among entomopathogenic B. sphaericus strains.     

Numerical classification and identification of Bacillus sphaericus including some strains pathogenic for mosquito larvae

Ninety-one strains of Bacillus sphaericus, including representatives of all the established DNA homology groups, related round-spored and oval-spored species, and six strains pathogenic for mosquito larvae, were examined for 155 characters. Numerical analyses (Jaccard coefficient/average linkage clustering) based on the 88 variable features revealed 14 clusters at the 79% similarity level that contained more than one strain and 17 single member clusters. All insect pathogenic strains were recovered in a single cluster and the classification was in accord with an established classification based on DNA sequence homology. Two frequency matrices for probabilistic identification were constructed and tested. A comprehensive matrix comprising 14 mesophilic, round-spored taxa and 27 tests gave good results for identification of hypothetical median organisms, cluster overlap and identifications of representative strains (based on data generated in the classification study). Reference strains for the 14 taxa and eight additional insect pathogenic strains were examined for the 27 tests and were correctly identified with high scores using this matrix. A second matrix comprising seven taxa and 13 tests also performed well in the theoretical evaluation and correctly identified the reference strains and insect pathogenic strains.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

Phenotypic characterization of Bacillus thuringiensis and Bacillus cereus

One hundred and thirty-seven strains of Bacillus thuringiensis and 35 strains of Bacillus cereus were tested for the presence or absence of 99 traits. An analysis of these data indicated that strains of B. thuringiensis were indistinguishable from B. cereus, except for their ability to produce parasporal crystals. This conclusion was based on a comparison of the phenotypic properties of B. thuringiensis and B. cereus, as well as on the results of numerical analyses of the data which grouped strains into clusters on the basis of phenotypic similarity. In the resulting dendrograms, strains of B. thuringiensis and B. cereus were interspersed, exhibiting no tendency to segregate. In addition, with the exception of serovar israelensis, strains on B. thuringiensis belonging to the same flagellar serovar showed little or no tendency to group in different clusters. A comparison of the phenotypic differences between serovars indicated that the greater the number of strains in the serovars, the fewer, if any, phenotypic traits separating them. This suggests that the properties reported to differentiate serovars can be attributed to the internal phenotypic diversity of the species. Characterization of 10 mosquitocidal strains of Bacillus sphaericus indicated that the traits employed in this study readily distinguished these highly related organisms from strains of B. thuringiensis and B. cereus.     

Extracellular serine protease synthesis by mosquito-pathogenic strains of Bacillus sphaericus

Nine strains of Bacillus sphaericus toxic to mosquito larvae produced haloes of hydrolysis when cultured on casein-nutrient agar. In batch culture, protease synthesis by B. sphaericus BSE 18 occurred during exponential growth and was repressed by high concentrations of peptone. Extracellular protease from this bacterium showed optimal activity at about pH 10.2, was inhibited by phenylmethylsulphonyl chloride and chymostatin but not soybean trypsin inhibitor or EDTA. Hydrolysis of N -CBZ-glycine-nitrophenyl ester was consistent with the major enzyme being a serine protease.     

Growth and spore germination factors of various strains of Bacillus sphaericus

The work was aimed at studying the requirements of sixteen Bacillus sphaericus strains with a different larvicidal activity in amino acids and some other compounds necessary for their growth and spore germination. Most of the strains were found to require arginine, glutamate, methionine, threonine, serine, glycine, alanine and lysine, but they did not assimilate phenylalanine and proline. Arginine, methionine and glutamate were shown to be the most effective inductors of spore germination. Specific differences were detected in the requirements of virulent and avirulent strains. Glucose repressed both spore germination and spore formation.     

Purification and some properties of cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus

An intracellular cyclodextrin-hydrolyzing enzyme from Bacillus sphaericus E-244 isolated from soil was purified to a homogeneous state by means of Triton X-100 extraction, DEAE-Sepharose column chromatography, hydrophobic and molecular-sieve HPLC. The enzyme was estimated to have an Mr of 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 144,000 by HPLC gel filtration on TSK gel G 3000 SW. It had a pH optimum of 8.0, and the enzyme, stable at 25 degrees C and pH 5.5-9.5 for 24 h, was inactivated at 50 degrees C for 10 min. The enzyme hydrolyzed beta-cyclodextrin more effectively than linear maltooligosaccharides such as maltopentaose, maltohexaose and maltoheptaose or polysaccharides such as starch, amylopectin, amylose and pullulan.     

Isolation and identification of Bacillus sphaericus strains pathogenic for mosquito larvae

Three selective media for the isolation of Bacillus sphaericus have been compared. BATS medium and a formulation employing adenosine as the principal carbon source were the most effective for the recovery of spores of strain 1593. Anthranilic acid as the principal carbon source was less efficient. Eighty-four strains were isolated from mud samples using these media and were identified by computer. Identifications were confirmed for representative strains using DNA sequence homology. Most were B. sphaericus sensu stricto or members of an unnamed group. However, one strain (BSE 18) was identified as the DNA homology group IIB and this organism was found to be highly toxic toward larvae of Culex pipiens. Southern hybridization of BSE 18 DNA to a probe prepared from the cloned toxin gene from strain 1593 revealed that BSE 18 contained a typical gene for the 41.9-kDa toxin.     

Bioassay of three strains of Bacillus sphaericus on field-collected mosquito larvae.

Bacillus sphaericus strains 1593, 1404, and SSII-1 were assayed for infectivity against field-collected larvae of Psorophora columbiae, Culex nigripalpus, and Aedes taeniorhynchus in southwest Florida. Results indicate that all three strains are highly active against the Psorophora and Culex species. A. taeniorhynchus is also susceptible but requires higher dosages to achieve lethal responses. Tests were also conducted on the rate of infection and the differences in susceptibility of different instars to B. sphaericus. These tests indicate that nearly 75% of the mortality that occurs in the course of exposure to B. sphaericus occurs within 48 hr post-incubation with the bacteria. Furthermore, our tests indicate P. columbiae larvae decrease in susceptibility to the Bacillus with increase in larval age (instar). This investigation shows B. sphaericus to be a feasible biological control agent that warrants further study.     

Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB

Bacillus sphaericus produces a mosquitocidal binary toxin composed of two subunits, BinA (42 kDa) and BinB (51 kDa). Both components are required for maximum toxicity against mosquito larvae. BinB has been proposed to provide specificity by binding to the epithelial gut cell membrane, while BinA may be responsible for toxicity. To identify regions in BinB responsible for receptor binding and for interaction to BinA, we used six BinB shorter constructs derived from both the N-terminal and the C-terminal halves of the protein. All constructs expressed as inclusion bodies in Escherichia coli, similarly to the wild-type protein. A marked decrease in larvicidal activity was observed when BinA was used in combination with these BinB constructs, used either individually or in pairs from both N and C-halves of BinB. Nevertheless, immunohistochemistry analyses demonstrate that these constructs are able to bind to the epithelium gut cell membrane, and in vitro protein-protein interaction assays revealed that these constructs can bind to BinA. These results show that fragments corresponding to both halves of BinB are able to bind the receptor and to interact with BinA, but both halves are required by the toxin to exhibit full larvicidal activity.     

Purification and characterization of cystathionine gamma-synthase type II from Bacillus sphaericus

Cystathionine gamma-synthase type II, which catalyzes L-cystathionine synthesis from O-acetyl-L-homoserine and L-cysteine was purified from Bacillus sphaericus (IFO 3536) in seven steps. The purified enzyme appeared to be homogeneous by the results of polyacrylamide electrophoresis and ampholyte electrofocusing. The enzyme is a typical pyridoxal-P dependent enzyme, has a molecular mass of 165 kDa and consists of four subunits identical in molecular mass. The enzyme catalyzed the gamma-replacement reaction and the elimination reaction was hardly detected even when a large amount of enzyme was added. In the replacement reaction, O-acetyl-L-homoserine and the following thiol compounds: L and D-cysteine, L and D-homocysteine, sodium sulfide, various alkyl and aryl mercaptans, acted as the most suitable substrate to produce L-cystathionine and the corresponding S-substituted L-homocysteine derivatives.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.     

Laboratory evaluation of three mosquito pathogenic strains of Bacillus sphaericus isolated in Egypt

Three strains of Bacillus sphaericus H-5a5b designated Ghar. 1 & 10, Ghar. 2 & 20, and Ghar. 3 & 30 were tested for growth, virulence, and larvicidal activity against Culex pipiens in the laboratory. Incubation temperature was positively correlated (r = 0.91) to the rate of bacterial growth (strain Ghar. 2 & 20). All three strains retained their virulence through 25 successive transfers on nutrient agar. Acetone powder preparations showed high larvicidal activity against C. pipiens, although second instar larvae were more susceptible than fourth instars to all three strains. The most active strain was Ghar. 2 & 20 with LC50 values of 0.51 mg/liter (second instar) and 1.62 mg/liter (fourth instar) after 48 hr of exposure. Mortality rates in fourth instar larvae exposed to an acetone powder form of strain Ghar. 2 & 20 were significantly greater at higher than at lower temperatures.     

New high-toxicity mosquitocidal strains of Bacillus sphaericus lacking a 100-kilodalton-toxin gene.

Five new high-toxicity mosquitocidal strains of Bacillus sphaericus were isolated in Singapore. They all belong to phage group 8 and have binary toxin (51.4- plus 41.9-kDa) genes located on the chromosome but lack a 100-kDa-toxin gene. These strains of B. sphaericus constitute a new subgroup, as only two weakly toxic strains in phage group 8 have previously been described and all the known high-toxicity strains have both binary toxin and 100-kDa-toxin genes.     

Evaluation of an rRNA-targeted oligonucleotide probe for the detection of mosquitocidal strains of Bacillus sphaericus in soils: characterization of novel strains lacking toxin genes

Abstract: Colonies of Bacillus sphaericus on primary isolation have been identified using an oligonucleotide probe targeted to a specific region of the 16S rRNA. Of 3440 colonies from soil samples from Brazil, 57 hybridized to the probe in colony blots but when purified DNA was used in slot-blots the probe was more specific and only 27 isolates hybridized. Of these, 20 strains were confirmed as members of DNA homology group IIA (potential mosquitocidal strains) by ribotyping and isoenzyme analysis. However, none of these strains was toxic to Anopheles or Culex larvae, nor did they contain recognized toxin genes. This is the first demonstration of such non-pathogenic strains of B. sphaericus DNA homology group IIA and their common occurrence suggests that pathogenicity is not an important contribution to the success of these bacteria in the environment. Similar screening of strains from Scottish soils indicated that B. sphaericus DNA homology group IIA strains were less common in this habitat and none were recovered on this occasion.     

Purification and characterization of the larvicidal toxin of Bacillus sphaericus 1593M

We present here a procedure for purifying the larvicidal toxin from sporulating cells of Bacillus sphaericus 1593M and describe some of the biochemical and biophysical properties of this toxin. The procedure involves solubilization of the cell-wall/membrane bound toxin by sonication of cells followed by repeated rounds of freezing and thawing at 50 degrees C. Further purification involved Sephadex G-100 and DEAE Sephacel chromatography. We show by Sephadex G-100 chromatography that at pH 7.5 the smallest active form of the toxin has an Mr of 38,000 and that this toxin can reversibly aggregate to molecular forms of a size higher than 2 X 10(5) Mr. By shifting the pH from 7.5 to 8.5 only the aggregated forms can be observed.     

Serological classification of Bacillus sphaericus strains on the basis of toxicity to mosquito larvae

Summary A toxicity study of 54 Bacillus sphaericus strains isolated from vectors or breeding sites has led to a relatively homogeneous grouping of mosquito pathogenic strains into five H-serotypes among the nine serotypes determined. Each serotype seems to be characterized by a different level of toxicity and a classification of these five serotypes can be made on the basis of this toxicity. Within these serotypes, a scale of toxicity has been tentatively fixed and an arbitrary limit of toxicity suggested.