Plasmid transformation of Bacillus sphaericus 1593

Plasmids pUB110 and pBC16 were introduced by protoplast transformation into the entomocidal bacterium Bacillus sphaericus 1593. Transformants expressed the antibiotic resistance determinants present on the plasmid and exhibited sporulation frequencies and larvicidal toxicities which were equivalent to those characteristic of the parent strain. These transformations constitute the first report of genetic transfer in B. sphaericus.     

Localization of a mosquito-larval toxin of Bacillus sphaericus 1593.

Cell-free wall, membrane, and cytoplasmic fractions were prepared from Bacillus sphaericus 1593, which exhibited toxic activity against larvae of the mosquito Culex pipiens var. quinquefasciatus. Breakage of 12- to 14-h cells by sonication or French pressure cell yielded toxic material which could be assayed in a standard mosquito larva bioassay. When sporulating cells of strain 1593 were fractionated, the majority of the toxic activity was localized in the cell wall rather than in the plasma membrane or cytoplasm. The toxin located in the bacterial cell wall was relatively stable, in that activity was unaffected by treatment with trypsin, pronase, CHCl3-CH3OH-water, Triton X-100, 8 M urea (30 min), heat (80 degrees C, 12 min), sonication, refrigeration, lyophilization, or freezing. Activity was destroyed by boiling for 10 min or by 0.01 N NaOH. Only about 1.0% of the activity present in purified cell walls could be recovered by a 2-h extraction with 8 M urea or 3 M guanidine hydrochloride. A comparison of the toxicity of a cell-free cell wall fraction with that of a sample consisting entirely of heat-stable spores indicated that the spore preparation was about 10 times more active.     

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.     

Identification, cloning and sequence analysis of the Bacillus sphaericus 1593 41.9 kD larvicidal toxin gene

A number of strains of the widespread aerobic soil bacterium, Bacillus sphaericus, possess crystalline inclusions of a toxin lethal to a variety of insect (larvae) which are vectors of major tropical diseases. Partial amino acid sequence data from one strain, B. sphaericus 2362 have permitted us to design oligonucleotide probes for identifying the toxin gene in the closely related B. sphaericus 1593. The gene was found to be contained within an EcoRI-HindIII fragment and was cloned in its entirety in the bacterial plasmid pUC12. The DNA sequence was determined together with the upstream and downstream controlling elements, and a sequence of 370 amino acids was deduced for the toxin protein. This is the first reported sequence of a B. sphaericus toxin gene and will facilitate further work in characterizing the genes from other strains of different virulence and host range. The data do not support the suggestion that the toxin is derived by proteolysis of a protoxin precursor.     

Alkaline extraction of toxin from spores of the mosquito pathogen, Bacillus sphaericus strain 1593

Toxin was extracted from spores of the mosquito pathogen Bacillus sphaericus strain 1593 using 0.05 M NaOH. The molecular weight of this toxin was 35000-54000. Toxic activity of this extract was resistant to a variety of enzymes including subtilisin, but was degraded by pronase. Antiserum produced to 1593 spore toxin neutralized spore toxin and cytoplasmic toxin activity, but did not react with Bacillus thuringiensis var. israelensis crystal toxin, nor did var. israelensis toxin antiserum react with B. sphaericus toxin. Crystal like parasporal inclusions accompanying the B. sphaericus 1593 spores were removed by NaOH extraction.     

Conjugal transfer of pAMβ1 in Bacillus sphaericus 1593

Abstract The plasmid pAMβ1 has been successfully transferred from Streptococcus faecalis to Bacillus sphaericus by the method of filter mating. Subsequently, transfer of this conjugative plasmid has also been demonstrated between derivatives of B. sphaericus 1593, though at reduced frequencies. The transfer process was determined to be unaffected by either endogenous restriction enzyme activity of the recipient or the presence of exogenous DNase.     

Expression of the larvicidal gene of Bacillus sphaericus 1593M in the cyanobacterium Anacystis nidulans R2

Summary A 3.6 kb HindIII DNA fragement from Bacillus sphaericus 1593M was cloned and expressed in Escherichia coli and B. subtilis using pHV33 as shuttle vector and in the cyanobacterium Anacystis nidulans R2 with pUC303 as shuttle vector. The level of toxin activity of the respective recombinant plasmids pGsp04 and pGsp12 against Culex mosquito larvae was found to be the same in Escherichia coli and in the cyanobacterium.     

Comparative studies of the mosquito-larval toxin of Bacillus sphaericus SSII-1 and 1593

Two strains of Bacillus sphaericus. SSII-1 and 1593, were bioassayed for toxic activity against second-instar larvae of the mosquito Culex pipiens quinquefasciatus. It was found that strain 1593 developed a level of toxicity 3000 times that of strain SSII-1. Although the toxic activity of B. sphaericus SSII-1 was relatively unchanged throughout growth, an increase in activity of strain 1593 occurred as the bacteria began to sporulate. Strain differences were examined by (i) growth cycle experiments, (ii) bioassays of the toxicity of oligosporogenous mutants, and (iii) manganese limitation experiments. The toxin of strain 1593 was shown to be more stable than that of strain SSII-1. Unlike the spores of strain SSII-1, the spores of B. sphaericus 1593 were found to be highly toxic. Thin sections of SSII-1 or 1593 cells did not reveal the presence of any inclusion body that might be related to toxicity.     

Isolation and characterization of Pseudomonas putida R-prime plasmids

A number of enhanced chromosome mobilizing (ECM) plasmids derived from the wide host range plasmid R68 have been used to construct R-prime plasmids carrying a maximum of two map minutes of the Pseudomonas putida PPN chromosome, using Pseudomonas aeruginosa PAO as the recipient. For one ECM plasmid, pMO61, the ability to form R-primes did not correlate with the ability to mobilize chromosomes in intrastrain crosses, suggesting that different mechanisms are involved. Physical analysis of one R-prime showed that 3.5 kb of chromosomal DNA had been inserted between the tandem IS21 sequences carried by the parent ECM plasmid.     

An aerotaxis transducer gene from Pseudomonas putida

An aerotaxis gene, aer, was cloned from Pseudomonas putida PRS2000. A P. putida aer mutant displayed an altered aerotactic response in a capillary assay. Wild-type P. putida clustered at the air/liquid interface. In contrast, the aer mutant did not cluster at the interface, but instead formed a diffuse band at a distance from the meniscus. Wild-type aer, provided in trans, complemented the aer mutant to an aerotactic response that was stronger than wild-type. The P. putida Aer sequence is similar over its entire length to the aerotaxis (energy taxis) signal transducer protein, Aer, of Escherichia coli. The amino-terminus is similar to redox-sensing regulatory proteins, and the carboxy-terminus contains the highly conserved domain present in chemotactic transducers.     

Delineation of the minimal portion of the Bacillus sphaericus 1593M toxin required for the expression of larvicidal activity

The two genes of Bacillus sphaericus 1953M coding for the 51.4-kDa and 41.9-kDa proteins are both required for the expression of the active larvicidal toxin in Escherichia coli. The minimal size of the active peptide of the 41.9-kDa toxin was defined by in vitro deletion analysis of the gene and found to consist of 338 amino acids (38.3 kDa). N-terminal deletions past the Ile18 residue and C-terminal deletions past the His352 residue result in the loss of toxic activity and rapid degradation of such modified toxins by host proteases. The minimal active 38.3-kDa peptide produced in E. coli seems to mimick the stable processed form of the toxin found in larval midguts. However, it still requires the action of the synergistic 51.4-kDa protein for the larvicidal activity.     

Fate of Bacillus sphaericus 1593 and 2362 spores used as larvicides in the aquatic environment.

Dry powders produced from insecticidal Bacillus sphaericus strains 1593 and 2362 were applied against Culex tarsalis and Anopheles franciscanus mosquito larvae in small-plot field trials. Good control of C. tarsalis, but not of A. franciscanus, was produced at 0.1 and 0.2 lb/acre [ca. 0.122 and 0.244 kg/ha]. B. sphaericus spores settled rapidly from upper water layers and accumulated in bottom muds. Control of third/fourth-instar C. tarsalis larvae was maintained through day 4 after testing and was related to the presence of at least 100 spores per ml in the upper water layer 2 days earlier. B. sphaericus was shown to recycle in dead larvae both in the laboratory and in the field, producing an increase of 100- to 1,000-fold in spore numbers. There was no evidence of recycling in treated water nor of significant spore persistence upon reflooding of ponds after a very hot, dry period.     

Effect of UV Light on Spore Viability and Mosquito Larvicidal Activity of Bacillus sphaericus 1593

UV light from a germicidal lamp rapidly reduced the viability of Bacillus sphaericus 1593 spores, but insecticidal activity was resistant to inactivation by continuous exposure to UV light for 4 h.     

Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus strain 1593 on media using a byproduct from a monosodium glutamate factory

Two newly developed media, H4 and H7, were found to be highly suitable for culturing Bacillus thuringiensis subsp. israelensis and B. sphaericus, respectively. These media contained 0.05% K₂HPO₄ and 4% HDL (H4 medium) or 0.05% K₂HPO₄ and 7% HDL (H7 medium); HDL is the by-product from a monosodium glutamate factory. Tests to compare endospore formation and toxicity values of B. thuringiensis subsp. israelensis in H4 medium and nutrient broth supplemented with salts and glucose (NBSG) medium were carried out in a 3-liter fermentor. The viable cell count and LC₅₀ value of B. thuringiensis subsp. israelensis in H4 medium at 48 hr were 2.5 × 10⁸ cells/ml and 10^?7.2 (dilution), respectively, while those in NBSG medium were 1.6 × 10⁸ cells/ml and 10^?6.5, respectively. In the case of B. sphaericus grown in H7 medium, the number of cells and LC₅₀ value were found to be 1.4 × 10⁹ cells/ml and 10^?7.8, respectively. B. sphaericus grown in nutrient broth supplemented with salt and yeast extract (NBSY) were found to produce 6.4 × 10⁸ cells/ml and an LC₅₀ value of 10^?6.8. The toxicity of B. thuringiensis subsp. israelensis was tested against Aedes aegypti larvae, while that of B. sphaericus was tested against Culex quinquefasciatus. The cost of 10 liters of medium for production of B. thuringiensis subsp. israelensis and in B. sphaericus and H4 and H7 was $0.02 and $0.03, respectively. The cost of these newly developed media was much less than that of NBSG medium ($7.05 per 10 liters) for cultivation of B. thuringiensis subsp. israelensis and NBSY medium ($11.67 per 10 liters) for cultivation of B. sphaericus.     

Functional Complementation of Nontoxic Mutant Binary Toxins of Bacillus sphaericus 1593M Generated by Site-Directed Mutagenesis

Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B. sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.     

Effect of plasmids from various incompatibility groups on the development of bacteriophages specific to Pseudomonas aeruginosa and Pseudomonas putida

The aim of this work was to study the effect of plasmids belonging to different incompatibility groups on the growth of bacteriophages in Pseudomonas aeruginosa and Pseudomonas putida strains. The growth of bacteriophages was shown to be limited most often due to the presence in cells of plasmids belonging to the P-2 incompatibility group. Plasmids of the Inc P-2 group differed from one another in the spectrum of bacteriophages whose growth they limited. Phages whose growth was suppressed in strains containing plasmids of the P-5, P-9 or P-10 incompatibility groups were found. Some plasmids showed no specific interaction with bacteriophages. The plasmids investigated differed in the studied trait in P. aeruginosa and P. putida cells. In contrast to P. aeruginosa PAO, P. putida PpGI plasmid containing cells did not maintain the growth of donor-specific bacteriophages and, to a lesser degree, limited the growth of phages specific for P. putida PpGI.     

Identification of the functional site in the mosquito larvicidal binary toxin of Bacillus sphaericus 1593M by site-directed mutagenesis

To study the mode of action of the binary toxin (51- and 42-kDa) of Bacillus sphaericus, amino acid residues were substituted at selected sites of the N- and C-terminal regions of both peptides. Bioassay results of the mutant binary toxins tested against mosquito larvae, Culex quinquefasciatus, revealed that most of the substitutions made on both peptides led to either decrease or total loss of the activity. Furthermore, receptor binding studies carried out for some of the mutants of the 42-kDa peptide showed mutations in N- and C-terminal regions of the 42-kDa peptide did not affect the binding of the binary toxin to brush border membrane vesicles of mosquito larvae. One of the mutants having a single amino acid substitution at the C-terminal region ((312)R) of the 42-kDa peptide completely abolished the biological activity, implicating the role of this residue in membrane pore formation. These results indicate the importance of the C-terminal region of the 42-kDa of binary toxin, in general, and particularly the residue (312)R for biological activity against mosquito larvae.