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.     

An analysis of the genes encoding the 51.4- and 41.9-kDa toxins of Bacillus sphaericus 2297 by deletion mutagenesis: the construction of fusion proteins

A series of deletion mutants have been constructed, in which varying numbers of amino acids have been deleted from both the N- and C-termini of both the 51.4- and 41.9-kDa toxins of Bacillus sphaericus. The results show that between 34-39 and 52-54 amino acids respectively at the N- and C-termini of the 51.4-kDa protein, are not essential for toxicity. In the case of the 41.9-kDa protein, the removal of only 7 amino acids from the C-terminus abolishes toxicity whilst at least 17 amino acids can be deleted from the N-terminus without loss of toxicity. A fusion protein with the 51.4-kDa derived sequence N-terminal to the 41.9-kDa sequence yielded a protein of Mr 87 kDa which was not toxic by itself. When supplemented with cells expressing only the 51.4-kDa protein, toxicity was restored. In contrast, another fusion protein, in which the gene order was reversed, was shown to be fully active in toxicity assays.     

Genetic determinants of host ranges of Bacillus sphaericus mosquito larvicidal toxins.

The 51.4-kDa-41.9-kDa binary toxin produced by different strains of Bacillus sphaericus shows differential activity toward Culex quinquefasciatus, Aedes atropalpus, and Aedes aegypti mosquito larvae. The patterns of larvicidal activity toward all three mosquito species and growth retardation in A. aegypti have been shown to be due to the 41.9-kDa protein. By using mutant toxins expressed in Escherichia coli, insecticidal activity and growth retardation correlated with amino acids centered around position 100 of the 41.9-kDa protein. In its response to these toxins, A. atropalpus resembled C. quinquefasciatus rather than its congener, A. aegypti.     

The 42- and 51-kilodalton mosquitocidal proteins of Bacillus sphaericus 2362: construction of recombinants with enhanced expression and in vivo studies of processing and toxicity.

After site-directed mutagenesis, the genes coding for the 42- and 51-kilodalton (kDa) mosquitocidal proteins of Bacillus sphaericus 2362 were placed under the regulation of the aprE (subtilisin) promoter of the Bacillus subtilis vector pUE (a derivative of pUB18). The levels of expression of the gene products in B. subtilis DB104 and B. sphaericus 718 were assessed by bioassays with larvae of Culex pipiens and by Western immunoblots. The results indicated that a higher amount of protein was produced in B. subtilis DB104. Electron microscopic examination of B. subtilis DB104 and B. sphaericus 718 containing the 42- and 51-kDa proteins indicated that amorphous inclusions accumulated in the former species and that crystals identical in appearance to that found in B. sphaericus 2362 were produced in the latter. Strains producing only the 42- or the 51-kDa protein were not toxic to larvae of C. pipiens. A mixture of both strains, a single strain producing both proteins, or a fusion of the 51- and the 42-kDa proteins was toxic. The amount of B. subtilis DB104 containing the 42- and the 51-kDa proteins necessary to kill 50% of the larvae of C. pipiens was 5.6 ng (dry weight) of cells per ml. This value was significantly lower than that for B. sphaericus 2362 (14 ng [dry weight] per ml). Larvae consuming purified amorphous inclusions containing the 42-kDa protein degraded this protein this protein to primarily 39- and 24-kDa peptides, whereas inclusions with the 51-kDa protein were primarily degraded to a protein of 44 kDa. Past studies involving purified proteins from B. sphaericus 2362 indicate an associate of toxicity with the 39-kDa peptide. The results presented here suggest that the 44-kDa degradation product of the 51-kDa protein may also be required for toxicity.     

Novel Bacillus thuringiensis binary insecticidal crystal proteins active on western corn rootworm, Diabrotica virgifera virgifera LeConte

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.     

Sequence analysis of the mosquitocidal toxin genes encoding 51.4- and 41.9-kilodalton proteins from Bacillus sphaericus 2362 and 2297.

The nucleotide sequences of a 3,479-base-pair HindIII DNA fragment from Bacillus sphaericus 2362 and a 2,940-base-pair fragment from strain 2297 were determined; only minor differences were detected between them. Each contained two open reading frames coding for proteins of 51.4 and 41.9 kilodaltons. Both proteins were required for toxicity to larvae of the mosquito Culex pipiens.     

Cloning of the gene for the larvicidal toxin of Bacillus sphaericus 2362: evidence for a family of related sequences.

During sporulation, Bacillus sphaericus 2362 produces a parasporal crystalline protein which is toxic for the larvae of a number of mosquito species. Using the Escherichia coli cloning vector lambda gt11, in which gene products of the inserts may be fused to beta-galactosidase, we isolated 29 bacteriophages which produced peptides-reacting with antiserum to crystal protein. On the basis of restriction enzyme analyses of the recombinants and Ouchterlony immunodiffusion experiments with induced lysogens as a source of antigens, the recombinants were assigned to three groups, designated A, B, and C. Group A consisted of three clones which appeared to express all or part of the B. sphaericus toxin gene from their own promoters and one clone producing a beta-galactosidase-toxin fusion protein. The host cells of two induced recombinant lysogens of this group were toxic to larvae of Culex pipiens. A cell suspension containing 174 ng (dry weight) of the more toxic recombinant per ml killed 50% of the larvae. Both recombinants formed peptides with molecular sizes of 27, 43, and 63 kilodaltons (kDa). The antigenically related 27- and 43-kDa peptides were distinct from the 63-kDa peptide, which resembled crystals from sporulating cells of B. sphaericus in which antigenically distinct 43- and 63-kDa proteins are derived from a 125-kDa precursor. A 3.5-kilobase HindIII fragment from recombinants having toxic activity against larvae was subcloned into pGEM-3-blue. E. coli cells harboring this fragment were toxic to mosquito larvae and produced peptides of 27, 43, and 63 kDa. The distribution of the A gene among strains of B. sphaericus of different toxicities suggested that it is the sole or principal gene encoding the larvicidal crystal protein. The two recombinants of group B and the 23 of group C were all beta-galactosidase fusion proteins, suggesting that in E. coli these genes were not readily expressed from their own promoters. The distribution of these two genes in different strains of B. sphaericus suggested that they do not have a role in the toxicity of this species to mosquito larvae.     

On the respective roles of the two proteins encoded by the Bacillus sphaericus 1593M toxin genes expressed in Escherichia coli and Bacillus subtilis

The 3.6 kb HindIII DNA fragment of B. sphaericus 1593M chromosomal DNA bears two genes encoding two polypeptides of 41.9 kDa (protein "42") and 51.4 kDa (protein "51"). DNA fragments carrying only one of these two genes when expressed in E. coli yield products that are inactive towards Culex larvae. The larvicidal activity is recovered when Triton X-100 treated E. coli cells containing each one of the two genes are incubated together. In E. coli these two polypeptides are acting synergistically. The protein "51" appears to be involved in the maturation of protein "42" for expression of the larvicidal activity. In B. subtilis however the toxicity is expressed by cells carrying only the gene coding for protein "42". There is no need of the "51" gene product for the maturation of the "42" polypeptide, suggesting that the maturation is most likely accomplished by host enzymes.     

Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins.

In the course of sporulation, Bacillus sphaericus produces an inclusion body which is toxic to a variety of mosquito larvae. In this review we discuss the general biology of this species and concentrate on the genetics and physiology of toxin production and its processing in the midgut of the larval host. The larvicide of B. sphaericus is unique in that it consists of two proteins of 51 and 42 kDa, both of which are required for toxicity to mosquito larvae. There is a low level of sequence similarity between these two proteins, which differ in their sequences from all the other known insecticidal proteins of Bacillus thuringiensis. Within the midgut the 51- and 42-kDa proteins are processed to proteins of 43 and 39 kDa, respectively. The conversion of the 42-kDa protein to a 39-kDa protein results in a major increase in toxicity; the significance of the processing of the 51-kDa protein is not known. In contrast to the results with mosquito larvae, the 39-kDa protein is alone toxic for mosquito-derived tissue culture-grown cells, and this toxicity is not affected by the 51-kDa protein or its derivative, the 43-kDa protein. Comparisons of larvae from species which differ in their susceptibility to the B. sphaericus toxin indicate that the probable difference resides in the nature of the target sites of the epithelial midgut cells and not in uptake or processing of the toxin. A similar conclusion is derived from experiments involving tissue culture-grown cells from mosquito species which differ in their susceptibility to the B. sphaericus toxin.     

Isolation and purification of a Campylobacter upsaliensis autolysin.

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS)-PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.     

Phylogenetic analysis and heterologous expression of surface layer protein SlpC of Bacillus sphaericus C3-41

The surface layer protein encoding genes from five mosquito-pathogenic Bacillus sphaericus isolates were amplified and sequenced. Negative staining of the S-layer protein extracted from the cell wall of wild-type B. sphaericus C3-41 was prepared. It showed a flat-sheet crystal lattice structure. Two genes encoding the entire and N-terminally truncated S-layer protein (slpC and DeltaslpC respectively), were ligated into plasmid pET28a and expressed in Escherichia coli. SDS-PAGE revealed that about 130 KD and 110 KD proteins could be expressed in the cytoplasm of recombinant E. coli BL21(pET28a/slpC) and E. coli BL21(pET28a/DeltaslpC) respectively. Furthermore, an intracellular sheet-like or fingerprint-shape structure was investigated in two recombinant strains, which expressed SlpC and DeltaSlpC protein respectively, by ultrathin microscopy study, but bioassay results suggested that the S-layer protein of wild B. sphaericus C3-41 and recombinant E. coli BL21 (pET28a/slpC) have no direct toxicity against mosquito larvae. These results should provide information for further understanding of the function of S-layer protein of pathogenic B. sphaericus.     

Purification of the larvicidal toxin of Bacillus sphaericus and evidence for high-molecular-weight precursors

Crystals were purified from spore-crystal complexes of Bacillus sphaericus 2362 by disruption in a French pressure cell followed by centrifugation through 48% (wt/vol) NaBr. Crystals from such preparations had a 50% lethal concentration of 6 ng of protein per ml for the larvae of the mosquito Culex pipiens. When subjected to polyacrylamide gel electrophoresis under denaturing conditions, the proteins in B. sphaericus crystals migrated in positions corresponding to 43, 63, 98, 110, and 125 kilodaltons (kDa); solubilization of the crystal at pH 12 with NaOH eliminated all but the bands at 43 and 63 kDa. Since NaOH-solubilized preparations were toxic to mosquito larvae, these proteins were purified to electrophoretic homogeneity and antiserum was obtained to each. Analysis of the two purified proteins indicated that the 43-kDa protein was toxic to mosquito larvae (50% lethal concentration, 35 ng of protein per ml), whereas the 63-kDa protein was not. Further differences between them were their amino acid compositions, their lack of immunological cross-reactivity, their opposite net charges at pH 7.5, and their susceptibility to digestion by larval midgut proteases (the 63-kDa protein was highly susceptible, whereas the 43-kDa protein was not). The sequence of the 40 N-terminal residues of the 43-kDa protein was determined and found to contain a high percentage of hydrophobic amino acids. The sequence of the 63-kDa protein could not be determined, since it had multiple N termini. By electrophoretically separating the crystal proteins and then electroblotting onto nitrocellulose paper and visualizing the bands with antisera to the 43- and 63-kDa proteins in conjunction with an immunoblot assay, it was found that the high-molecular-mass crystal proteins (98 to 125 kDa) contained antigenic determinants of both proteins. These results suggested that the lower-molecular-weight crystal proteins detected in polyacrylamide gels after electrophoresis under denaturing conditions were derivatives of one or more of the higher-molecular-weight crystal proteins. In vivo studies of the products of crystal degradation by larvae of Culex pipiens indicated that the high-molecular-weight proteins and the 63-kDa antigenic determinants were rapidly degraded and that a 40-kDa protein related to the 43-kDa toxin persisted for the duration of the experiment (4 h). Some of the studies performed with B.sphaericus 2362 were extended to strains 1593, 1691, and 2297 of this species with results which indicated a high degree of similarity between the crystal proteins of all these larvicidal strains.     

Implications of high-molecular-weight oligomers of the binary toxin from Bacillus sphaericus

The mosquito-larvicidal binary toxin produced by Bacillus sphaericus is composed of BinB and BinA, which have calculated molecular weights of 51.4 and 41.9 kDa, respectively. NaOH extracts of B. sphaericus spores were analyzed using SDS-PAGE. Stained gels showed bands with molecular weights corresponding to those of BinB and BinA as well as two additional bands at 110 and 125 kDa. The matrix-assisted laser desorption/ionization mass spectrum of the purified 110 and 125 kDa bands showed two peaks at 104,160 and 87,358 Da that are assigned to dimers of BinB and BinA, respectively. Mass spectral analysis of trypsin-digested 110 and 125 kDa bands showed peaks at 51,328, 43,523, 43,130, and 40,832 Da that assigned to undigested BinB, two forms of digested BinB and digested BinA, respectively. Dynamic light scattering studies showed a solution of the purified 110 and 125 kDa bands was comprised almost entirely (99.6% of total mass) of a particle with a hydrodynamic radius of 5.6+/-1.2 nm and a calculated molecular weight of 186+/-38 kDa. These data demonstrate that the binary toxin extracted from B. sphaericus spores can exist in solution as an oligomer containing two copies each of BinB and BinA.     

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.     

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent.

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.     

Tightly bound binary toxin in the cell wall of Bacillus sphaericus

We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.     

Purification of green fluorescent protein overexpressed by a mutant recombinant Escherichia coli

Green fluorescent protein was purified from sonicated recombinant Escherichia coli and its mutant obtained after exposure to UV light. The latter overexpresses green fluorescent protein. The two-step procedure consisted of a two-phase aqueous extraction with PEG/salt and precipitation of the proteins from PEG phase by free Zn2+. The recoveries of green fluorescent protein were 73 and 83% in the cases of recombinant E. coli and its mutant, respectively. The corresponding fold purifications were 24 and 9, respectively. In both cases, the purified protein showed a single band on SDS-PAGE corresponding to 28 kDa.     

Cloning and sequencing of the gene encoding a 125-kilodalton surface-layer protein from Bacillus sphaericus 2362 and of a related cryptic gene.

Using the vector pGEM-4-blue, a 4,251-base-pair DNA fragment containing the gene for the surface (S)-layer protein of Bacillus sphaericus 2362 was cloned into Escherichia coli. Determination of the nucleotide sequence indicated an open reading frame (ORF) coding for a protein of 1,176 amino acids with a molecular size of 125 kilodaltons (kDa). A protein of this size which reacted with antibody to the 122-kDa S-layer protein of B. sphaericus was detected in cells of E. coli containing the recombinant plasmid. Analysis of the deduced amino acid sequence indicated a highly hydrophobic N-terminal region which had the characteristics of a leader peptide. The first amino acid of the N-terminal sequence of the 122-kDa S-layer protein followed the predicted cleavage site of the leader peptide in the 125-kDa protein. A sequence characteristic of promoters expressed during vegetative growth was found within a 177-base-pair region upstream from the ORF coding for the 125-kDa protein. This putative promoter may account for the expression of this gene during the vegetative growth of B. sphaericus and E. coli. The gene for the 125-kDa protein was followed by an inverted repeat characteristic of terminators. Downstream from this gene (11.2 kilobases) was an ORF coding for a putative 80-kDa protein having a high sequence similarity to the 125-kDa protein. Evidence was presented indicating that this gene is cryptic.     

Factors and markers of virulence in Escherichia coli from human septicemia

A lethal and necrotic factor which causes cell multinucleation in HeLa cell cultures has previously been shown to be coded by the Vir plasmid of Escherichia coli. Using an absorbed rabbit antiserum which neutralized the Vir toxic properties, we have compared the SDS-PAGE immunoblots from laboratory and field strains which either produce or do not produce Vir toxicity. A single band of 110 kDa was found to be specifically associated with vir toxicity in E. coli strains. This antiserum also recognized the 115 kDa protein band which was previously identified as the cytotoxic necretozing factor (CNF) of certain E. coli strains. These results suggest that the toxin coded by the Vir plasmid is a protein of 110 kDa distinct from, but immunologically related to CNF.     

Sporulation-associated activation of Bacillus sphaericus larvicide.

Preparations of the larvicidal crystal from 46-h cultures of Bacillus sphaericus 2362 contain 125-, 110-, 63-, and 43-kilodalton (kDa) proteins (P. Baumann, B. M. Unterman, L. Baumann, A.H. Broadwell, S.J. Abbene, and R.D. Bowditch, J. Bacteriol. 163:738-747, 1985). The 63- and 43-kDa proteins, which have been purified, are not immunologically cross-reactive, and only the 43-kDa protein is toxic to mosquito larvae. Since antigenic determinants of the two smaller proteins have been detected in the higher-molecular-weight proteins (125 and 110 kDa), it has been suggested that the latter are precursors of the 43- and 63-kDa peptides. In the present study, purified 110-kDa protein was found to be toxic to the larvae of Culex pipiens (50% lethal concentration = 115 ng/ml). A luciferase-luciferin assay for intracellular ATP as well as an assay based on the exclusion of Trypan Blue by live cells indicated that the 110-kDa protein had no effect on tissue-culture-grown cells of C. quinquefasciatus, while cells exposed to the 43-kDa protein rapidly lost viability (50% lethal concentration = 54 microgram(s)/ml by the intracellular ATP assay). These findings suggested that the 110-kDa protein and, by extension, the 125-kDa protein are protoxins which are activated during sporulation by cleavage to a 43-kDa toxin. To further investigate the origins and relationships of the crystal proteins of B. sphaericus, we analyzed samples during the growth and sporulation of the culture. Synthesis of crystal proteins was initiated at the end of exponential growth and was completed after about 7 h.(ABSTRACT TRUNCATED AT 250 WORDS)