Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1

Abstract

A cosmid library was prepared from a partial BamHJ digest of total DNA from Bacillus sphaericus SSII-1. Two hundred fifty Escherichia coli clones were screened for toxicity against larvae of the mosquito Culex quinquefasciatus. One toxic clone, designated pKF2, was chosen for further study. Two toxic subclones, designated pXP33 and pXP34, obtained by ligating PstI-derived fragments of pKF2 into pUC18, contained the same 3.8-kb fragment, but in opposite orientations. Sequence analysis revealed the presence of an open reading frame corresponding to a 100-kDa protein and the 3' end of a further open reading frame having significant homology to open reading frames of transposons TnSOl and Tn2l. The sequence of the SSII-1 toxin was compared with those of known toxins and was found to show regional homology to those of ADPribosyltransferase toxins. The distribution of the toxin gene among other B. sphaericus strains was examined.Bacillus sphaericus is an aerobic spore-forming Bacillus species, several strains of which are pathogenic for mosquito larvae (13). B. sphaericus SSII-1 was isolated in 1973 from infected mosquito larvae collected in India (36). This strain was considerably more toxic than the other B. sphaericus strains known at that time. Early studies indicated that the toxin was retained within or attached to the bacterial cells and that the toxic activity was markedly unstable. Storage of cells under refrigeration or heating at 80°C resulted in the loss of activity. Toxin production occurred predominantly in the vegetative phase of growth before the onset of sporulation (27,29).Further studies on B. sphaericus SSII-1 declined rapidly with the discovery of strains, such as 1593 (36a), 2362 (40), and 2297 (41), which had higher toxicities. In contrast to strain SSII-1, these strains develop relatively stable, high toxicity at the onset of sporulation (7). To date, two types of toxin have been characterized in the highly toxic strains: a pair of proteins of 51.4 and 41.9 kDa associated with the parasporal crystal (3,5,19) and a toxin of 110 kDa related to a 125-kDa surface layer protein (5, 6). Genes encoding the 51.4-and 41.9-kDa proteins were shown to be absent from strain SSII-i (4). In the same study, Baumann et al. showed that sequences inserted in their group C clones were also absent from strain SSII-1. The group C clones were later shown to contain sequences corresponding to the gene encoding the 125-kDa protein (6). Davidson (12) Construction of the cosmid library. To prepare total genomic DNA from B. sphaericus SSII-1, we lysed cells in the presence of 1% sodium dodecyl sulfate and 0.1 mg of proteinase K per ml. DNA was purified from the lysate by the method of Ausubel et al. (2). In brief, hexadecyltrimethyl ammonium bromide was added, and the mixture was incubated at 65°C for 20 min prior to extraction with chloroformisoamyl alcohol. DNA was precipitated with ethanol from the aqueous phase and purified by banding on a cesium chloride gradient coqtaining ethidium bromide. DNA was partially digest...

Characterization and biological activity of a brazilian isolate of Bacillus sphaericus (Neide) highly toxic to mosquito larvae

“…The binary action of the 42 and 51 kDa peptides in the process of toxicity of B. sphaericus has been established (Broadwell et al 1990, Davidson et al 1990). The role of a third 100 KDa toxin previously characterized (Davidson 1983) which may be influencing the results obtained in this research has been established elsewhere (Thanabalu et al 1991).…”

Analysis of the catalytic site of the actin ADP‐ribosylating Clostridium perfringens iota toxin

“…4. Comparison of the amino acid sequences of the region adjacent to a glutamic acid residue suggested to be involved in catalysis of the ADP-ribosylation by various transferases (Lim, C. limosum ADP-ribosyltransferase [28]; C3N, C. botulinum C3 transferase [26]; C3P, C. botulinum C3 transferase [25]; EDIN, S. aureus transferase [27]; Cer, B. cereus transferase [23]; CT, cholera toxin [30]; LT, E. coli heat labile toxin [43]; MTX, mosquitocidal toxin from B. sphaericus [44]; PT, pertussis toxin [45]; T2, bacteriophage ADPribosyltransferase T2 [32]; mRT6, mouse homologue of the rat T cell alloantigen RT6 [46]; rMT, rabbit muscle ADP-ribosyltransferase [33]; ETA, Pseudomonas exotoxin A [47]; DT, diphtheria toxin [481.…”

Cytotoxicity and ADP-ribosylating activity of the mosquitocidal toxin from Bacillus sphaericus SSII-1: possible roles of the 27- and 70-kilodalton peptides

“…The N-terminal region of the Mtx protein was found to have significant regional homology to the catalytic subunits of ADP-ribosyltransferase (32) toxins, such as the pertussis toxin S1 subunit (26), and had no significant homology with the 51.4-and 41.9-kDa toxins from the high-toxicity B. sphaericus strains (32). In earlier studies (31), a 97-kDa protein, Mtx21 (amino acids 30 to 870), derived from the 100-kDa mosquitocidal protein (Mtx) of B. sphaericus SSII-1 by the deletion of the putative signal sequence, was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli and purified by affinity chromatography.…”

“…Early studies indicated that the toxin was very unstable, was produced predominantly before the onset of sporulation, and was associated with the cell pellet (23, 24). The genes encoding the 51.4-and 41.9-kDa binary toxin have been shown to be absent from B. sphaericus SSII-1 (4).To study the nature of the toxicity of B. sphaericus SSII-1, we have cloned a gene encoding a 100-kDa protein and have designated it the mix (mosquitocidal-toxin) gene (32). The N-terminal region of the Mtx protein was found to have significant regional homology to the catalytic subunits of ADP-ribosyltransferase (32) toxins, such as the pertussis toxin S1 subunit (26), and had no significant homology with the 51.4-and 41.9-kDa toxins from the high-toxicity B. sphaericus strains (32).…”

“…To study the nature of the toxicity of B. sphaericus SSII-1, we have cloned a gene encoding a 100-kDa protein and have designated it the mix (mosquitocidal-toxin) gene (32). The N-terminal region of the Mtx protein was found to have significant regional homology to the catalytic subunits of ADP-ribosyltransferase (32) toxins, such as the pertussis toxin S1 subunit (26), and had no significant homology with the 51.4-and 41.9-kDa toxins from the high-toxicity B. sphaericus strains (32).…”