The presence of two cry-like genes first identified in Clostridium bifermentans subsp. malaysia CH18 was investigated in Clostridium species including 12 subspecies of Clostridium bifermentans, 13 strains of other members of Clostridia genus, and 13 different subspecies of Bacillus thuringiensis. Oligonucleotides designed to amplify the two toxin genes, cmb71 and cmb72, were used. We found that these genes are present in 80% of the Clostridium bifermentans strains tested and in 8% of the other Clostridium and Bacillus thuringiensis strains.
A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.
Bacillus thuringiensis (Bt), an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. However, little is known about the receptor molecules that bind parasporins and the mechanism of anti-cancer activity. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit preferential cytotoxic activity for human leukaemic T cells (CEM-SS) but is non-cytotoxic to normal T cells or other cancer cell lines such as human cervical cancer (HeLa), human breast cancer (MCF-7) and colon cancer (HT-29) suggesting properties similar to parasporin. In this study we aim to identify the binding protein for Bt18 in human leukaemic T cells.
Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91bp downstream) called cbm72, is 1857bp long and encodes a 71727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462bp) and cbm17.2 (459bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426bp and 1022bp downstream from cbm72, respectively. They encode 17189-Da and 17451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.