Crystal proteins from Bacillus thuringiensis serovar. medellin

Abstract

Colombian strains 163-131 and 24-726 of the Bacillus thuringiensis serovar. medellin (Btmed), serotype H-30, are very toxic to mosquito larvae. Strain 24-726 was serologically and biochemically characterized. It is almost identical to the reference-strain 163-131. The parasporal inclusion of Btmed strain 163-131 was analysed by electron microscopy. The crystal protein matrix was very similar to that observed in B. thuringiensis serovar. israelensis (Bti). Aedes aegypti, Anopheles albimanus and Culex quinquefasciatus larvae were exposed to 500× the half-lethal concentration (LC50) of Btmed strains, Bti strain 1884 and B. thuringiensis serovar. morrisoni (Btm) strain PG-14. Mortality of Aedes aegypti occurred within approx. 60 min with the four strains, whereas C. quinquefasciatus mortality was three times slower with Btmed than with strains 1884 and PG14. The onset mortality of Anopheles albimanus starts when other species are already dead. The thermolabilities of the mosquitocidal activities of the crystal proteins were tested by incubation of cultures of 20 min at various temperatures. Btmed lost all mosquitocidal activity at 73°C, and 1884 and PG-14 at 79°C. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of the crystals purified from strain 163-131 shows polypeptides at 100 kDa, multiple bands at 80, 75, 70, 67, and 65 kDa, and two doubles at 40–41 and 28–30 kDa. Immunodetection with antibodies raised against Bti toxins shows cross-reaction between the 30-kDa and to a lesser extent the 28-kDa polypeptides of Btmed crystals and Cyt A of Bti. A slight response is observed with the 65-kDa Btmed to serum raised against Cry IV D of Bti.