Crystallization and preliminary X-ray diffraction studies of the 51 kDa protein of the mosquito-larvicidal binary toxin fromBacillus sphaericus

Abstract

Certain strains of Bacillus sphaericus produce a highly toxic mosquito-larvicidal protein during sporulation which is active against vectors of dengue, encephalitis and malaria. This toxin is initially expressed as 51 and 42 kDa proteins and is converted to 43 and 39 kDa proteins, respectively, which form the active heterodimer complex. For a better understanding of the toxicity mechanism at the molecular level, the 51 kDa protein of the binary toxin of B. sphaericus strain 2297 was expressed as a glutathione-S-transferase fusion protein and puri®ed by af®nity chromatography. Protein crystals were grown from an amorphous precipitate in ®ve months using the hanging-drop vapor-diffusion method. The protein crystals were dissolved and were found to be composed of a proteolytically modi®ed 45.2 kDa derivative similar to the active form of this protein. The crystals form in space group P4 3 2 1 2 (or P4 1 2 1 2) and diffract to 2.6 A Ê , with unit-cell dimensions a = b = 133.48, c = 69.76 A Ê .

The bacterium, Lysinibacillus sphaericus, as an insect pathogen

“…Oligomerisation of BinA and BinB occurs in solution to give a BinA 2 BinB 2 tetramer (Smith et al, 2005). Some progress towards elucidating a 3-dimensional structure for the Bin proteins has been made with the crystallisation of the BinB protein alone (Chiou et al, 1999) and a BinA/BinB co-crystal (Smith et al, 2004) but, to date, no crystal structure is available. The availability of such a structure will aid in understanding receptor binding, mechanism of action and the structural importance of residues shown to influence toxicity and target range of the Bin toxin.…”

Microorganisms in Biological Pest Control — A Review (Bacterial Toxin Application and Effect of Environmental Factors)

“…Production of entomopathogenic toxin is a biochemical changes that accompany the morphological changes during sporulation. When fresh medium (or a pulse of a carbon source) is made available, the spore will germinate and produce a vegetative rod-shaped cell [53,54] Because B. subtilis, like other Gram-positive eubacteria, lacks an outer membrane, many of these proteins are directly secreted into the growth medium. In most cases, these secreted proteins are enzymes involved in the hydrolysis of natural polymers such as proteases, lipases, carbohydrases, DNases and RNases.…”

Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB

“…The 3D structure of the toxin is not available, although crystals of BinB (Chiou et al, 1999) and binary toxin (Smith et al, 2004) have been reported. Using EMBOSS pairwise alignment (http://www.…”