However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship find more between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron

microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation. Bacillus sphaericus is a Gram-positive, spore-forming aerobic bacterium (Charles et al., 1996). A number of highly toxic strains of B. sphaericus

can synthesize two crystalline mosquito-larvicidal proteins of 42 kDa (BinA) and 51 kDa (BinB) during sporulation (Baumann et al., 1985). The two proteins act together to function as a binary toxin (Broadwell et al., 1990). Bacillus sphaericus is considered one of the most successful

microbial larvicide MLN8237 and has been commercialized over the past decade (Berry, 2011). Besides being an important bio-insecticide for mosquito control, Methamphetamine B. sphaericus has several important phenotypic properties, including being incapable of polysaccharide utilization and having exclusive metabolic pathways for a wide variety of organic compounds and amino acids (Russell et al., 1989; Han et al., 2007). Bacillus species undergo dramatic morphological, physiological and biochemical changes during sporulation and these changes have been studied in great detail in Bacillus subtilis (Hilbert & Piggot, 2004). In response to starvation, B. subtilis initiates a developmental process by forming an asymmetric septation that divides the bacterium into two asymmetric compartments, the mother cell and forespore. The smaller, forespore compartment develops into the spore, whereas the larger mother cell nurtures the developing forespore. Initially, the forespore and mother cell lie side by side; subsequently, the mother cell engulfs the forespore in a phagocytosis-like process. The engulfed forespore exists as a free-floating protoplast within the mother cell and is enveloped by two membranes, the peptidoglycan cortex layer and the protein coat layer. Ultimately, the spore is released into the environment by lysis of the mother cell. Due to the considerable interest in the use of B.