Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had K m values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.Bacillus sphaericus is an aerobic, mesophilic, spore-forming bacterium with terminal swollen sporangia and spherical spores. As a consequence of the specific toxicity to mosquito larvae of binary toxin (Bin) and mosquitocidal toxins (Mtxs) produced during the sporulation and vegetative stages, respectively, some toxic strains have been widely used for many years as biopesticides in the field in mosquito control programs.B. sphaericus can metabolize a wide variety of organic compounds and amino acids, but it is unable to use hexoses and pentoses as unique carbon sources (20). The specific metabolic limitation of B. sphaericus hampers its potential industrial development due to the high costs of the proteinaceous media used for toxin production compared to the costs of alternative media based on starch or molasses. Consequently, studies elucidating the whole metabolic potential of B. sphaericus are essential for genetic improvement of this bacterium.Previous research indicated that the inability of B. sphaericus to metabolize carbohydrates could be attributed to its inability to transport glucose or sucrose into the cell (24) and the absence of key enzyme activities in the Embden-Meyerhof-Parnas (EMP) (glucokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase), hexose monophosphate pathway (phosphogluconate dehydratase), and Entner-Doudoroff (6-phospho-2-keto-3-deoxyglyconate aldolase) pathways (20). Recent studies revealed that B. sphaericus strain 2362 has a glucose transport system (2) and that it also has 6-phosphofructokinase activity; thus, it could use Nacetylglucosamine as a sole carbon source (3). However, there are no data available about the presence of gene and enzyme activities of glucose...

Expression of a chitinase gene, chiAC, from Bacillus thuringiensis in B. sphaericus 2297 using the binary toxin promoter yielded a recombinant strain that was 4,297-fold more toxic than strain 2297 against resistant Culex quinquefasciatus. These results show that this chitinase can synergize the toxicity of the binary toxin against mosquitoes and thus may be useful in managing mosquito resistance to B. sphaericus.Insects of dipteran species such as Culex spp., Anopheles spp., and Aedes spp. are responsible for the transmission of many infectious disease agents. Directing an attack against these mosquitoes is recognized as one of the more effective approaches for eradicating the threat from infectious diseases (4). As a gram-positive, spore-forming, aerobic, entomopathogenic bacterium, Bacillus sphaericus has been successfully used for mosquito control in the last decade. Its activity against target mosquito larvae is mainly due to the crystal toxin, commonly referred to as the binary toxin, as it consists of equimolar amounts of two proteins of 51 and 42 kDa. (1). However, high-level resistance against B. sphaericus binary toxin after intensive treatment has developed in mosquitoes (6), with resistance ratios ranging from 35-to 150,000-fold in the laboratory (5, 8) and from 10-to 100,000-fold in the field (15). Experiments testing binary toxin binding to the mosquito larval midgut in vitro have demonstrated that resistance of mosquitoes to B. sphaericus binary toxin has occurred mainly because of elimination of the toxin-binding site on the midgut brush border membrane fraction (1, 6). The appearance of high-level of resistance in mosquitoes is a threat to the future application of B. sphaericus as a mosquito control agent.On the basis of the sequence of the chi gene from B. thuringiensis subsp. israelensis (GenBank accession no. AF526379), two pairs of primers, chiAC-1-chiAC-3 and chiAC-2-chiAC-3, were designed for amplifying the chiAC open reading frame by PCR with different enzyme digestion sites introduced from strain T04A001 (3) (Table 1) as described elsewhere (17). After digestion with EcoRI and HindIII, the PCR fragment amplified with primers chiAC-1 and chiAC-3 was ligated with EcoRI/HindIII-digested plasmid pET28a and the resulting plasmid was transformed into Escherichia coli BL21. The recombinant E-pETC21 was selected on LB agar supplemented with kanamycin (50 g/ml). The 70-kDa ChiAC fusion protein was purified for preparation of rabbit anti-ChiAC with a His-Bind Resin chromatography kit by following the procedure provided by the manufacturer (Novagen) (9).For the expression of chiAC in B. sphaericus, a binary toxin promoter was amplified as described elsewhere (14) with primers pbinary-1 and pbinary-2. The amplified binary toxin promoter (ϳ0.5 kb) was digested with BamHI and SphI and then introduced into BamHI/SphI-digested vector pBU4, resulting in recombinant plasmid pBb. The open reading frame of ChiAC, obtained by PCR with primers chiAC-2 and chiAC-3, was inserted into pBb at the SphI and HindII...

The toxicities of Mtx1 toxin against dipteran and lepidopteran species have been evaluated in this study. It was shown that Mtx1 has little or no toxicity to the tested lepidopteran species, but has moderate-level toxicity to Aedes albopictus Skuse (Diptera: Culicidae) and high-level toxicity to both susceptible and binary toxin-resistant Culex quinquefasciatus Say (Diptera: Culicidae). The LC(50) values of Mtx1 against a susceptible C. quinquefasciatus colony SLCq and two resistant colonies RLCq1/C3-41 and RLCq2/IAB59 selected in the laboratory with Bacillus sphaericus (Mayer & Neide) strains C3-41 and IAB59 respectively were 0.508, 0.854 and 0.675 mg L(-1) respectively. The data indicate that Mtx1 has a different mode of action from the binary toxin, and that it could be an alternative toxin to delay or overcome resistance development to binary toxin in C. quinquefasciatus.