Glycolysis And The Involved Enzymes In Bacillus Sphaericus

Bacillus sphaericus is a Gram-positive, spore-forming, aerobic mosquito pathogenicbacterium. Due to its specific activity against mosquito larvae, B. sphaericus has beensuccessfully used for mosquito control worldwide. Except N-acetylglucosamine, all B.sphaericus could not metabolize any sugars as carbon resource, thus hindering the furtherapplication of B. sphaericus as larvicidal agent because of high production cost offormulations. Previous research indicated that the inability of B. sphaericus to metabolizecarbohydrates could be attributed to the absence of key enzyme activities in the EMP(glucokinase, glucose phosphate isomerase, phosphofructokinase), HMP(phosphogluconate dehydratase) and ED (6-phospho-2-keto-3-deoxyglyconate aldolase)pathways. Unfortunately, little was known on this special sugar metabolism in B.sphaericus untill now. In this research, the glycolysis related enzymes-glucokinase,phosphofructokinase and phosphate glucose isomerase in B. sphaericus had been studied.Our results confirmed the presence of a glucokinase encoding gene glcK on thebacterial chromosome and the expression of glucokinase during the vegetative growth ofB. sphaericus strains. Furthermore, the glcK was cloned from strain C3-41 and expressedin Escherichia coli. Biochemical analysis revealed that this gene encoded a protein withmolecular weight of 33 kDa and the purified recombinant glucokinase had a K_m value of0.52 mM and 0.31 mM for ATP and glucose, respectively. It has been proved this ATPdependent glucokinase could also phosphorylate fructose and mannose. Sequencealignment of glcK indicated that it had a ATP-binding motif in the N-terminal and aα-helix-turn-α-helix DNA-binding motif in the C-terminal, and these conservatived motifs had a high similiary with Glck of other bacterial, and it belongs to the ROKprotein family.A phosphofructokinase encoding gene pfk was found to be widely distributed in B.sphaericus, it had a sigle copy on chromosome and was composed of 960 bp nucleitidesencoding a protein about 42 kDa. Futhermore, a pfk ORF was cloned from B. sphaericusstrain C3-41 and expressed in E. coli. The expression of pfk in recombinant E. coli straincould complement the PFK activity of a pfk mutated E. coli strain DF1020. And the PFKsequece analysis showed it had the conservative amino acids-binding sites and anATP-bingding domain.Genomic sequence of B. sphaericus C3-41 and the specific PCR analysis revealedthat all B. sphaericus strains lacked phosphoglucose isomerase gene pgi, and could notproduce phosphoglucose isomerase activity. It is postulated that the absence of pgi in B.sphaericus might be one of the reasons for bacterial inability to metabolize carbohydrates.For restoring the EMP pathway of B. sphaericus, a pgi ORF was cloned from B. cereusATCC 33018, and then have it expressed in B. sphaericus 2297 and 2362 under thecontrol of binary toxin promoter and cry3A promoter seperately. All recombinant B.sphaericus strains could expressed PGI during bacterial vegetative and spore stage. Evenif the recombinant could not grow and develop well on a inorganic medium with sugar asa carbon resource as expected, the cell extracts could convert glucose into acid in vitro.Primary anonatation result of the C3-41 genome sequence revealed that B.sphaericus lacked EⅡin the PEP-PTS system as well as the dominance ofoligopiptide-specific binding protein and permease protein in ATP-Binding Cassette(ABC) transporter. Supported by our experimental results, it is obvious that the interruptof EMP in B. sphaericus was ascribed to the absence of pgi gene and PGI production.And the partly absence of transporter system, or some effect of the metabolism regulatedsystem likely result in the inability of sugar metabolism in B. sphaericus recombinants.Our findings provide additional data to further elucidate the specific metabolicpathway and for genetic-improvement of B. sphaericus for further mosquito control.