Molecular Characterization of a Glucokinase with Broad Hexose Specificity from Bacillus sphaericus Strain C3-41

Abstract

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...

Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison with Those of Closely Related Bacillus Species

“…strain NRRL B-14905 lack an additional 17 enzymes involved in carbohydrate metabolism in B. subtilis (data not shown). The absence of a pgi gene from B. sphaericus was reported previously by other authors (28), and the introduction of a heterologous pgi gene into this bacterium was able to restore polysaccharide utilization in vitro but not in vivo (B. Han, unpublished data). Thus, the absence of key metabolizing enzymes and a sugar transport system may explain the metabolic inactivity toward glucose, fructose, and most polysaccharides.…”

“…israelensis, which is the other major bacterium used in the biological control of mosquitoes, B. sphaericus offers a distinct advantage, having higher levels of efficacy and environmental persistence (50,66). Besides being an important bioinsecticide for mosquito control, B. sphaericus has several important phenotypic properties, including those of being incapable of polysaccharide utilization and having exclusive metabolic pathways for a wide variety of organic compounds and amino acids (3,28,54,59,72).…”

Identification and Regulation of the N -Acetylglucosamine Utilization Pathway of the Plant Pathogenic Bacterium Xanthomonas campestris pv. campestris

“…These glucokinase family proteins (PF02685 in the Pfam database [5]) possess a ROK domain (PF0480) and might be members of group B of the hexokinase superfamily (27). Although the physiological functions of most members of this group are unknown, several enzymes belonging to this family have been shown to phosphorylate glucose and other hexoses (8,21). Our work provides the first evidence of a role in phosphorylation of GlcNAc for this protein family.…”

“…campestris growth on GlcNAc-containing media, this protein can substitute for NagK-IIA in GlcNAc phosphorylation. The glucokinase family protein GlcK from Bacillus sphaericus has been reported to have substrate ambiguity, phosphorylating not only glucose but also fructose and mannose (21). It is thus possible that GlcNAc is an alternative substrate for NagK-IIB.…”

Optimization of Bio-Mosquitocidal Production using Local Bacterial Strains of Bacillus Sphaericus

“…It has several important phenotypic properties, including those of being incapable of polysaccharide utilization and having exclusive metabolic pathways for a wide variety of organic compounds and amino acids [4,5].…”