Regulation Of Sugar Transport And Metabolism In Oral Bac

Dental & Craniofacial Research

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Abstract

Dietary sucrose is a major contributor to the etiology of dental caries. This disaccharide, (comprised of glucose and fructose molecules) provides the precursors for glycan synthesis, that facilitates adherence of Streptococci to the tooth surface. The localized production of lactic acid (via the microbial fermentation of sucrose) causes demineralization of surface enamel, and initiation of tooth decay. Positional change of the O-glycosidic linkage between C1 of the glucose moiety and the remaining five -OH groups of the fructose ring, yields the five stereo- isomers of sucrose: trehalulose, turanose, maltulose, leucrose and palatinose. Prior to commencement of our research program, it was generally assumed the microorganisms were unable to metabolize the isomers of sucrose. This belief, and the fact that the isomers are comparatively sweet, has encouraged the use of these ?non-cariogenic? compounds as substitutes for sucrose in various food products. Surprisingly, and contrary to expectation, we have found that many bacterial species from both Gram-positive and Gram-negative genera (including, Klebsiella pneumoniae, Bacillus subtilis, Clostridium acetobutylicum and Fusobacterium mortiferum) readily grow on these five isomeric disaccharides. Bacteria with the capacity to metabolize the isomers of sucrose invariably possess two chromosomal genes that encode: (i) a membrane-localized phosphoenolpyruvate (PEP)-dependent transporter that facilitates the simultaneous translocation and phosphorylation of the isomers, and (ii) a unique NAD/Mn(2+) -dependent phospho- alpha-glucosidase that catalyzes the intracellular hydrolysis of phosphorylated isomers to yield glucose 6-phosphate and fructose. In the case of Klebsiella pneumoniae, the two genes (aglA, aglB) and their products are designated AglA and AglB, respectively. Although prerequisite for growth of K. pneumoniae and other species, it was unclear whether expression of these two proteins alone, was sufficient for growth of microorganisms on the isomeric disaccharides. Of particular concern was the fact that these particular species lack a gene encoding EIIA(Agl), a third phosphorylatable protein required for functional activity of a PEP-dependent Agl: phosphotransferase system (PTS). Previously (but without proof), we suggested that an EIIA from a different PTS must substitute for the ?missing? EIIA(Agl) in order to complement activity of the PEP-dependent Agl transport system. To address this hypothesis, a collaborative investigation was initiated in 2006 with researchers at the University of Berne, Switzerland. For our studies, Escherichia coli K-12 was the organism of choice, because this enteric bacterium is unable to metabolize sucrose or its isomers, and is readily amenable to genetic manipulation. Importantly, strains of E. coli K-12 containing the necessary mutations, and purified components of the phosphotransferase system (HPr and Enzyme I) were made available by our collaborators. Our experimental approach was to first clone both aglA and aglB genes into an expression plasmid (pAP2). When transformed with this plasmid, E. coli K-12 grew readily on a wide variety of O-alpha-linked glucosides, including all isomers of sucrose. Subsequently, a mutant strain of E. coli K-12 lacking EIIA(Glc) of the glucose: PTS was also transformed with pAP2. This transformant failed to grow on any of the alpha-glucosides tested, thus suggestive of EIIA(Glc) participation in the transport of sucrose isomers. Confirmation for our hypothesis was obtained from in vitro experiments conducted with membrane preparations of the E. coli K-12 containing the AglA transporter from K. pneumoniae. When supplemented with the high-energy phosphoryl donor (phosphoenolpyruvate), HPr and EI, these cytoplasmic membranes failed to catalyze the phosphorylation of the isomeric compounds. However, upon addition of purified EIIA(Glc) to the reaction mixture, an immediate and rapid phosphorylation of the disaccharides was observed. We believe that AglA (transporter), AglB (phospho-alpha -glucosidase), and EIIA(Glc) of the glucose-PTS are necessary and sufficient, for the growth of microorganisms on sucrose isomers and related alpha-D-glucopyranosides.