FERMENTATION OF SUCROSE ISOMERS BY ORAL LEPTOTRICHIA The sequenced genome of L. buccalis ATCC 14201 revealed three contiguous genes at loci: Lebu_1525,1526 and 1527. The translation products of these genes exhibited significant homology with phospho-α-glucosidase (Pagl), a regulatory protein (GntR) and a phosphoenol pyruvate-dependent sugar transport protein (EIICB), respectively. In non-oral bacterial species, these genes comprise the sim operon that facilitates sucrose isomer metabolism. Growth studies showed that L. buccalis fermented a wide variety of carbohydrates, including four of the five isomers of sucrose. Growth on the isomeric disaccharides elicited expression of a 50kDa polypeptide comparable in size to that encoded by Lebu_1525. The latter gene was cloned, and the expressed protein was purified to homogeneity from Escherichia coli TOP 10 cells. In the presence of two essential cofactors (NAD+ and Mn2+ ion) the enzyme readily hydrolyzed p-nitrophenyl-alpha-glucopyranoside 6-phosphate (pNPalphaG6P), a chromogenic analog of the phosphorylated isomers of sucrose. By comparative sequence alignment, immuno- reactivity and signature motifs, the enzyme was assigned to the phospho-α-glucosidase (Pagl) clade of glycosylhydrolase Family 4. It is our contention that the products of Lebu_1527 and 1525, catalyze the phosphorylative translocation and hydrolysis of sucrose isomers in L. buccalis, respectively. Four genetically diverse, but 16S rDNA related species of Leptotrichia have recently been described: L. goodfellowii, L. hofstadii, L. shahii and L. wadei. Our determination of the phenotypic traits of these new species with respect to carbohydrate utilization yielded some unexpected findings. Whereas non-oral bacterial species invariably grow on all sucrose isomers, considerable variation was noted in the number(s) of isomers that supported growth of a particular species of Leptotrichia. For example, L. shahii metabolized all five isomeric compounds, L. buccalis fermented four of the sucro-disaccharides whereas, under the same conditions, L.wadei failed to grow on either maltulose or leucrose. At the molecular level, such isomeric discrimination presumably reflects subtle species-specific mutations or conformational changes in either the transport or intracellular Pagl proteins. The genetic basis for the variability of metabolic traits may be revealed by future comparative analyses when the genomes of the different species of Leptotrichia have been sequenced in their entirety. As genetic information accumulates in the Human Oral Microbiome Database (HOMD), it likely that genetic loci similar to the sim operon in Leptotrichia will be discovered in presently un-sequenced genomes of oral bacteria. In this context, the fermentation of presumed non-cariogenic disaccharides by oral microorganisms may be more widespread than currently envisaged. Our findings have been published in the peer-reviewed journal, Molecular Oral Microbiology. STRUCTURE AND FUNCTION OF PHOSPHO-BETA-GLUCOSIDASE (BGLA-2) FROM STREPTOCOCCUS PNEUMONIAE TIGR4. Streptococcus pneumonia is the major causative agent of acute pneumonia, otitis media, meningitis, and septicemia, which annually result in the deaths of millions worldwide. In the human host, S. pneumoniae encounters a variety of glyco-conjugates, including mucins, host defense molecules, and surface exposed glycans on epithelial cells. In common with other pathogenic microbes, S. pneumonia produces a variety of secreted or surface-associated glycosidases whose function(s) include the modification and hydrolysis of host glyco-conjugates. Genome sequencing, in combination with exploration of new virulence factors, suggests that a large number of glycosidases are necessary for maximum virulence of S. pneumoniae. BglA-2 is encoded by the gene Sp_0578 in the chromosomal DNA of S. pneumoniae TIGR4. After cloning of the gene in a high expression vector, BglA-2 (471 residues, Mr = 54,361) was purified to electrophoretic homogeneity. The natural substrates of BglA-2 include: cellobiose-6-phosphate, gentiobiose-6P, arbutin-6P, salicin-6P and related O-beta-linked disaccharide phosphates. These compounds are not commercially available, and accordingly were prepared enzymatically by procedures developed previously in our laboratory (J. Biol. Chem. 277: 34310-34321, 2002). Use of these novel compounds permitted substrate specificity and kinetic analyses to be conducted. Thermodynamic parameters including enthalpy changes, ligand affinity and stoichiometry of binding between substrates and BglA-2 were determined by isothermal titration calorimetry (ITC). Crystals of BglA-2 in native form, and in complex with a non-hydrolyzable analog (thio-cellobiose-6-phosphate), have been prepared, and the structure of the enzyme has been solved by X-ray (synchrotron) diffraction and resolution of phase by molecular replacement. Future experiments, involving site-directed mutagenesis and gene deletion, will address the pathogenic contribution(s) of BglA-2 to S. pneumoniae infection.
