Metabolism of sugars by genetically diverse species of oral Leptotrichia

Leptotrichia buccalis ATCC 14201 is a gram-negative, anaerobic rod-shaped bacterium resident in oral biofilm at the tooth surface. The sequenced genome of this organism reveals three contiguous genes at loci: Lebu_1525, Lebu_1526 and Lebu_1527. The translation products of these genes exhibit significant homology with phospho-a-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 50-kDa 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 TOP10 cells. In the presence of two cofactors, NAD+ and Mn2+ ions, the enzyme readily hydrolyzed p-nitrophenyl-a-glucopyranoside 6-phosphate (pNPaG6P), a chromogenic analogue of the phosphorylated isomers of sucrose. By comparative sequence alignment, immunoreactivity and signature motifs, the enzyme can be assigned to the phospho-a-glucosidase (Pagl) clade of Family 4 of the glycosyl hydrolase super family. We suggest that the products of Lebu_1527 and Lebu_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. The phenotypic traits of these new species, with respect to carbohydrate utilization, have also been determined.