The role of Gram-negative anaerobic microorganisms as causative or contributory agents in a variety of human diseases is well-documented. For example, the oral anaerobe Fusobacterium nucleatum, in addition to being the most abundant bacterium in periodontal pockets, is frequently involved in abscesses of the periodontium, head and neck infections, chronic sinusitis, otitis media and pleuropulmonary infections. Despite their obvious role in disease, the pathogenic mechanisms of Fusobacterium spp. are virtually unknown. For this reason we propose to develop the molecular genetic techniques necessary for the identification of virulence factors associated with oral anaerobes, particularly members of the genus Fusobacterium. In order to develop such a system, genes encoding metabolic traits must be characterized and incorporated as selectable markers in a plasmid- or transposon-based gene transfer system that is functional in Fusobacterium spp. Except for preliminary studies conducted in this laboratory, the aforementioned prerequisites for genetic analysis have yet to be met for Fusobacterium spp. We have succeeded in introducing broad host-range plasmids into F. mortiferum ATCC 25557 via conjugation. In addition, we have identified two systems unique to F. mortiferum (namely, the maltose and cellobiose phosphotransferase systems) which are eminently suitable as selectable metabolic traits for gene transfer studies. Utilization of a metabolic system as a marker avoids the limitations imposed on selection in strains that are naturally resistant to multiple antibiotics. Significant accomplishments include the purification of maltose-6P:6-phosphohydrolase (MalH) from F. mortiferum ATCC 25557; the cloning, expression in Escherichia coli, and sequence analysis of MalH; the purification of cellobiose-6P:6-phosphohydrolase from F. mortiferum ATCC 25557; the molecular cloning, sequencing, and expression of the gene pbgA, encoding P-b-glucosidase, and the gene pbgB, encoding endo-(1,4)-beta-glucanase, from F. mortiferum ATCC 25557; and organization of current transposon mutagenesis and conjugative systems for use in Fusobacterium spp. Elucidation of the biochemical steps and the mechanism of genetic regulation of the maltose and cellobiose phosphotransferase systems are prerequisite to their use as markers in a gene transfer system for oral anaerobes.
