Organisms of the genus Fusobacterium are important human and animal pathogens which are isolated frequently from liver and brain abscesses. F. nucleatum is isolated from normal subgingival dental plaque but its numbers increase dramatically in cases of gingivitis and periodontitis suggesting pathogenic potential. Little is known about the physiology of the fusobacteria and most published reports refer to these organisms as asaccharolytic although sugar use is easily demonstrated. Previous work in this laboratory has shown that glucose transport by F. nucleatum is dependent on energy provided by the fermentation of amino acids. In addition, we have shown extensive sugar use by F. mortiferum strongly suggesting that this organism can be used successfully as a model for genetic studies which are lacking in the fusobacteria. In the past year we have concentrated on the proof of the cellobiose phosphoenolpyruvate phosphotransferase (PTS) use in F. mortiferum. A cellobiose PTS assumes the intermediate formation of cellobiose-6-P and its subsequent hydrolysis by a specific hydrolase. Cellobiose-6-P and a variety of other beta disaccharide phosphates were made using an ATP kinase purified from Klebsiella pnemoniae. The beta glucoside phosphates had the predicted molecular weight when analyzed by mass spectrometry and were all hydrolyzed by the 54 KD protein from cellobiose grown F. mortiferum which we suggested as the cellobiose- 6-P hydrolase. In addition, an artificial beta glucoside phosphate was made from p nitrophenyl beta D glucoside [PNP beta GP]. The hydrolysis of PNP beta GP gave a yellow color which could be used to quantitate the hydrolase activity or be used qualitatively to follow the purification of the enzyme. The cellobiose phosphate hydrolase retained activity in air in contrast to the maltose-6-P hydrolase. Washed, cellobiose grown cells of F. mortiferum used limited amounts of cellobiose from a buffered suspension when incubated aerobically. The aerobic inhibition of cellobiose use was gradual, rather than abrupt, as in the maltose system of the same organism.
