Bordetella avium 197 produces a dimeric, 42.6 kDA protein ('osteotoxin') which is toxic for MC3Te-E1 osteogenic cells. Genetic analyses and enzymatic characterization of the purified protein established that the osteotoxic protein was beta- cystathionase (encoded by metC), an enzyme in the biosynthetic pathway of methionine, and that the protein was constitutively produced in the presence of methionine. We have proposed that beta-cystathionase contributes to degenerative changes in bone- forming osteoblast cells by cleaving cystine to thiocysteine, a product which donates sulfur and inactivates essential cellular enzymes. To determine the role of individual amino acids in toxicity and catalysis of cystine, the B. avium metC gene was subjected to oligonucleotide-directed, site-specific mutagenesis. The metC gene was introduced into pET-11d, resulting in a 30-fold increase in production of betacystathionase in E. coli SA2821 as compared to B. avium 197. Substitution of alanine for lysine at position 214, the putative co-factor (pyridoxal 5'-phosphate) binding site, resulted in a 95% reduction in beta-cystathionase activity, indicating that lysine residue 214 is involved in catalysis but is not essential for enzyme activity. Replacement of cysteine residues 117 and 309 with alanine resulted in >90% reduction in enzyme activity while substitution of cysteine residues 88 and 279 had a minor effect on enzyme activity. In order to develop plasmid vectors for genetic transfer and mutagenesis, native plasmids were isolated from fusobacterium species. It was determined that F. nucleatum and F. russii contain one plasmid while F. necrogenes contains two plasmids. We were unable to isolate plasmids from F. periodonticum, F. mortiferum, F. ulcerans, F. sulci, F. necroforum, F. varium, F. perfoetans, F. gonidiaformans, F. simiae, or F. naviforme. A 10 kb plasmid from F. necrogenes was isolated and characterized by restriction enzyme analysis. This plasmid will serve as the basis for shuttle vector development in future studies.
