The Gram-negative species Fusobacterium nucleatum (Fn) is a heterogeneous species of anaerobic bacteria important in Dental plaque biofilms and infections with significant societal impact, including periodontitis. Genomic DNA sequence is available for three strains of Fn, representative of the major subspecies with distinct phenotypes. Our laboratory first reported transformation of Fn. Recent developments include reliable transformation of two Fn strains with second generation shuttle plasmids, and the demonstration of site-directed mutagenesis in two Fn chromosomal genes. The greatest promise for investigations of microbial properties lies in molecular manipulations conducted in host strains with defined genomes. We propose to further develop systems of genetic transfer for Fn focusing on strains with available genomic sequence.
In Specific Aim 1 we will test the hypothesis that DNA restriction prevents or limits efficient transformation and mutagenesis in Fn, using insertional inactivation of selected Fn restriction endonucleases, or cloning of Fn methyltransferases in E. coil for use in pre-methylating DNA as a strategy to overcome restriction barriers.
In Specific Aim 2 we will test the hypothesis that the novel replicon of a native fusobacterial plasmid pFN3 will provide the basis for an alternate stable vector system with a distinct host range. The pFN3 replicon will be characterized and its host range tested in comparison to previously described Fn replicons. Finally, sequence analyses suggest that native fusobacterial plasmids may be mobilizable by conjugation.
In Specific Aim 3 we will test the hypothesis that conjugative gene transfer can be used in Fn. This may be of particular benefit in bypassing host restriction barriers; because single stranded DNA, typically resistant to restriction endonucleases, is transferred to the recipient strain. These studies will enable molecular analyses in representative strains of a diverse and important indigenous pathogen, and will thus provide a critical foundation for future studies of ecology and pathogenesis.
