Reporter gene systems for Fusobacterium nucleatum

Fusobacterium nucleatum is an oral pathogen that has been shown to have a role in periodontal disease.  F. nucleatum is an autochthonous member of the oral bacterial community, its numbers have been shown to increase with disease onset.  F. nucleatum has been shown to be involved in biofilm formation in the oral cavity and production of proteins that inhibit and kill immune cells.  Due to the importance that F. nucleatum plays in periodontal disease three F. nucleatum genomes have been sequenced.  To help investigate F. nucleatum pathogenesis we constructed reporter gene systems using luciferase and green fluorescent protein (GFP).  Objectives:  The focus of this research was to develop reporter systems in F. nucleatum for studying gene expression and enabling microscopic visualization of bacterial cells.  Methods:  Several F. nucleatum metabolic genes promoters were cloned and fused with luciferase or GFP reporter genes in an F. nucleatum shuttle plasmid.  Expression levels for the luciferase reporter fusions were assessed using a luciferase assay.  F. nucleatum cells were incubated in the presence of oxygen before visualization or measurement of the reporter product.  Results:  Reporter constructs using F. nucleatum promoters resulted in expression of reporter genes in both E. coli and F. nucleatum.  Expression of reporters was higher in E. coli than in F. nucleatum.  Reporter genes using the Butyrl-CoA promoter showed 8-fold higher luciferase activity than reporter genes using major outer membrane protein (FomA) and 100-fold higher activity than RNA polymerase (RpoB) promoters.  Conclusions:  The GFP reporter system will allow visualization of F. nucleatum during cell activities including adhesion, cell invasion, pathogenesis, or biofilm formation and permit more precise quantification of cell numbers than culture methods.  The luciferase reporter will enable quantification of gene expression during these assays.

Research supported by Washington Dental Service, (W.S.) C3 Scientific Corporation (W.S.) and USPHS Grant DE12639 (S.K.H.)