The Borrelia burgdorferi Hbb protein shows sequence similarity to members of the Escherichia coli HU/integration host factor (IHF) family of DNA accessory factors. We have overexpressed the hbb gene product in E. coli and purified the protein to near homogeneity. Biochemical analyses have revealed that Hbb has unique properties and is neither a strict HU nor IHF analogue. Hbb was found to bind specifically to a site in the putative origin of DNA replication between dnaA and dnaN. DNA footprinting studies have shown that this site is unrelated to the consensus sequence recognized by IHF proteins. Hbb induces a dramatic bend (> 126 degrees ) at this site and was also shown to restrain negative supercoils efficiently upon DNA binding. These features of the protein suggest that Hbb may act as a DNA accessory factor that facilitates the assembly of higher order protein-DNA complexes, such as those involved in DNA replication, transcription, recombination, packaging and perhaps other DNA metabolic processes unique to Borrelia.