Unravelling the interactions of nucleoid-associated proteins with DNA using single-molecule biophysical techniques

Nucleoid-associated proteins are a large family of proteins that are involved in both structuring and regulating the bacterial genome. Individually they only make small changes to DNA but work in tandem to organise the nucleoid into a compact and organised structure. In this thesis I look at three of these proteins: the DNA-binding proteins, H-NS and StpA, and the integration host factor (IHF). H-NS and StpA were investigated using tethered-particle motion and optical tweezers. They form filaments along DNA, and can also bridge DNA. I show that these filaments are permanently altered by biologically-relevant (piconewton) forces. Mutants of StpA, with different dimerisation behaviour, demonstrate the importance of oligomerisation in the force-dependent behaviour of these filaments beyond DNA binding. Atomic force microscopy (AFM) was used to study IHF, to determine the angles by which it bends DNA. IHF appears to have two non-specific partial bending modes as well as the canonical bending mode that requires a consensus sequence. Additionally, large clusters of DNA were seen, formed by IHF bridging DNA, providing evidence for IHF in the stabilisation of extracellular DNA in biofilms and a potential role in nucleoid organisation. Individually these proteins only make small changes to DNA and so axial optical tweezers were developed to be able to perform force spectroscopy on DNA that is hundreds of base pairs long. This will allow for sequence specific behaviour to be investigated, and the isolation of single binding sites as with AFM.