A biochemical and biophysical study on cell division proteins from Staphylococcus aureus and biofilm proteins from Bacillus subtilis.

Abstract

Part I Cell division in bacteria is tightly regulated by a multiprotein complex called the divisome. Proteins in the divisome couple cell division and growth, ensuring that a single copy of the chromosome is present in each resulting daughter cell, and preventing more than one instance of division from occurring at any one time. This thesis concerns a combination of biophysical and biochemical techniques used to study the cell division proteins DivIVA, Stk1 and GpsB from Staphylococcus aureus. A model of the solution molecular envelope of DivIVA is derived by small-angle X-ray scattering and compared to a previously proposed model of the protein from Bacillus subtilis. The molecular mechanisms of DivIVA oligomerization are probed through use of size-exclusion chromatography coupled multi-angle light scattering on various truncations of the protein. The structure of the N-terminal domain of S. aureus GpsB is solved and used to rationalise the interaction between GpsB and PBP4. Attempts are made to determine an interaction network between the cell division proteins and members of the peptidoglycan and wall-teichoic acid synthesis machinery by several biochemical and biophysical assays. Part II Biofilms are communities of sessile bacteria that form on a wide variety of natural and manmade surfaces, sometimes at a detriment to human health. Bacteria in biofilms are held together by a viscous extracellular matrix consisting of polysaccharides, lipids, proteins, and extracellular DNA (eDNA). Species of Bacillus are known to secrete two structurally similar endonucleases, Nuclease A and B (NucA & NucB), into their environment as a means of taking up eDNA either to enhance their genetic diversity, or for metabolic purposes, respectively. As a mechanism of protection from self-induced genome degradation, NucA is co-expressed with a proposed inhibitor, Nin. A combination of biophysical/chemical techniques are used to probe the interaction between NucA/B and Nin from Bacillus subtilis. In vitro studies show that NucA and NucB bind to Nin and that Nin inhibits their endonuclease activity. The affinity of the interactions between NucA or NucB and Nin are probed and found to be sub-nanomolar. The structures of NucA/NucB in complex with Nin are solved by X-ray crystallography, revealing the mechanism of inhibition by Nin, and allowing for the calculated dismantling of the complexes by site-directed mutagenesis.