Characterisation of Lipopolysaccharide O-antigen Acetyltransferases in the Bacterium Salmonella

Characterisation of Lipopolysaccharide O-antigen Acetyltransferases in the Bacterium Salmonella Reyme Herman MSc by Research University of York Biology March 2015 Abstract Bacteria are known to possess various modifications on the cell envelope for a variety of reasons, including pathogenicity and survivability. Components on the Gram-negative bacteria cell envelope such as the lipopolysaccharide (LPS) and peptidoglycan, among others, are commonly modified in a multitude of ways. In Salmonella, the LPS itself can possess different modifications on the three components of the molecule; the O-antigen, core polysaccharide and lipid A. This study focuses on two LPS O-antigen acetyltransferases, namely OafA and GtrC family II proteins, which target different sugar residues within the O-antigen. Findings from this project suggest that OafA and GtrC family II proteins are highly similar and possess two domains in the form of an N-terminal inner membrane bound acyltransferase 3 domain and a C-terminal SGNH hydrolase domain. SGNH hydrolase domains possess a characteristic α/β/α fold. In addition, a catalytic triad of serine, aspartate and histidine is also present with the latter being separated by a maximum of two amino acids (DXXH). Using the GtrC family II protein from the invasive non-typhoidal Salmonella (iNTS) strain S. Typhimurium D23580, GtrCBTP1, as a model for GtrC family II proteins, the amino acids within the catalytic triad were individually replaced by alanine and the functions were analyzed by phage susceptibility. Each of the single replacement mutations resulted in the abrogation of the protein function, suggesting that each of the catalytic residues tested are important for protein function. To further describe these acetyltransferases, biochemical and biophysical experiments would be performed and these methods require pure samples of proteins. This study developed a method for the successful expression and purification of these membrane bound acetyltransferases with an overall yield of approximately 1.2 mg of the purified proteins per litre of bacterial culture. Expression of these proteins were performed in an E. coli based expression culture using an L-arabinose inducible vector which also introduced a deca-His tag on the C-terminal end of the expressed proteins. The methods described in this thesis for the expression and purification of OafA and GtrCBTP1 can be brought forward to aid in the preparation of these proteins for future biochemical and biophysical experiments.