Présentation sous forme d’affiche au congrès CSM (Canadian Society of Microbiology) à Laval le 26 mai 2003.

Paradis-Bleau, C., Sanschagrin, F., and Levesque, R.C. 2003. Molecular genomics and biochemical characterization of the FtsA and FtsZ cell division proteins from Pseudomonas aeruginosa . 53rd Annual Meeting of the Canadian Society for Microbiologists, Laval, Quebec. May 25 to 28, 2003.

The revolutionary era of antibiotics has been surpassed by the evolutionary capacity of microorganisms to develop resistance. The acute resistance of the opportunistic pathogen Pseudomonas aeruginosa to most classes of antibiotics is a good example. With the objective of developing new classes of antimicrobial agents, we must have a better understanding of essential prokaryotic mechanisms such as bacterial cell division. We studied FtsA and FtsZ, two essential and highly conserved proteins in bacteria. FtsA and FtsZ from P. aeruginosa were cloned in the pET30a overexpression vector and fused with a his tag at the C terminus. Overexpression of FtsA in E. coli BL21-(λDE3) led to the accumulation of the protein in inclusion bodies. To overcome this frequent problem, we developed a protocol permitting the isolation, refolding and production of a biologically active FtsA. We obtained 5 mg of 99% pure FtsA with a concentration of 0.5 mg/mL and N-terminal sequencing confirmed the identity of the protein. Overexpression of FtsZ permitted the accumulation of soluble proteins in the cytoplasm of E. coli BL21-(λDE3) and FtsZ was purified on a nickel affinity chromatography. We obtained 20 mg of 99% pure FtsZ with a concentration of 13.5 mg/mL with an identity confirmed by N-terminal sequencing. A UV cross-linking assay was used to study the FtsA binding of ATP and showed that FtsZ binds preferentially GTP among the 4 nucleotides used as substrates. We characterized the specificity of enzymatic activity of each enzyme through a TLC assay. Hydrolysis of nucleotide substrates was measured using P32-labelled nucleotides. We noted that FtsA hydrolyses predominantly ATP and weakly GTP, CTP and TTP. It was also noted that FtsZ hydrolyses predominantly ATP and weakly GTP, CTP and TTP. This information could be useful for the development of screening assays for analysis new inhibitors of prokaryotic cell division.