Essentiality of Methionine Aminopeptidase in Staphylococcus Aureus

This dissertation, "Essentiality of Methionine Aminopeptidase in Staphylococcus Aureus" by Chi-wai, Bonnie, Wong, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Essentiality of methionine aminopeptidase in Staphylococcus aureus Submitted by Wong Chi Wai Bonnie for the degree of Master of Philosophy at the University of Hong Kong in December 2004 Staphylococcus aureus, a Gram positive pathogen, is a major cause of potentially life-threatening infections acquired in hospitals and in the community. These infections include endocarditis, wound and surgical infections, and bloodstream infections. S. aureus has developed resistance to most classes of antimicrobial agents after their introduction into clinical use, leaving vancomycin as the last line of defense for staphylococcal infections. However, the recent emergence of vancomycin resistant S. aureus strains calls for the need to identify novel targets for the development of antimicrobial therapies. Methionine aminopeptidases (MetAPs), encoded by the map gene, are enzymes responsible for the catalytic removal of the N-terminal methionine from newly formed peptides, if the second residue has a small and uncharged side-chain. In Gram negative bacteria, such as Escherichia coli and Salmonella typhimurium, and yeast Saccharomyces cerevisiae, map has been demonstrated to be essential. In Gram positive bacteria, the essentiality of map has not been illustrated experimentally. The crucial role of map in S. aureus was examined in the present study. Deletion of map in S. aureus resulted in lethality, leading to the conclusion that map is essential. Mutant strains of S. aureus were constructed by fusing a tetracycline-inducible promoter xyl/tetO to map, whereby map gene expression could be controlled by the presence or absence of an inducer anhydrotetracycline (ATc) and growth dependence on ATc could be monitored. In vitro analysis of the xyl/tetO promoter revealed that it was transcriptionally "leaky." To tighten up the system, an additional tetO sequence was inserted into the promoter region to create a xyl/tetO promoter, which was coupled to the insertion of additional nucleotides in the spacing region between the ribosome-binding site (RBS) and the start codon (AUG) of map. Six genetically modified strains of S. aureus were constructed; three of them, designated RKC124, RKC126, and RKC168, possessed 12, 16, and 20 RBS - ATG spacing nucleotides for map, respectively, in the background of the xyl/tetO promoter. The genetic construct of each mutant was verified by PCR, sequencing, and Southern hybridization experiments. Growth experiments in liquid cultures of RKC124, RKC126, and RKC168 demonstrated that as the RBS - ATG spacing for map increased, growth was increasingly delayed in the absence of inducer ATc. When ATc was absent, strain RKC168 grew only to pinpoint colonies after incubation for 16 hours. It was further determined that the possession of a map gene harbored in a multicopy plasmid and transcribed from the S. aureus native promoter was able to relieve the tetracycline dependence of RKC168, indicating that map, and not the expression of another gene located downstream from map, is essential for cell growth. The experimental demonstration of the essentiality of map in S. aureus has validated map as an attractive target for the development of broad-spectrum antibacterial and antifungal therapeutics. The conditional map mutant will aid future studies of the physiological role of MetAPs and discovery of MetAP-specific antibiotics. DOI: 10.