| An indispensable part of the cell envelope, the bacterial cell wall, is a vitally important part of virtually all bacteria. Many proteins found in the bacterial cell envelope are involved in biosynthesis of the cell wall, its remodeling and recycling or protection of this structure from antibacterial agents (beta-lactamases). The significance of these processes came to fore soon after introduction of penicillin to clinical use in 1940s.;Currently, many of the antibiotics used in clinical practice target different aspects of cell wall biosynthesis. The biggest class in this group of antibiotics is that of beta-lactams. Exposed to beta-lactam antibiotics, bacteria quickly develop new mechanisms to neutralize these harmful substances and to preserve the cell wall integrity. One such mechanism is production of beta-lactamases, enzymes that hydrolyze beta-lactam antibiotics. Three classes of these enzymes are believed to have evolved from ancestral penicillin-binding proteins (PBPs), enzymes responsible for bacterial cell wall biosynthesis. Although both PBPs and Gram-negative beta-lactamases operate in the periplasm, PBPs are anchored to the cytoplasmic membrane, but beta-lactamases are not. It is believed that beta-lactamases shed the membrane anchor in the course of evolution. The significance of this event remains unclear. Our attempt to demonstrate potential influence of the membrane anchor on the overall biological efficiency of beta-lactamases is going to be discussed in this dissertation.;In some Gram-positive bacteria expression of beta-lactamases is induced by beta-lactam antibiotics. Arguably, the most clinically significant pathogen from this group is Staphylococcus aureus. Expression of the beta-lactamase, encoded by blaZ, is regulated by two genes, blaR1 and blaI. Our efforts towards cloning, expression of these genes, and elucidation of the blaZ expression regulation are one of the subjects of this dissertation.;In our search for answers about regulation of the cell wall biosynthesis, remodeling, and recycling we turned our attention to the low-molecular-weight PBP 5 and six lytic transglycosylases found in E. coli. Our findings about biochemical functions and kinetics properties of these enzymes are reported in this dissertation. |
