<p>Peptidoglycan (PG) remodelling and cell division are two important cellular processes that are the major target of antibiotics. Due to rising resistance, the need for new antibiotics today has never been greater. Therefore it is important to fill the gaps in our understanding of these two important processes in order to discover new and promising antibiotic targets. Peptidoglycan synthesis and remodelling is a highly coordinated event that involves a wide number of enzymes and processes which are not well understood. N-acetylmuramoyl-L-alanine amidases, whose function is to cleave the amide linkage between the stem peptides and the lactyl moiety of N-acetylmuramic acid, is a major class of PG-active proteins. Their role in daughter cell separation during cell division is well established in <em>Escherichia coli</em> however little is known about it in other systems. Using enzymatic assays we characterize AmiC as a novel amidase in <em>Pseudomonas aeruginosa. </em>Through mutational analysis and microscopy we show that AmiB and AmiC are required for daughter cell separation. A deletion of both enzymes results in a cell chaining phenotype with abnormal cell morphology. Transmission electron microscopy reveals that the double mutant is arrested at the septal peptidoglycan separation step. In addition to cell chaining, the ∆<em>amiB/amiC</em> mutant exhibits a significant increase in susceptibility to antibiotics. We also demonstrate that the LysM motif of AmiB is not required for its role in cell separation. Furthermore, the <em>amiB</em> mutant has significantly shorter cells than the wildtype indicating an additional role for the enzyme in the cell. Lastly, through a novel bioinformatics strategy we identify PA5047 as a potential PG amidase.</p> / Bachelor of Science (BSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/13376 |
| Date | 10 1900 |
| Creators | Al-Saigh, Sarra |
| Contributors | Burrows, Lori, Biochemistry |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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