ENZYMOLOGY OF BACTERIAL CELL MEMBRANES

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The objective of this project is to study how the enzymes of the cytoplasmic membrane of Micrococcus luteus, a gram positive bacterium, effect the biosynthesis of teichuronic acid which is covalently linked to the peptidoglycan. A series of intermediates containing undecaprenyl phosphate as carrier lipid effect teichuronic acid biosynthesis by sequential elongation of the carbohydrate chain of teichuronic acid. Modified cell preparations will be used to determine if some of the intermediates can transfer the putative teichuronic acid chain to peptidoglycan. Concomitant peptidoglycan synthesis may also be required. Another objective is to isolate and chemically characterize the unique linkage region oligosaccharide which joins teichuronic acid to peptidoglycan. An enzymatic and chemical degradation scheme based on the presumed structure of the linkage region will be followed by purification. Methylation analysis, mass spectrometry and nuclear magnetic resonance spectroscopy will be used for characterization. The glycosyltransferase which is involved in teichuronic acid chain elongation will be purified so that the mechanism can be determined by which glucosyl residues are incorporated into the polymer with retention of anomeric configuration while the alternate residues of the polymer are incorporated with inversion of configuration. Teichuronidase, an enzyme which degrades teichuronic acid, will be isolated from an organism which can utilize teichuronic acid as growth substrate. Characterization of the enzyme will follow. Immunoelectron microscopy utilizing antibodies directed against teichuronic acid will be used to evaluate the location of teichuronic acid in the cell wall and sites of synthesis in the cytoplasmic membrane. Proteus myxofaciens, a gram negative bacterium, produces an extracellular gel which will be isolated and characterized by methylation analysis, mass spectrometry and nuclear magnetic resonance spectroscopy.