The objectives of this project were to develop a high performance liquid chromatographic method for separating the pigment esters mixture, to determine the locations of the pigment moiety in the isolated esters using pholosiphases, and to characterize the pigment-protein complex and determine its distribution in other bacteria. Saponification of the two pigment esters 1 and 2 with aqueous KOH yielded two free pigments on TLC plates developed by two solvent systems. The fasters moving of these two free pigments co-chromatographed with the one free pigment produced from each pigment ester by phospholipase A2 treatment. This suggests that the pigment molecule is a methoxy derivative of xanthomonadin and is esterified to the 2-position of the glycerol moiety of each pigment ester. No free pigment was released from phospholipases C and D treatment of the two pigment esters, indicating that pigment is not esterified to the sorbitol or phosphate moiety of pigment esters 1 or 2.

Carotenoid pigments in W. dermatitidis, the first pathogenic, dematiaceous fungus in which carotenoid pigments nave been reported, are located primarily (81%) in lipid organelles which floated on the surface of the supernatant fraction of lysed cells. Pigment in this fraction could be extracted with ethyl ether without prior treatment with acetone indicating the pigment is unbound in the lipid organelle. Eight percent remains after exhaustive ether extraction and is recovered after the sample is treated with acetone indicating this fraction is non-covalently bound to proteins in the membranes associated with the lipid organelle. The remaining pigment (about 12%) represents contamination of the supernatant with the lipid organelles.

Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis. Bacterial ATCases are divided into three classes, A, B and C. Class A ATCases are largest at 450-500, are. dodecamers and represented by Pseudomonas ATCase. The overlapping pyrBC' genes encode the Pseudomonases ATCase, which is active only as a 480 kDa dodecamer and requires an inactive pyrC'-encoded DHOase for ATCase activity. ATCase has been studied in two non-pathogenic members of Mycobacterium, M. smegmatis and M. phlei. Their ATCases are dodecamers of molecular weight 480 kDa, composed of six PyrB and six PyrC polypeptides. Unlike the Pseudomonas ATCase, the PyrC polypeptide in these mycobacteria encodes an active DHOase. Moreover, the ATCase: DHOase complex in M. smegmatis is active both as the native 480 kDa and as a 390 kDa complex. The latter lacks two PyrC polypeptides yet retains ATCase activity. The ATCase from M. phlei is similar, except that it is active as the native 480 kDa form but also as 450,410 and 380 kDa forms. These complexes lack one, two, and three PyrC polypeptides, respectively. By contrast,.ATCases from pathogenic mycobacteria are active only at 480 kDa. Mycobacterial ATCases contain active DHOases and accordingly. are placed in class A1 . …

Olestra is, a sucrose polyester, a noncaloric fat substitute, made from sucrose and several fatty acid esters. It has been approved by the FDA as a food additive used in preparing low-fat deep-frying foods such as savory snacks. Available literature on olestra was evaluated that had both positive and negative connotations. Clinical trials in numerous species of animals including humans were conducted to determine if olestra would affect the utilization and absorption of macro- and micronutrients; the effects of olestra on growth, reproduction, or its toxicity were also examined. The roles of olestra as a fat substitute, how it could effect on humans and the environment, and the potential impacts from its use in large amounts were assessed. Olestra can be removed from the environment by aerobic bacteria and fungi which may be isolated from activated sludge and soils.

An aspartate transcarbamoylase (ATCase) gene from Bordetella pertussis was amplified by PCR and ligated into pT-ADV for expression in Escherichia coli. This particular ATCase (pyrB) was an inactive gene found adjacent to an inactive dihydroorotase (DHOase) gene (pyrC'). This experiment was undertaken to determine whether this pyrB gene was capable of expression alone or if it was capable of expression only when cotransformed with a functional pyrC'. When transformed into E. coli TB2 pyrB-, the gene did not produce any ATCase activity. The gene was then co-transformed into E. coli TB2 pyrB- along with a plasmid containing the pyrC' gene from Pseudomonas aeruginosa and assayed for ATCase activity. Negative results were again recorded.