Michael A. Gealt

The sterols and fatty acids from purified flagella ofChlamydomonas reinhardi

Purified flagella of the eukaryotic algaChlamydomonas reinhardi have a sterol composition (55% ergosterol [24β-methylcholesta-5,7,22-trans-trien-3β-ol] and 45% 7-dehydroporiferasterol [24β-ethylcholesta-5,7,22-trans-trien-3β-ol]) identical of that of the whole algal cell. Fatty acids isolated fromC. reinhardi flagella were identified as 16∶0, 18∶0, 18∶1, 18∶2 and 18∶3. Whole cell fatty acids included 14∶0, 16∶2 and 16∶3 in addition to those found in the flagella. Triunsaturates comprised 22.9% of the flagellar fatty acids and 76.4% of those from the whole cell.

Ergosterol and Lanosterol from Aspergillus nidulans

Ergosterol was identified as the major free sterol of Aspergillus nidulans by thin-layer chromatography, alumina column chromatography, gas-liquid chromatography, high-performance liquid chromatography, UV spectroscopy, proton magnetic resonance spectroscopy and mass spectral analysis. Lanosterol, the initial cyclized precursor of ergosterol, was identified as a minor component of the free sterols. In the steryl ester material, however, lanosterol was usually more abundant than ergosterol, suggesting that the esters serve as storage compounds for the membrane sterol precursors.

The sterols of growth and stationary phases of Aspergillus nidulans cultures.

The accumulation of 4-desmethyl and 4,4-dimethyl sterols, as well as the triterpenoid beta-amyrin, was analysed during both exponential and stationary phases of Aspergillus nidulans growth. Throughout growth, the amount of 4-desmethyl sterol was proportional to the cellular dry weight, while the dimethyl sterols and beta-amyrin stopped accumulating after day 2. The sterols were found primarily as the free alcohol and not as fatty acid esters, the glycosides, or acyl glycosides. The amount of beta-amyrin in stationary phase cultures was affected by the concentrations of Mg2+ and Cu2+.

Growth Characteristics of Saccharomyces cerevisiae and Aspergillus nidulans when Biotin is Replaced by Aspartic and Fatty Acids

When either aerobic or anaerobic cultures of Saccharomyces cerevisiae were supplemented with aspartic and fatty acids in place of biotin, stationary phase populations were very small compared with those obtained in the presence of biotin. Similarly, these acids failed to fulfil the role of biotin-requiring strain of Aspergillus nidulans. Furthermore, a requirement for saturated fatty acid was found with anaerobically cultured S. cerevisiae. Cells were fragmented when biotin was replaced by aspartic and oleic acids alone, while cellular integrity was maintained, but with only slight growth, when biotin was replaced by oleic and palmitic acids together with aspartate. The importance of biotin in the growth of A. nidulans was particularly pronounced in the presence of glucose. In a medium containing glucose, growth ceased when biotin was replaced by aspartate and Tween 80 (a source of saturated and unsaturated fatty acids), but such replacement permitted a very small amount of growth to occur in the absence of glucose.

Recombinant plasmid gene transfer in amended soil

Genetically engineered E. coli amended into a non-sterile soil microcosm were able to mobilize their recombinant plasmid DNA into a plasmidless E. coli recipient. This transfer required participation of a mobilizer bacterium containing a self-transmissible (conjugative) plasmid. Mobilization also depended on mob sequences present on the non-conjugative recombinant plasmid. Sequences from the non-conjugative plasmid pHSV106, which contains the herpes simplex virus thymidine kinase gene, were identified by DNA hybridization in recipient cells isolated from the soil.

Vectors & Fomites An Investigative Laboratory for Undergraduates

One need not look very far to find relevant news items concerning man and the microbial world. The media of the past decade have been filled with stories of newly emerging and reemerging disease agents (Ebola, Hantavirus, tuberculosis), modern epidemics (HIV, herpesvirus), as well as the many accomplishments of the recombinant DNA revolution. The problem has always been making microbiology relevant to undergraduate students. Human microbiological concerns, in one form or another, have revolved around the transfer of microbial agents from one organism to another. Researchers want to know how to avoid transfer of a disease agent or how to prevent the spread of the products of genetic engineering. The history of microbial analysis is full of studies detailing how a microbe gets from one place to another. In many cases the transfer occurred because of interaction with an invertebrate, such as a tick (lyme disease) or mosquito (encephalitis). In other cases transfer occurs from the surface of a contaminated needle (HIV). Transfer agents are referred to as fomites (nonliving) or vectors (living). A review of laboratory manuals utilized in introductory general microbiology courses indicates few exercises dealing with laboratory model systems to demonstrate vector or fomite activities. The purpose of this exercise is to allow students to discover the action of vectors without exposing them to infectious agents. We also use this exercise as a means to introduce genetically engineered mi-

Isolation of beta-amyrin from the fungus Aspergillus nidulans.

The pentacyclic triterpene alcohol beta-amyrin, which is commonly found in plants, was isolated from wild-type cultures of the ascomycete fungus Aspergillus nidulans. The isolated beta-amyrin was characterized by TLC, GLC, and HPLC and produced identical mass and 1H NMR spectra to those of authentic beta-amyrin. This material was isolated from static (non-shaking) cultures.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase of Aspergillus nidulans

Abstract The 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) of the filamentous fungus Aspergillus nidulans has been analyzed to further characterize the regulatory aspects of ergosterol biosynthesis. The supernatant contained approximately 95% of the total recovered activity following centrifugation (8000g) of homogenized cells. Following ultracentrifugation (105, 000g), total activity was evenly distributed between the supernatant and pellet, although specific activity was 2.5-fold higher in the pellet. The enzyme possessed a pH optimum at pH 7.0, along with a second peak at pH 5.5. The V max was 328 pmol NADPH oxidized per minute, and the apparent K m for dl -HMG-CoA was 28.5 μ M . Consistent with the pattern of sterol production, HMGR specific activity was highest during rapid growth and decreased during stationary phase. This pattern is different from that observed in Saccharomyces cerevisiae and suggests that the regulation of ergosterol biosynthesis in A. nidulans is quite different from that observed in yeast. Addition of exogenous ergosterol (1–10 μg/ml) to the medium (in the presence of the sterol synthesis inhibitor micronazole) caused at 31–36% decrease in HMGR specific activity in culture extracts. This reaction to ergosterol (or a metabolic by-product) suggested that A. nidulans may utilize a regulatory response analogous to that reported in animal cells.

Lipids and Lipoidal Mycotoxins of Fungi

The presence of lipids in fungi is necessary for maintenance of proper membrane structure and function, storage of energy in the form of triglycerides, and the production of specialized molecules, e.g., hormones and toxins. Species of fungi are able to establish themselves in virtually all types of environments, including on and in both animals and plants. Under most conditions the immune system of animals is capable of controlling systemic fungal infections, which is fortunate because the similarity in physiology that exists between the fungus and its host makes treatment and management of the disease state most difficult. Immunosuppressed states due to disease, e.g, acquired immunodeficiency syndrome (AIDS), or immunosuppressive therapy, e.g., that administered in conjunction with organ transplants, have led to a dramatic increase in the frequency of certain types of fungal infection. Chief among these diseases are those caused by Candida and Aspergillus species, both of which are found abundantly in nature.

Sterol Metabolism in Aspergillus Species

The functional complexity of the eukaryotic membrane requires a similar structural complexity. One of the major components of the membrane is the sterol molecule, such as cholesterol (in animals), sitosterol (in plants), and ergosterol (in fungi). These neutral lipid molecules act not only as a bulk lipid, but also act to stabilize the fluidity of the membrane structure, thus allowing for protein function under conditions such as high or low temperature under which the phospholipid hydrocarbon chains might either gel or become too fluid for proper physiological functioning.