Extraction, Characterization, and Cellular Localization of the Lipids of Staphylococcus aureus

Satisfactory extraction and assay procedures have been developed for the lipids of Staphylococcus aureus. The following lipids have been characterized in detail: the vitamin K(2), which is shown to exist as isoprenologues with side chains of 35, 40, and 45 carbon atoms; monoglucosyldiglyceride and diglucosyldiglyceride, which account for all the carbohydrate in the lipid extracts; the lysyl ester of phosphatidyl glycerol, phosphatidyl glycerol, and cardiolipin, which account for 98% of the phosphate in the lipid extract. The extraction procedure removes 98% of the total bacterial fatty acids. Acidification of the medium before harvest and refluxing in isopropanol are critical in the extraction procedure for the maximal recovery of lysyl-phosphatidyl glycerol and the glucolipids. The lipids have been shown to be a part of the same membrane as the respiratory pigments.     

Genetics and physiology of colicin-tolerant mutants of Escherichia coli

A series of colicin-tolerant (tol) mutants of Escherichia coli K-12, which adsorbed colicins but were not killed by them, were isolated and studied genetically and physiologically. Three major classes of mutants were found: tol II, tolerant to colicins A, E1, E2, E3, and K; tol III, tolerant to A, E2, E3, and K; and tol VIII, tolerant to E1 only. The sites of tol II and tol III mutations mapped near the gal region (gene order: tol-gal-bio) and were cotransduced with gal by P1. In heterozygous diploids, tol(+) was dominant over tol; tol II and tol III gave full complementation. All the tol mutations that mapped near gal rendered the bacteria more fragile during growth and hypersensitive to deoxycholate and to ethylenediaminetetraacetic acid. The tol VIII mutation mapped between str and his. These mutants were extremely sensitive to deoxycholate and were also hypersensitive to methylene blue, acridines, and various other compounds. The sensitivity is attributed to increased uptake due to selective alteration of the permeability barrier. The colicin-tolerant mutations are interpreted as affecting some components of the cytoplasmic membrane which mediate between the adsorbed colicin molecules and the target sites of their biochemical effects in the bacterial cell.     

Formation of Extracellular Sphingolipids by Microorganisms: IV. Pilot-Plant Production of Tetraacetylphytosphingosine by Hansenula ciferrii

Tetraacetylphytosphingosine (TAPS) formation by the F-60-10 mating type strain of the yeast Hansenula ciferrii, previously observed on agar plates, has been shown to take place in submerged cultures. The optimal conditions for TAPS formation, and the correlation of TAPS production and sugar utilization under aerobic conditions, were studied in 10-liter fermentors. For each gram of glucose consumed, 5 mg of TAPS were formed; for each gram of yeast solids produced, 15 mg of TAPS were synthesized. A 750-liter pilot-plant run yielded 175 g of crude TAPS, which were obtained by hexane extraction of centrifuged yeast cells.     

Induced Autolysis of Aspergillus oryzae (A. niger group): IV. Carbohydrates

The examination of substances formed during induced autolysis by Aspergillus niger was continued in this work, which dealt in particular with carbohydrates. The autolysate contained a large amount of d-glucose (14 to 20% dry wt) and traces of glycolic aldehyde, dihydroxyacetone, ribose, xylose, and fructose. It also contained glycopeptides (about 10% dry wt), which were split from the cell wall during autolysis and which differed from one another in their level of polymerization and their composition. They were constituted by glucose and mannose, glucose and galactose, or mannose, glucose, and galactose (mannose being the most abundant in this case), and amino acids (chiefly alanine, serine, glutamic acid, and aspartic acid). During autolysis, only a part of the cell wall was dissolved, since it retained its shape. Upon further chemical hydrolysis, it produced mostly glucose and glucosamine, and smaller amounts of mannose, galactose, and amino acids. Presumably, glucomannoproteins and glucogalactoproteins were present in the intact cell as a macromolecular complex, constituting, together with chitin, the major part of the cell wall of Aspergillus.     

POLYESTER-METHACRYLATE EMBEDMENTS FOR ELECTRON MICROSCOPY

It has been found that tissues fixed for electron microscopy and dehydrated in acetone can be embedded in mixtures of n-butyl methacrylate and polyester resin. Activation with 1 per cent tert-butyl hydroperoxide followed by 12 to 48 hours at 60°C produces blocks that section well with glass knives. The ribbons are cleared of methacrylate by heat (200–250°C for 1 hour) and/or immersion in organic solvents (CCL₄, acetone-ether). After removal of the methacrylate the residual polyester matrix provides thermostable and insoluble support for the tissue. Its insolubility permits staining by immersion of cleared preparations in organic solvents carrying heavy metal compounds in solution. Clearing by heat stabilizes section-grid relationships. The removal of volatile materials by clearing substantially reduces contamination of both specimen and microscope. Tissue fine structure is well preserved in these preparations.