Enrichment of bacteriorhodopsin with isotopically labeled amino acids by biosynthetic incorporation in Halobacterium halobium

The growth of Halobacterium halobium was optimized in a chemically defined synthetic medium. Arginine, isoleucine, leucine, lysine, methionine, tyrosine, and valine were found to be essential for growth. Optimal growth rates and cell yields were obtained when the medium was also supplemented with the nonessential amino acids alanine, asparagine, glutamic acid, glycine, phenylalanine, proline, serine, and threonine. The complete synthetic medium supported the same maximum growth rate, cell yield, and production of the integral membrane protein bacteriorhodopsin as was obtained in a complex peptone-based growth medium. Using this defined synthetic medium, isotopically labeled bacteriorhodopsin was produced with several 13C-enriched amino acids. The yield of 13C-labeled bacteriorhodopsin was greater than 35 milligrams of purified protein per litre of cell culture. Key words: bacteriorhodopsin, biosynthetic isotopic labeling, synthetic culture medium, nuclear magnetic resonance.

Components in brain heart infusion selective for chromogenic variants of Staphylococcus aureus

Large numbers of chromogenic variants were isolated from cultures of a parent strain of Staphylococcus aureus growing in the dialysate but not in the residue of brain heart infusion (Difco). Gas–liquid chromatographic analysis of the dialysate detected 18 amino acids in this medium. Large numbers of chromogenic variants also were isolated from 13 of 18 synthetic media deficient in a single amino acid but not in the complete synthetic medium containing all 18 amino acids. Gas–liquid chromatographic analysis detected marked quantitative differences in the amino acid metabolites present in a complete synthetic medium and the synthetic medium deficient in arginine after growth for 12 days. The data suggest that differences in the amino acid metabolism of the parent and chromogenic variants could account for the population changes observed in brain heart infusion.

THE ROLE OF A PROTEASE IN SPORULATION OF PENICILLIUM JANTHINELLUM

Although peptidase A, the trypsinogen-activating acid protease of Penicillium janthinellum, is formed during the post-logarithmic phase of growth and while the organism is actively sporulating, there is no direct correlation between the production of the enzyme and sporulation. The evidence for this is threefold: (a) in surface cultures acridine orange almost completely suppresses enzyme formation but reduces sporulation to a much smaller extent; (b) 6-ethyl-thiopurine decreases spore formation but stimulates the production of peptidase A; (c) transfer of mycelium in the log phase from a complete, synthetic medium to a medium lacking nitrate induces the formation of high enzyme levels without affecting the spore count.The possibility of an indirect connection between sporulation and peptidase production is not ruled out.

STUDIES ON THE NUTRITION OF PHYTOPHTHORA CRYPTOGEA

Phytophthora cryptogea, cause of a root disease of alfalfa, grew well over a wide range of pH values (5.6–7.2) on a synthetic medium containing sucrose as the carbon source. The fungus also grew well on glucose and soluble starch. L-Asparagine was the most favorable source of nitrogen, but failed to support growth of a variant obtained from a single germinated oospore. Low concentrations of CaCl2∙2H2O stimulated growth of P. cryptogea, P. drcehsleri, P. parasitica, and P. boehmeriae. The minimal concentration of thiamine for growth was between 12 and 25 μg/liter. Soil extract, representing 50% or 90% of the volume of the synthetic medium, did not supply the thiamine requirement or stimulate growth of the fungus. Alfalfa root extract, alone or added to the synthetic medium, not only satisfied the thiamine requirement of the fungus but produced about 30% more mycelial growth than the complete synthetic medium.

LATENT VIRAL INFECTION OF CELLS IN TISSUE CULTURE

Mouse fibroblasts (L cells) fail to support the growth of psittacosis virus (6BC strain) if they are maintained on a medium containing only inorganic salts and glucose for 2 days prior to infection. Virus propagation can be stimulated by the addition of a synthetic medium containing amino acids, water-soluble vitamins, glutamine, glucose, and inorganic salts. By omitting single amino acids from the complete synthetic medium, tyrosine, threonine, methionine, isoleucine, phenylalanine, tryptophan, leucine, valine, and cysteine or cystine were found to be essential for stimulation, while lysine, arginine, histidine, hydroxyproline, proline, glutamic acid, aspartic acid, serine, alanine, and glycine were not essential. The cells on deficient media showed varying degrees of degenerative changes, but there was little correlation between ability to support psittacosis virus growth and morphologic condition of the cells. Glucose is also an essential component of the medium for viral growth, but the absence of glutamine had no effect on stimulation of virus propagation. L cell cultures maintained on media deficient in phenylalanine or tryptophan for 2 days before infection were also found to be incapable of supporting virus growth. The implications of this study in latent viral infections are discussed.