Synthetic Complete Medium Research Articles

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.

Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed.

An examination of adaptive reversion in Saccharomyces cerevisiae.

Reversion to Lys+ prototrophy in a haploid yeast strain containing a defined lys2 frameshift mutation has been examined. When cells were plated on synthetic complete medium lacking only lysine, the numbers of Lys+ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. An examination of the distribution of the numbers of early appearing Lys+ colonies from independent cultures suggests that the mutations to prototrophy occurred randomly during nonselective growth. In contrast, an examination of the distribution of late appearing Lys+ colonies indicates that the underlying reversion events occurred after selective plating. No accumulation of Lys+ revertants occurred when cells were starved for tryptophan, leucine or both lysine and tryptophan prior to plating selectively for Lys+ revertants. These results indicate that mutations accumulate more frequently when they confer a selective advantage, and are thus consistent with the occurrence of adaptive mutations in yeast.

Disruption analysis of metallothionein-encoding genes in Candida glabrata

Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT- II a, an amplified locus, and MT- II b, a single-copy gene, to determine the roles of various MT genes in CuSO 4 resistance in C. glabrata. The concentration of CuSO 4 required to inhibit the growth by 50% (IC 50) of a C. glabrata strain harboring an amplified MT- II a locus and a single-copy MT- II b and MT- I genes was 7 mM in a synthetic complete medium. The IC 50 decreased to approx. 1 mM when the amplified MT- II a locus was deleted. The disruption of the MT- II b gene decreased the IC 50 further to 0.1 mM. The CuSO 4 resistance in a strain lacking both of the MT- II genes was attributable to MT- I; no evidence was found for the production of (γEC) nG isopeptides. The comparison of the nucleotide sequence of MT- II b to that of MT- II a revealed the same coding sequence with differences in the 5′ region. However, substantial differences were found in the 3′ region. MT- II b was expressed since we were able to purify the protein from the strain that had an intact MT- II b gene, but a deleted MT- II a gene. In addition, CuSO 4 resistance was provided by MT- II b. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT- I in the presence or absence of the MT- II genes.

Effect of plasmid size and medium on growth kinetics and plasmid copy number in Saccharomyces cerevisiae

Saccharomyces cerevisiae cultures were selected to determine the effects of plasmid size on host growth kinetics and plasmid copy number. A complete synthetic medium was verified and the effects of yeast nitrogen base without amino acids medium and leucine selection were established for the strain. The dependence of copy number on the plasmid size, medium, and oxygen availability was also measured. This study was part of a comprehensive effort to elucidate the behavior of recombinant yeast.

Investigations of aneuploidy-inducing chemical combinations in Saccharomyces cerevisiae

For several years we have been investigating combinations of chemicals for their ability to induce aneuploidy. Earlier published results indicated that combinations of certain chemicals showed a potentiation effect while other combinations did not. We have continued to explore this phenomenon and report additional findings in this communication. Combinations of ethyl acetate and methyl ethyl ketone showed a potentiation effect as did 1-methyl-2-pyrrolidinone-nocodazole combinations. Combinations that did not show a potentiation effect were 2-pyrrolidinone-nocodazole and 1-methyl-2-pyrrolidinone-ethyl acetate. We also found that nocodazole, which is a potent inducer of aneuploidy in yeast extract-peptone-dextrose (YEDP) medium but not in synthetic complete (SC) medium, showed a potentiation effect with ethyl acetate in SC medium. This effect in SC medium is similar to that previously reported for nocodazole with ethyl acetate in YEPD medium. When nocodazole was dissolved in 1-methyl-2-pyrrolidinone as a concentrated stock solution, a potentiation effect occurred even at low concentrations of the solvent.

Changes in ribosomal properties during adenylate deprivation in the cells of Kluyveromyces lactis.

In an adenine-requiring mutant strain of the yeast, Kluyveromyces lactis, the intracellular content of ATP is one-third to one-fifth that in a prototrophic wild strain under growing conditions. The quantitative differences becomes rather small in resting stationary-phase cells. Temporary changes in the two-dimensional protein patterns of mutant ribosomes occur when the ATP content is lowest during the transition phase of growth. The transfer of exponentially growing cells to a synthetic complete medium void of adenine induces the same changes in mutant ribosomes within several hours. Identification of ribosomal proteins by two-dimensional gel electrophoresis indicated all changeable proteins (at least five proteins) to belong to 40S ribosomal subunits. The mutant ribosomes prepared from the transition-phase cells have much lower activity (below 60%) for poly(U)-directed polyphenylalanine synthesis than those in exponentially growing or resting stationary-phase cells. Thus, changes in ribosomal components associated with the differences in ribosome activity in a cell-free system were noted in the adenylate-deprived cells of K. lactis.

Polypeptide synthesis during lytic induction of phage 11 of Staphylococcus aureus.

Staphylococcus aureus phage 11 was induced to replicate by treatment of lysogens with mitomycin C. Lysis was complete in 80 min at 37 degrees C in complete synthetic medium. Labelling with L-[3H]lysine showed a stimulation of protein synthesis during phage replication, followed after 30 min by shut-down of host protein synthesis while virion protein synthesis continued. The synthesis of approximately 16 virus-specific proteins was detected during replication, with seven of these recovered in purified virions after lysis. The remainder appeared to be synthesized earlier in phage replication and thus may represent precursors of virion proteins, or regulatory proteins, or enzymes associated with phage replication. Protein synthesis during replication of three suppressible morphological (head, tail) mutants of phage 11 did not differ significantly from that seen in the wild-type lysogen, indicating that the mutation in each case affected a protein whose synthesis was not detectable, or which was synthesized but did not fulfil its role in virion maturation or assembly. However, in a suppressible 'early' mutant which did not lyse when treated with mitomycin C, the synthesis of the 'late' (virion) proteins (with one exception) did not occur. 'Early' proteins were apparently made normally in this mutant, and there was no shut-down of host protein synthesis. This mutant phage presumably encodes a defective 'early' protein involved in the regulation of replication, or a key precursor polypeptide of 'late' protein synthesis. The morphological mutants provided a means to analyse and tentatively to allocate six of the virion proteins to head or tail/baseplate structures.

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.

Optimal growth of Streptococcus cremoris HP in milk is related to β- and ϰ-casein degradation

Initiation of growth and the growth rate of Streptococcus cremoris HP in a complete synthetic medium supplemented with an enzymatic digest of casein appeared to be inhibited by (di)hydrogen phosphate. The inhibitory effect of the inorganic phosphate fraction was counteracted by β-glycerophosphate. Growth experiments involving different caseins or combinations of caseins as the only source of nitrogen (and essential amino acids) were performed in an adapted medium in which optimal growth was expected to depend only on the type of nitrogen source. Maximal growth occurred on a combination of β-casein and a relatively low concentration of χ. It approximated the growth on milk added to the medium. These results and those showing the capacity of the organism to grow on milk-derived fractions suggest that in milk it is mainly the soluble (β-) casein fraction together with the easily accessible hydrophilic part of micellar χ, which maintain optimal growth.

Heptakis(2,6-O-dimethyl)beta-cyclodextrin: a novel growth stimulant for Bordetella pertussis phase I.

The effect of cyclodextrins on the growth of Bordetella pertussis Tohama phase I in synthetic medium was evaluated. The addition of cyclodextrins, especially heptakis(2,6-O-dimethyl)beta-cyclodextrin (Me beta CD), to a complete synthetic medium such as Stainer-Scholte medium gave the same number of individual colonies and growth rates as those on Bordet-Gengou medium. Furthermore, with the addition of Me beta CD, growth inhibition by fatty acids such as oleic or palmitic acid was overcome and normal cell growth was observed. This modified Stainer-Scholte medium, designated as cyclodextrin solid medium (CSM), supported excellent growth of 20 lyophilized clinical isolates. Serotypes of the organisms after 10 passages on this CSM plate were not changed. These results suggest that Me beta CD is a significant growth stimulant and CSM is one of the most suitable synthetic media for culture of B. pertussis phase I.

Chemically defined medium for high yields of sterigmatocystin.

Isolate of Aspergillus versicolor strain produced 138 micrograms/ml of sterigmatocystin in a complete synthetic medium containing sucrose, salts, 1-phenylalanine, and Ca-pantothenate. The SSP (sucrose salts phenylalanine) medium apparently provided all necessary ingredients for the production of high levels of sterigmatocystin. For optimal sterigmatocystin formation, the amounts of sucrose and 1-phenylalanine were found to be 200 g and 5 g per liter, respectively. When Ca-pantothenate (0.01 g per liter) added, much higher amounts of sterigmatocystin were recovered, whereas CaCl2 addition (0.01%) drastically reduced the yield. The high levels of sterigmatocystin were recovered in the cultures which incubated stationary at 26 to 29 degrees C for over 12 days. Seven strains or isolates tested yielded high levels of sterigmatocystin in the SSP medium, whereas in each other media such as YES medium and rich medium only one isolate yielded highest amount of sterigmatocystin was found.

Development of Defined, Minimal, and Complete Media for the Growth of Hyphomicrobium neptunium

A complete synthetic medium containing 15 amino acids, a minimal synthetic medium (GAMS) containing 4 amino acids, and a supplemented minimal medium (GAMS + calcium pantothenate) have been developed for the cultivation of Hyphomicrobium neptunium ATCC 15444. Depending on the complexity of the synthetic media, generation times were approximately 2 to 3 times longer, and maximum cell densities were 0.3 to 0.9 log(10) lower than in ZoBell marine broth 2216. The fates of C-labeled amino acids in GAMS were monitored. Results suggested that H. neptunium was auxotrophic for methionine, utilized glutamic acid as a primary energy source, and readily anabolized and catabolized serine and aspartic acid. Individual amino acid concentrations above 125 mM induced prolonged lag periods, whereas only methionine was not growth limiting at a concentration as low as 2 mM.

Growth and maintenance of HeLa cells in serum-free medium supplemented with hormones.

HeLa cells grow in a nutritionally complete synthetic medium (Ham's F12) supplemented with insulin, transferrin, hydrocortisone (aldosterone), fibroblast growth factor, and epidermal growth factor. This hormone-supplemented medium supports clonal growth, long-term cultivation, and a growth rate equal to that of serum-supplemented medium. The omission of any one of the five components results in less than maximal cell growth.

Isolation and characterization of yeast mutants defective in intermediary carbon metabolism and in carbon catabolite derepression

Yeast mutants deficient in the constitutive ADHI (adc 1) were used for the isolation of mutants with deficiencies of the intermediary carbon metabolism, and of mutants defective in carbon catabolite derepression. Mutants were recognized by their inability to grow on YEP-glycerol and/or on ethanol synthetic complete medium. They were either defective in isocitrate lyase (ic11), succinate dehydrogenase (sdh1), or malate dehydrogenase (mdh1, mdh2), mdh-mutants could not uniformely be appointed to one of the known MDH isozymes. Homozygous mdh and sdh1 diploids are unable to sporulate. Three gene loci could be identified by mutants pleiotropically defective in many or all of the enzymes tested In ccr 1 mutants, derepression of isocitrate lyase, fructose-1,6-diphosphatase, ADHII and possibly of the cytoplasmic MDH is prevented, whereas the mitochondrial TCA-cycle enzymes, succinate dehydrogenase and malate dehydrogenase, are not significantly affected. CCR2 and CCR3 have quite similar action spectra. Both genes are obviously necessary for derepression of all enzymes tested. It could be shown that ccr1, ccr2 and ccr3 mutants are not respiratory deficient.

Photorespiration in diatoms. IV. Two pathways of glycolate metabolism in synchronized cultures of Cylindrotheca fusiformis.

Cylindrotheca fusiformis is shown to be able to convert glycolate to glycerate via tartronic semialdehyde as well as by the better known route involving transamination to glycine. Enzymes related to photorespiration were compared in light-dark synchronized cultures of C. fusiformis kept in continuous light in a complete synthetic seawater medium or starved for nitrogen or silicon. Glycolate oxidation remained constant throughout the cell cycle and was unaffected by starvation. Transamination of glyoxylate was stimulated by light, inhibited during nitrogen starvation, and dramatically stimulated by reintroduction of nitrate to the medium. Glyoxylate carboligase was also stimulated by light and inhibited during nitrogen-starvation but only partially recovered activity after reintroduction of nitrate.

Non-equivalence of yepd and synthetic complete media in yeast reversion studies

Non-equivalence of yepd and synthetic complete media in yeast reversion studies

Non-equivalence of YEPD and synthetic complete media in yeast reversion studies

In yeast reversion studies, assay of the total number of cells is made by plating irradiated cells on agar plates containing yeast extract, peptone and dextrose (YEPD) medium. The number of revertants are scored by plating cells on synthetic complete (SC) medium deficient in the particular nutrient for which the reversion is tested. In this procedure equivalence for cell survival between the YEPD and the SC media is always assumed. However it is shown in this paper that this assumption is valid only up to dose levels where cell killing is not significant. At high doses, survivals on the two media differ significantly from each other for both high and low LET radiations. This difference influences the slope of the reversion frequency curve at high doses. Since the reversion frequency is expressed with reference to the number of survivors after a given radiation dose, it is essential to see that the same chance of survival is offered to the reverted and unreverted cells. Even though reversion is reported to vary linearly with dose, it is found that this linearity is restricted only to dose levels where cell killing is not significant. At higher doses, the reversion frequency varies in a very complex manner with dose for both high and low LET radiations. The complexity depends further on the reference medium chosen.

The vitamin requirements of some oral streptococci

Of the 18 oral streptococci studied, 6 were cariogenic strains of Streptococcus mutans, while the other 12 were of unknown cariogenicity. All strains were grown aerobically and the vitamin requirements were determined by omitting in turn each of 8 B-group vitamins from a complete synthetic medium. Single transfer was used to show the non-essential nature of some of the vitamins. Pantothenate and nicotinic acid were essential or stimulatory for the growth of all strains but none required folic or p-amino-benzoic acids. The use of 2 other synthetic media suggested that the vitamin requirements of most strains were not influenced by the composition of the medium. With the possible exception of Streptococcus salivarius, the vitamin requirements do not indicate species or group divisions, nor do they appear to be related to cariogenicity. Moreover, these observations cannot account for the preferential colonization of various sites in the oral cavity by the streptococcal species.

Fractionated serum dialysate and synthetic media for culturing 2- and 4-cell rabbit embryos.

Embryos in the 2- and 4-cell stage were cultured for 4 days in a simple glucose-salt solution with 1.5% crystallized bovine serum albumin (BSA) to which were added fractions of freeze-dried bovine blood serum dialysate separated on G-25 and G-10 Sephadex columns. Development of 1389 embryos was compared in 35 treatments. The fractions which promoted blastocyst formation had a molecular weight of less than 700. Most of the amino acid content of the unfractionated dialysate was found in the active fractions, thus implicating amino acids as being important additions to the simple synthetic medium. In the absence of BSA no blastocysts formed in the simple synthetic medium containing serum dialysate, or in a “complete medium” formed by adding a complement of amino acids, vitamins, and trace minerals to the simple medium. The addition of serum dialysate to the complete medium resulted in 4.5% of the embryos reaching the expanding blastocyst stage as contrasted with 69.8% when 1.5% BSA was added. The combined addition of serum dialysate and BSA to the simple medium resulted in 22.6% expanding blastocysts. These studies suggest the need for a macromolecular component (BSA) and indicate that the serum dialysate did not provide as optimum a balance of amino acids and other nutrients as were provided by the complete synthetic medium.