Synthetic Minimal Medium Research Articles

Effect of different K+ concentrations on Cryptococcus neoformans phenoloxidase activity.

Melanin synthesis in Cryptococcus neoformans, catalyzed by phenoloxidase activity, is one of the oldest virulence factors known. However, until now, the relationship between melanin production in C. neoformans and its virulence has been poorly understood. Among different chemical compounds only Fe3+ and Cu2+ cations enhance the phenoloxidase activity in C. neoformans. A few reports in the literature describe the influence of different cations on C. neoformans phenoloxidase activity, excluding iron. In this study, 13 C. neoformans strains isolated from AIDS patients and 7 from bird droppings (B.D.), were examined in order to clarify the effect of different K+ concentrations on phenoloxidase activity. A new solid and liquid caffeic acid minimal synthetic medium (MSM-CAF) containing only caffeic acid and ferric citrate with different potassium concentrations was used to evaluate C. neoformans phenoloxidase activity. In the MSM-CAF solid medium the degree of brown pigmentation on the agar plates was read on days 1, 2 and 3 of incubation, and the pigmentation of the C. neoformans strains was classed into 5 categories. The brown pigment of the liquid MSM-CAF test tubes were checked after 24 hours of incubation by measuring the optical density (O.D.) at 480 nm. Three C. neoformans AIDS and B.D. strains, randomly chosen, were tested for phenoloxidase activity, according to the modified protocols of Polacheck et al., Torres-Guerrero et al. and Rhodes. According to the results obtained, it has been observed that K+ does not activate the phenoloxidase activity in the C. neoformans AIDS and B.D. strains. In particular, with an increase in potassium concentrations in the MSM-CAF solid and liquid medium, there was a corresponding inhibition of the phenoloxidase activity on both the C. neoformans AIDS and B.D. strains.

Properties of a hydrophobin isolated from the mycoparasitic fungus Verticillium fungicola.

Verticillium fungicola, isolated from Agaricus bisporus (commercial mushroom), produced significant extracellular hydrophobin when grown for 7 days in a static liquid culture of synthetic minimal medium. The hydrophobin was purified by precipitation with ammonium sulphate (80% saturation), Sephadex G-100 gel filtration, and hydroxyapatite column chromatography. The purified protein yielded a single band in polyacrylamide gel electrophoresis under native conditions, with an apparent molecular mass of 70 +/- 4 kDa, and also another single band in SDS-PAGE, with a molecular mass of 7 +/- 3 kDa. Molecular mass determined with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) resulted in 7563.9 m/z. The same protein was extracted from the V. fungicola mycelium. Analysis of the amino acid composition revealed the presence of about 50% hydrophobic residues, detecting at least six cysteines, evaluated as cystines, and no free sulfhydryl groups. The protein did not show any glycosylation. On the basis of similarities in hydropathy patterns and solubility characteristics, V. fungicola hydrophobin can be included as a new member of Class II hydrophobins.

Screening of soil bacteria for production of biocleaner.

Soil bacteria were studied for the production of biodegradable cleaning agents. Among 86 bacterial strains resistant to liquid paraffin, 58 showed hemolytic activity. These strains were cultured, and the supernatant of culture broths was evaluated for cleaning activity against a dirty porcelain tile. Potent activity was exhibited in 18 strains. The lowest value of surface tension was obtained from Bacillus sp. NKB03 suggesting the presence of a biosurfactant. Aeromonas sp. NKB26c and Bacillus cereus NKB46b exhibited enzymatic cleaning activity. A cleaning efficiency of 82% was achieved when using a mixture of supernatants from culture broths of Bacillus sp. NKB03 and Aeromonas sp. NKB26c in synthetic minimal media. The cleaning efficiency using this mixture was higher than that of sodium dodecyl sulfate. These results suggest that a mixture of supernatants from culture broths of Bacillus sp. NKB03 and Aeromonas sp. NKB26c has potential for commercial use as a biocleaner.

Mass-murder deletion of 19 ORFs from Saccharomyces cerevisiae chromosome XI.

Nineteen open reading frames (ORFs) in the left arm of chromosome XI of the yeast Saccharomyces cerevisiae were inactivated. This was done by producing single-gene or contiguous-gene deletions in haploid and diploid strains. Four deletions are lethal to the corresponding haploid strains, and two result in a failure to grow on a rich glycerol medium. Complementation experiments showed that five of the six identified phenotypes were due to deletion of a single gene (ORFs YKL173w, YKL172w, YKL165c, YKL154w are essential, and YKL160w is required for growth on glycerol medium). One of the phenotypes observed on glycerol medium was not suppressed by the corresponding deleted genes. None of the other deletions, covering 13 ORFs in all, gave rise to any obvious phenotype when the cells were grown at three different temperatures on rich glycerol or glucose medium or on minimal synthetic medium.

Disruption of six ORFs onSaccharomyces cerevisiae chromosome X: theYJL069c gene of unknown function is essential to cell viability

The short flanking homology PCR strategy (Wach et al., 1994) was used to disrupt six open reading frames (ORFs) on chromosome X of diploid strains (FY1679 and W303) of the yeast Saccharomyces cerevisiae. Two of the six ORFs analysed (YJL069c and YJL066c) display no similarity to known sequences. Three others (YJL065c, YJL068c, and YJL070c) are similar to those respectively encoding the DNA polymerase epsilon subunit c, human esterase D and rat AMP deaminase 1. YJL071w has recently been identified as the ARG2 gene coding for acetylglutamate synthase. Inactivation of the YJL069c gene proved lethal and the yjl071w haploid disruptants were auxotrophic for arginine. For the four other gene inactivations, neither the heterozygous deletion diploids nor the corresponding haploid deletion mutants displayed any special phenotype when grown on rich glycerol or glucose medium or on synthetic minimal medium at three different temperatures, or on media containing compounds interfering with nucleic acid or protein synthesis. Mating and sporulation efficiencies were the same for the viable disruptants as for wild-type cells. The six kanMX4 disruption cassettes were cloned into the pUG7 vector and each of the cognate wild-type genes was inserted into the pRS416 centromeric plasmid. All strains and plasmids have been deposited in the EUROFAN collection (EUROSCARF, K. -D. Entian, Frankfurt, Germany).

News & notes: production, purification, and properties of an endo-1, 3-beta-glucanase from the basidiomycete Agaricus bisporus.

Agaricus bisporus H 25 produced extracellular endo-1, 3-beta-glucanase when grown in a static culture at 25 degrees C in a minimal synthetic medium supplemented with A. bisporus cell walls plus fructose. Endo-1,3-beta-glucanase was purified 17.85-fold from 20-day-old culture filtrates by precipitation at 80% ammonium sulfate saturation, Sephadex G-75 gel filtration, and preparative PAGE followed by electroelution. The purified enzyme yielded a single band in both native and SDS-polyacrylamide gels with a molecular mass of 32 kDa (SDS-PAGE) and 33.7 kDa (MALDI-MS), showing an isoelectric point of 3.7. The enzyme was active against beta-1,3- linkages and, to a lesser extent, against beta-1,6-, exhibiting an endohydrolytic mode of action and a glycoprotein nature. Significant activities of the endo-glucanase against laminarin and pustulan were observed between pH 4 and 5.5, and between 40 degrees and 50 degrees C for laminarin, and between 30 degrees and 50 degrees C for pustulan. The optimum pH and temperature were 4.5 and 45 degrees C for both substrates.

Cleavage of L-leucine-containing dipeptides by Clostridium butyricum

The ability of Clostridium butyricum cultures to hydrolyze three L-leucine-containing dipeptides (Leu-Leu, Leu-Gly and Gly-Leu) in a synthetic minimal medium is demonstrated by using gas chromatography coupled with mass spectrometry. The 13C nuclear magnetic resonance and a labeled dipeptide L-[1- 13C]Leu-Gly were used to confirm this activity.

RESUMPTION OF RAPID PROLIFERATION FROM LAG PHASE IN CULTURES OFSACCHAROMYCES CEREVISIAEIN POOR NUTRIENT CONDITIONS. EFFECT OF SURFACE AND INTRACELLULAR SIGNALLING MECHANISMS

Saccharomyces cerevisiaewas inoculated into a dilute synthetic minimal medium with glycerol as the carbon source. The number of live cells in the cultures was determined by colony counts on agar plates. Untreated control cells had doubled in number about once at the end of the first week and had gone through eight doublings by the end of the second week. Addition of either 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP) or human recombinant insulin, made the cells go through 12 and 10 doublings, respectively, by the end of the first week. In contrast, 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP) had only slight stimulating effects on cell multiplication, but if it was combined with phorbol-12-myristate-13-acetate (PMA) the cells went through about 12 doublings during the first week. Addition of LY 83583, an inhibitor of soluble guanylate cyclase, prevented cell proliferation. Further addition of 8-bromo-cGMP bypassed this inhibition. Singly, bradykinin or PMA did not affect cell multiplication. However, when these two compounds were combined, the cells went through about 10 doublings during the first week. Neither bradykinin, nor PMA had any releasing effect on the inhibition of LY 83583. These results indicate the existence of several routes leading to cell proliferation in wildtypeS. cerevisiaecells.

Clk1, a serine/threonine protein kinase-encoding gene, is involved in pathogenicity of Colletotrichum lindemuthianum on common bean.

A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes.

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'-->3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.

Mcs4, a two-component system response regulator homologue, regulates the Schizosaccharomyces pombe cell cycle control.

The Schizosaccharomyces pombe cdc2-3w wee1-50 double mutant displays a temperature-sensitive lethal phenotype termed mitotic catastrophe. Six mitotic catastrophe suppressor (mcs1-6) genes were identified in a genetic screen designed to identify regulators of cdc2. Mutations in mcs1-6 suppress the cdc2-3w wee1-50 temperature-sensitive growth defect. Here, the cloning of mcs4 is described. The mcs4 gene product displays significant sequence homology to members of the two-component system response regulator protein family. Strains carrying the mcs4 and cdc25 mutations display a synthetic osmotic lethal phenotype along with an inability to grow on minimal synthetic medium. These phenotypes are suppressed by a mutation in wee1. In addition, the wis1 gene, encoding a stress-activated mitogen-activated protein kinase kinase, was identified as a dosage suppressor in this screen. These findings link the two-component signal transduction system to stress response and cell cycle control in S. pombe.

Purification and characterization of an acidic endo-β-1,4-xylanase from the tomato vascular pathogen Fusarium oxysporum f. sp. lycopersici

Fusarium oxysporum f. sp. lycopersici produced at least one acidic and one basic xylanase when grown on synthetic minimal medium supplemented with tomato vascular tissue as the sole carbon source. An acidic endo-β-1,4-xylanase (EC 3.2.1.8) was purified to apparent homogeneity from cultures of the fungus grown on xylan, using preparative isoelectric focusing. The enzyme denominated XYL1 had a M r of 40 kDa upon SDS PAGE and a pI of 3.7. The pH and temperature optima were 4.5 and 40°C, respectively. The enzyme was N-glycosylated. The N-terminal amino acid sequence (Glu, Val, Ala, Pro, Val, Phe, Asn, Ile, Asp, Asp, Lys) showed no homology with other known xylanases.

Influence of fructose on Candida albicans germ tube production.

The influence of different fructose concentrations (5, 3, 1 and 0 g/l) was tested on Germ Tube (GT) production by Candida albicans strain AS3P, using a Minimal Synthetic Medium (MSM) without (NH4)2SO4. The results obtained showed good GT production in the presence of all the different fructose concentrations and in the absence of any nitrogen source. The greatest GT production was obtained with 3 g/l of fructose vs 1 g/l of glucose, after 4 hr of incubation. On the other hand fructose consumption was lower than that of glucose at all concentrations over the 4 hour period. The data obtained may suggest that fructose is metabolized in a different way from glucose for GT production by C. albicans.

Glucose influence on germ tube production in Candida albicans.

The influence of different glucose concentrations was tested in minimal synthetic medium on Candida albicans strain. After 18 hours of starvation, germ-tube (GT) production, amount of consumed glucose, oxygen and the pH of the medium were checked every hour from the beginning through the end of the experiment. Optimal GT production was obtained with 1 g/l of glucose. At this concentration the greatest glucose and oxygen consumption were also noted. No pH variations in the medium were observed in all of the glucose concentrations used. At 3 and 5 g/l glucose concentrations a lower GT production were obtained. The Crab-tree effect might interfere with GT production when glucose concentration is higher than 1 g/l. This data may support the hypothesis that GT production is strictly glucose dependent.

A vitamin-free minimal synthetic medium for Cryptococcus neoformans.

The use of a simple synthetic medium is essential for study on the growth and physiology of Cryptococcus neoformans. In the present study, a minimal synthetic liquid medium (MSM) was tested for the growth of 23 C. neoformans strains. This medium contained a low concentration of glucose, ammonium sulphate and inorganic salts with a pH value of 4.5, but no amino acids or vitamins. The strains were starved for 4 days to eliminate nutrients which might have been carried over from their pre-culture medium. Then, they were inoculated in the MSM as an initial OD of 0.020 at 550 nm and incubated at 37 degrees C for 20 days. Cell growth was generally monitored daily by measuring the absorbance at 550 nm. The medium supported the growth of the strains tested and gave an average final OD of 0.500. The results obtained indicate that C. neoformans may be autotrophic with respect to vitamins and in particular to thiamine. The MSM medium is easy to prepare and store. It is highly reproducible and useful for studies on the growth and physiology of C. neoformans.

The Yeast Proprotein Convertase Encoded by YAP3 Is a Glycophosphatidylinositol-anchored Protein That Localizes to the Plasma Membrane

The yeast YAP3 gene encodes an aspartyl endoprotease that cleaves precursor proteins at selected pairs of basic amino acids and after single arginine residues. Biosynthetic studies of this proprotein processing enzyme indicate that Yap3 is predominantly cell-associated and migrates as a approximately 160-kDa protein on SDS-polyacrylamide gel electrophoresis. Nearly equal amounts of Yap3 are immunodetected in a-haploid, alpha-haploid, and a/alpha-diploid yeast, demonstrating that the expression of YAP3 is not mating type-specific. As shown by endoglycosidase H treatment, which drastically reduces both the estimated molecular mass and the heterogeneity of the protein on SDS-polyacrylamide gel electrophoresis (68 versus 160 kDa), the oligosaccharides N-linked to the protein are subjected to extensive outer chain mannosylation. Outer chain sugar mannosylation takes place in the Golgi apparatus and is commonly found on yeast secreted glycoproteins and/or cell wall mannoproteins. Treatment of the total yeast membranes with chemical agents known to disrupt protein-protein and protein-lipid interactions reveal that Yap3 is membrane-associated. Based upon the release of the membrane-bound form by bacterial phosphatidylinositol phospholipase C digestion and metabolic labeling of the protein with myo-[3H]inositol, Yap3 owes its association with the membrane to the addition of a glycophosphatidylinositol anchor. The cellular localization of Yap3 has been addressed by subcellular fractionation studies. In both differential centrifugation of intracellular organelles and sucrose density gradients, the bulk of Yap3 at steady state co-localizes with the plasma membrane azide-insensitive ATPase. Furthermore, consistent with the transport of Yap3 to the plasma membrane, the endoprotease sediments with secretory vesicles which accumulate at restrictive temperature in the late secretory mutant sec1-1. We therefore conclude that the endoprotease encoded by YAP3 is a glycophosphatidylinositol-anchored protein, which can process substrates both intracellularly and at the cell surface.

Purification and Partial Characterization of aβ-1,3-Glucanase Secreted by the MycoparasiteStachybotrys elegans

A β-1,3-glucanase secreted by Stachybotrys elegans, when grown on minimal synthetic medium containing Rhizoctonia solani cell wall fragments, was purified to homogeneity. The purification method involved ammonium sulfate precipitation and ion-exchange and size-exclusion chromatographies. The molecular mass of the enzyme was estimated under denaturing conditions to be about 94 kDa. The enzyme had an optimum pH of 5.0 and was most active between 40 and 50°C. Except for Mn2+, the enzyme activity was not sensitive to the metal ions tested and Km of 0.18 mg/ml was estimated for laminarin as a substrate. Cell wall lytic activity of purified β-1,3-glucanase was tested on actively growing R. solani hyphae. β-1,3-Glucanase induced morphological changes such as hyphal tip swelling, bursting, leakage of cytoplasm, and formation of numerous septae.

Nutritional factors affecting nisin production by Lactococcus lactis subsp. actis NIZO 22186 in a synthetic medium

L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin‐producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth‐stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.

II. Yeast sequencing reports. A putative P‐type Cu2+‐transporting ATPase gene on chromosome II of Saccharomyces cerevisiae

We have sequenced a gene on the right arm near the telomere of chromosome II of Saccharomyces cerevisiae which codes for a putative P-type cation-transporting ATPase (PCA1). The gene codes for a 1216 amino acids protein. The PCA1 gene expresses a 3.5 kb message in both haploid and diploid cells when grown in glucose-based rich medium YPD. The gene product is most similar at the C-terminal region to a human copper-transporting ATPase and Enterococcus hirae copper-transporting ATPases and also an N-terminal dithiol region that was proposed to be a 'metal-binding motif'. Cells lacking PCA1 display no obvious phenotype when tested under standard conditions: whereas they cease growth much earlier than the isogenic wild-type cells in a minimal medium with high copper concentration. Overexpression of PCA1 under GAL1/10 promoter in yeast cells causes poor growth. We also show that yeast strains carrying PCA1 in multiple copies grow slower than isogenic wild-type strains in a minimal synthetic medium containing 0.3 mM-CuSO4.

The Mu gem operon: its role in gene expression, recombination and cell cycle.

Two genes, gemA and gemB, belong to the gem operon located in the semi-essential early region of bacteriophage Mu. The product of gemA modulates the expression of various host genes, including cell division and DNA replication genes. In addition, GemA is also responsible for decreasing host DNA gyrase activity and for DNA relaxation. The product of gemB is involved in Mu late gene transcriptional transactivation. Phage mutants such as Mu gem2ts have strong effects on the bacterial host: i) infected bacteria become unable to grow in minimal synthetic medium and behave phenotypically as relA- mutants; ii) survivors of the infection are re-programmed in their cell cycles, with synchronous cell divisions, cyclical waves of DNA relaxation and recoiling and; iii) Mu gem2ts prophages excise precisely their DNA from the initial integration site and re-integrate in other non-randomly distributed sites. Neither the phage transposase nor the host RecA protein are implicated in this process.