Stimulation of Yeast Sporulation by Glycerol

S ummary : Glycerol stimulated sporulation of Saccharomyces cerevisiae Hanson, especially when the cells were precultured in a complex growth medium instead of a chemically defined medium. Optimum spore yields occurred with 1–4% of glycerol but some were produced in 16% glycerol. Sporulation in glycerol was much less sensitive to ammonium sulphate inhibition than it was in acetate. Growth occurred with glycerol as sole carbon source and glutamic acid as sole nitrogen source, but not with ammonium sulphate as the sole nitrogen source.     

Physiological and environmental effects on metabolic flux change caused by heterologous gene expression inEscherichia coli

Expression of theZymomonas mobilis pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) genes inEscherichia coli has been known to reduce acetate accumulation by shifting carbon flow from acetate to ethanol. In this study, we investigated the effects of physiological and environmental conditions on the metabolic flux alteration caused by the expression of thepdc andadh genes. In the batch cultures, no significant differences, regardless of medium composition, were found in growth rate and glucose uptake rate between the host strains and the recombinant strains expressing thepdc andadh genes. In the continuous cultures performed with glucose minimal medium, however, the recombinant strains gave more biomass than the host strains at the same specific growth rates. On the contrary, in the continuous cultures with complex medium, the host strains yielded more biomass than the recombinant strains. Analysis of the culture supernatants revealed that the effect of thepdc andadh expression on byproduct formation was more significant at low specific growth rates than at high specific growth rates. This study suggests that physiological and environmental conditions should be carefully considered and precisely defined in assessing the effects of heterologous gene expression on metabolic activities of recombinantE. coli.     

Influence of hydrogen acceptors on growth and energy production ofProteus mirabilis

The generation time ofP. mirabilis in defined and in complex medium is shorter in the presence of hydrogen acceptors than in their absence. In the presence of hydrogen acceptors the molar growth yield for glucose and the acetate production are strongly increased. From the molar growth yield and the acetate production Y_ATP in defined medium was calculated as 5.5 g/mole, whereas in complex medium a value of 12.6 g/mole was obtained. The molar growth yield, the acetate production, the amount of hydrogen acceptor reduced and Y_ATP were used to calculate P/2e⁻ratios for phosphorylation coupled to electron transfer to oxygen, nitrate and tetrathionate as respectively 2.80; 1.48 and 1.23 in defined medium. Under anaerobic conditions in the presence of nitrate or tetrathionate as hydrogen acceptor in complex medium a bend in the growth curve is observed. In the period of rapid growth the P/2e⁻ratio for nitrate reduction is of the same magnitude as that in defined medium, however much lower P/2e⁻ratios are found during the subsequent period of slow growth. The P/2e⁻ratios for tetrathionate reduction in complex medium for both growth periods are lower than those in defined medium. Most probably these results indicate that during this period growth and energy production are uncoupled. Under aerobic conditions in complex medium a constant Y_O value of 32.2 g/atom O is found during a short period of the growth curve. Afterwards when the cell density increases a steady decrease of Y_O is observed.     

Synthesis of Sterols In Herpetomonas Samuelpessoai: Influence of Growth Conditions*

Ergosterol was the only sterol detected in Herpetomonas samuelpessoai grown in a defined, lipid-free medium. When cultivated in a complex medium, this flagellate was found to contain 6 additional sterols. As measured by incorporation of L-[Me-¹⁴C]methionine, in the absence of acetate, the sterol synthesis was greater at 28 C than at 37 C; in the presence of acetate, however, this synthesis was greater at 37 C. When [2-¹⁴C]acetate was used as the sterol precursor, the synthesis level at 37 C exceeded that at 28 C.     

Effects of temperature and medium composition on the ethanol tolerance ofBacillus stearothermophilus LLD-15

The effects of temperature (60°–70°C) and medium composition (complex and defined) on ethanol tolerance ofBacillus stearothermophillus LLD-15, an L-lactate dehydrogenase mutant have been determined in shake flasks under aerobic conditions. In all cases, there was complete inhibition of growth in the presence of 6%v/v ethanol.B. stearothermophillus LLD-15 was found to be less tolerant to ethanol at 70°C than at 60°C and also less tolerant to ethanol in a defined medium than in a complex medium.     

Metabolism of Aspartate by Propionibacterium freudenreichii subsp. shermanii: Effect on Lactate Fermentation

More than 90% of the aspartate in a defined medium was metabolized after lactate exhaustion such that 3 mol of aspartate and 1 mol of propionate were converted to 3 mol of succinate, 3 mol of ammonia, 1 mol of acetate, and 1 mol of CO₂. This pathway was also evident when propionate and aspartate were the substrates in complex medium in the absence of lactate. In complex medium with lactate present, about 70% of the aspartate was metabolized to succinate and ammonia during lactate fermentation, and as a consequence of aspartate metabolism, more lactate was fermented to acetate and CO₂ than was fermented to propionate. The conversion of aspartate to fumarate and ammonia by the enzyme aspartase and subsequent reduction of fumarate to succinate occurred in the five strains of Propionibacterium freudenreichii subsp. shermanii studied. The ability to metabolize aspartate in the presence of lactate appeared to be related to aspartase activity. The specific activity of aspartase increased during and after lactate utilization, and the levels of this enzyme were lower in cells grown in defined medium than levels in those cells grown in complex medium. Under the conditions used, no other amino acids were readily metabolized in the presence of lactate. The possibility that aspartate metabolism by propionibacteria in Swiss cheese has an influence on CO₂ production is discussed.     

Pigment production in chemostat cultures of Streptococcus bovis

Summary Streptococcus bovis 2B formed bright red cell clumps in glucose-limited defined medium under certain nonsteady state growth conditions. Scanning electron micrographs showed that clumped cells were more rounded than those from complex medium. Clumped cells appeared to be bound to one another at the cell surface and there was no sign of a polysaccharide matrix. Freeze fractured cells showed that the inner membrane particle distribution was different in the clumped cells from fast growing cells in complex or defined medium. Clumping is potentially a mechanism to decrease transport by reducing the cell surface area. It appears to be a nonsteady state phenomenon, occurring in situations leading to unbalanced growth.     

Acetate,methanol and carbon dioxide as substrates for growth of Methanosarcina barkeri

Methanosarcina barkeri grows in defined media with acetate, methanol or carbon dioxide as carbon sources. Methanol is used for methanogenesis at a 5 times higher rate as compared with the other substrates. M. barkeri can use the substrates simultaneously, but due to acidification or alkalification of the medium during growth on methanol or acetate, respectively, growth and methanogenesis may stop before the substrates are exhausted. Growth and methanogenesis on methanol or acetate are inhibited by the presence of an excess of H₂; the inhibition is abolished by the addition of carbon dioxide, which probably serves as an essential source of cell carbon, in the absence of which methano-genesis ceases.     

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic <Emphasis Type="Italic">Cobetia marina</Emphasis> strain L-2

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5°C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5–15°C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5°C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5°C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5°C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5°C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.     

Fermentation parameters of Peptostreptococcus productus on gaseous substrates (CO,H2/CO2)

Intrinsic growth and substrate uptake parameters were obtained for Peptostreptococcus productus, strain U-1, using carbon monoxide as the limiting substrate. A modified Monod model with substrate inhibition was used for modeling. In addition, a product yield of 0.25 mol acetate/mol CO and a cell yield of 0.034 g cells/g CO were obtained. While CO was found to be the primary substrate, P. productus is able to produce acetate from CO₂ and H₂, although this substrate could not sustain growth. Yeast extract was found to also be a growth substrate. A yield of 0.017 g cell/g yeast extract and a product yield of 0.14 g acetate/g yeast extract were obtained. In the presence of acetate, the maximum specific CO uptake rate was increased by 40% compared to the maximum without acetate present. Cell replication was inhibited at acetate concentrations of 30 g/l. Methionine was found to be an essential nutrient for growth and CO uptake by P. productus. A minimum amount of a complex medium such as yeast extract (0.01%) is, however, required.     

大肠杆菌DH5α耐乙酸突变株的选育及其代谢特性研究

大肠杆菌DH5α是基因工程常用的宿主菌之一,但由于对代谢副产物乙酸十分敏感,影响外源基因的表达效率。为了提高E. coli DH5α乙酸耐受力,采用60Co诱变结合连续培养,逐步提高稀释率和乙酸钠选择压力,于含乙酸钠平板进一步筛选,得到5株对乙酸耐受能力显著增强的突变菌株,具有良好的遗传稳定性,其中DA19显示最强的耐受性能。DA19与DH5α相比,在复合培养基YPS和YPS2G中菌体浓度分别提高17％和5％,最大比生长速率分别提高8％和27％,产乙酸分别减少为6％和59％;在基本培养基中的细胞浓度提高24倍,在含10g/L乙酸钠培养基中达到的细胞浓度与不加乙酸钠DH5α的细胞浓度相当。     

Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus

A chemically defined medium has been developed for anaerobic cultivation of hyaluronic-acid(HA)-producing Streptococcus zooepidemicus, which contains 11 amino acids essential to growth, and glutamine as a principal nitrogen source. The final HA concentration, the specific rate of HA production and HA-to-glucose yields were similar for growth in the chemically defined medium relative to growth in complex medium. Consequently cells cultivated on chemically defined medium can be expected to have similar activity regarding HA synthesis as compared to cells grown on complex media. However, the specific growth rate and volumetric HA production rate were found to be less favourable in the chemically defined media. Received: 4 October 1996 / Accepted: 25 October 1996     

Increase in the production of β-carotene in recombinant Escherichia coli cultured in a chemically defined medium supplemented with amino acids

Escherichia coli DH5α strain was selected as the recombinant host, and a chemically defined medium supplemented with amino acids was used instead of a complex medium for the efficient production of β-carotene. In a fed-batch culture using glycerol with a chemically defined medium supplemented with amino acids, the concentration, specific content, and productivity of β-carotene were 2,470 mg/l, 72 mg/g cells, and 77 mg/l h after 32 h, respectively. These values were, respectively, 43, 33, and 26 % higher than those obtained using the complex medium. This is the highest β-carotene production that has been reported among the recombinant cells to date.     

Mechanisms of acetate formation and acetate activation in halophilic archaea

The halophilic archaea Halococcus (Hc.) saccharolyticus, Haloferax (Hf.) volcanii, and Halorubrum (Hr.) saccharovorum were found to generate acetate during growth on glucose and to utilize acetate as a growth substrate. The mechanisms of acetate formation from acetyl-CoA and of acetate activation to acetyl-CoA were studied. Hc. saccharolyticus, exponentially growing on complex medium with glucose, formed acetate and contained ADP-forming acetyl-CoA synthetase (ADP-ACS) rather than acetate kinase and phosphate acetyltransferase or AMP-forming acetyl-CoA synthetase. In the stationary phase, the excreted acetate was completely consumed, and cells contained AMP-forming acetyl-CoA synthetase (AMP-ACS) and a significantly reduced ADP-ACS activity. Hc. saccharolyticus, grown on acetate as carbon and energy source, contained only AMP-ACS rather than ADP-ACS or acetate kinase. Cell suspensions of Hc. saccharolyticus metabolized acetate only when they contained AMP-ACS activity, i.e., when they were obtained after growth on acetate or from the stationary phase after growth on glucose. Suspensions of exponential glucose-grown cells, containing only ADP-ACS but not AMP-ACS, did not consume acetate. Similar results were obtained for the phylogenetic distantly related halophilic archaea Hf. volcanii and Hf. saccharovorum. We conclude that, in halophilic archaea, the formation of acetate from acetyl-CoA is catalyzed by ADP-ACS, whereas the activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS.Abbreviations. Hc. Halococcus - Hf. Haloferax - Hr. Halorubrum - Hb. Halobacterium An erratum to this article can be found at     

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling^-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.     

Effects of metabolites present during growth of Tetrahymena pyriformis on the subsequent secretion of lysosomal hydrolases

Tetrahymena were grown in proteose-peptone medium supplemented with glucose, mannose, fructose, galactose, acetate, succinate, or pyruvate and then washed and resuspended in a non-nutrient salt solution and the amounts of 7 acid hydrolases secreted into the medium in a one hour incubation were measured. Cells that had been grown in the presence of glucose secreted about half the amounts of acid phosphatase, β-N-acetylglucosaminidase and acid protease as did control cells grown in unsupplemented medium. Pyruvate was about as effective as glucose and both were slightly more effective than acetate or fructose. Succinate had little effect. Similar experiments showed that α-mannosidase, β-fucosidase, and β-galactosidase are secreted into the salt solution and that secretion is reduced by prior growth of the cells in medium supplemented with glucose or mannose but not galactose. Except for α-mannosidase, these reductions in amounts of hydrolase secreted were not accompanied by appreciable changes in intracellular activity, and therefore demonstrate a persistent effect of growth in the presence of certain metabolites on the subsequent secretion of lysosomal hydrolases. Since the inhibition of subsequent secretion depended on both the individual metabolite and the particular hydrolase examined, it appears that the effect of metabolites is not limited to a general inhibition of secretion but may differentially alter some properties of lysosomal subpopulations. A preliminary characterization of the secreted acid protease of Tetrahymena suggests that there may be two acid proteases released, since up to 25% of the activity was not inhibited by high concentrations of pepstatin, leupeptin, or chymostatin.     

Chemically defined and minimal media forBacteroides gingivalis

A chemically defined medium (BGDM) has been developed specifically forBacteroides gingivalis. The medium contains 4 amino acids, 5 mineral salts, cysteine hydrochloride as a reducing agent, and the growth factors hemin and menadione. Eight strains ofB. gingivalis have been subcultured repeatedly in this medium with no apparent changes in colonial or cellular morphology. The metabolic end products of strains grown in this medium were reproducible and yielded patterns similar to those produced by cells cultured in complex media. The growth rates were about 50% slower than those of cells grown in a complex medium, and the growth rate constants ranged between 0.013 and 0.067 H^–1. When the defined medium was supplemented with protein hydrolysates such as trypticase, proteose peptone, bactocasitone, or yeast extract, at concentrations up to 1.0%, growth increased. No such growth increase was observed in the medium supplemented with casamino acids. Thus a minimal medium can be formulated by adding one of the growth-enhancing protein hydrolysates to the defined medium at varying concentrations depending upon the growth yield required.     

Induction of multispored asci in two-spored strains of Saccharomyces cerevisiae by amitrole.

Although growth of two yeast strains characterized by consistent production of two diploid spores per ascus was inhibited in complex presporulation media containing amitrole, a fraction of the cells produced were able to form asci with more than two spores after transfer to acetate sporulation medium. Cells grown in a defined presporulation medium containing amitrole did not acquire this ability. The increase in spore numbers per ascus is attributed either to the induction by amitrole in growth medium of cells with more than one nucleus or to the restoration of normal meioses in the multispored asci.     

Reduction of trimethylamine N-oxide by Escherichia coli as anaerobic respiration.

E. coli was found to grow anaerobically on lactate in the presence of trimethylamine N-oxide (TMANO), reducing it to trimethylamine. Anaerobic growth on glucose was promoted in the presence of TMANO. When a culture grown in complex medium was transferred to defined medium, growth on glucose and ammonia took place in the presence of TMANO after consumption of complex nutrients introduced with the preculture, in contrast to growth in nitrate respiration. The amounts of ethanol, succinate, and lactate among the fermentation products were decreased and that of acetate was increased in the presence of TMANO. Formate generation was much reduced at pH 7.4, whereas stoichiometric formation of formate was observed in the absence of TMANO. Cells grown anaerobically in the presence of TMANO had a higher activity of amine N-oxide reductase than cells grown under other conditions. The content of cytochrome-558 was elevated in the presence of TMANO during growth in complex medium. Cytochrome c-552 found in cells grown in diluted complex medium or defined medium in the presence of TMANO was oxidized by TMANO in cell extracts. The molar growth yield on glucose was higher in the presence of TMANO than in its absence and lower than that in the presence of nitrate.