NUTRITIONAL REQUIREMENTS OF THE CHYTRID KARLINGIA ASTEROCYSTA,AN OBLIGATE CHITINOPHILE

The nonfilamentous chytrid, Karlingia asterocysta, has been isolated in pure culture on chitin media and the nutritional requirements of a single-spore clone investigated. The fungus displayed an absolute requirement for chitin or preformed N-acetyl-d -glucosamine. This requirement could only be relieved partially by glucose in the presence of limiting acetyl glucosamine concentrations. Under similar conditions other carbohydrates were not utilized. Sulfate was used as a sulfur source and either nitrate or ammonium ion served as nitrogen sources, though growth was better with amino acids. The organism had a very low phosphate optimum (5 × 10^–5 m ) and was inhibited by concentrations at or above 1 × 10^–3 m . The optimal pH range extended from 6.0 to 7.5 and growth decreased rapidly at higher or lower pH values. Thiamine was required at a very low concentration; only 2 μg thiamine-HCl/liter were required for optimal growth. In a rich, agitated medium K. asterocysta completed a single growth cycle (i.e., plant generation) in 70 hr at 25 C.     

Glucose transport byAspergillus niger: the low-affinity carrier is only formed during growth on high glucose concentrations

The filamentous fungusAspergillus niger accumulates large levels of citric acid in the medium when grown under conditions favouring a high rate of sugar catabolism. With the aim of understanding the mechanisms involved in this process we investigated glucose transport in this fungus. To this end a medium was designed that enables growth of the fungus into a fine, hairy filamentous mycelium, suitable for transport studies. It was found thatA. niger contains a single, high-affinity glucose transporter when grown on a low (1% w/v) glucose concentration, but forms an additional low-affinity transporter when grown on a high (15% w/v) glucose concentration. Both glucose transporters exhibit decreased activities at low pH and are inhibited by citric acid. However, the activity of the low-affinity transporter is much less affected by these conditions. Two 2-deoxyglucose-resistant (dgr) mutants ofA. niger, which produce citric acid at a much lower rate than the parent strain, are impaired in the formation of the low-affinity transporter, but form the high-affinity transporter with higher activities. We conclude that the low-affinity glucose transporter takes part in the mechanism by whichA. niger responds to high extracellular glucose concentrations leading to citric acid accumulation.     

AMMONIA PRODUCTION IN UREA-GROWN CULTURES OF CHLORELLA ELLIPSOIDEA12

Production of ammonia by urea-grown Chlorella ellipsoidea was investigated. Ammonia was produced during the stationary growth phase in cultures with urea as sole nitrogen source and glucose as supplementary carbon source. Ammonia was produced only in medium containing excess urea and limiting amounts of glucose. Ammonia production was accompanied by increase in pH. In cultures with nitrate as sole nitrogen source and glucose as supplementary carbon source, growth and pH changes were similar to those in urea-glucose medium, but no ammonia was detected. Cultures grown in urea-acetate medium were similar to those grown in urea medium without additional organic carbon source. No ammonia was produced under these circumstances and growth was significantly lower than that achieved in glucose-supplemented cultures. C. ellipsoidea evidently produces an enzyme or enzyme system which forms ammonia from urea. This organism was reportedly urease-free because previous workers did not detect ammonia formation from urea. Our findings indicate that special circumstances are required to produce detectable amounts of ammonia from urea. These findings are in agreement with a recent report of urea-splitting, cofactor-requiring enzyme in cell-free extracts of Chlorella.     

The selective isolation ofMicromonospora from soil by cesium chloride density gradient ultracentrifugation

Summary A novel method is described for the selective isolation ofMicromonospora from mixed microbial populations in soil. Microorganisms were released from soil by sonication, and the bulk of the soil debris was discarded by low-speed centrifugation. The supernatant microbial suspension was concentrated and applied to a continuous, linear (1.1–1.6 g/ml) gradient of CsCl which was then centrifuged at high speed. The gradient was fractionated, and each fraction was diluted and plated on a medium devoid of antimicrobial agents.Micromonospora were found in the 1.35–1.42 g/ml density band. Occasionally,Bacillus species were also obtained in this density range, but other nonfilamentous bacteria or actinomycetes were usually not observed. This technique allowed the isolation of portions of the soilMicromonospora population which were suppressed by conventional isolation techniques employing heat and antibiotics.     

An essential role for the Escherichia coli DnaK protein in starvation-induced thermotolerance, H2O2 resistance, and reductive division.

During a 3-day period, glucose starvation of wild-type Escherichia coli produced thermotolerant, H2O2-resistant, small cells with a round morphology. These cells contained elevated levels of the DnaK protein, adjusted either for total protein or on a per-cell basis. Immunoprecipitation of [35S]methionine-labeled protein produced by such starving cells demonstrated that DnaK underwent continuous synthesis but at decreasing rates throughout this time. Glucose resupplementation of starving cells resulted in rapid loss of thermotolerance, H2O2 resistance, and the elevated DnaK levels. A dnaK deletion mutant, but not an otherwise isogenic wild-type strain, failed to develop starvation-induced thermotolerance or H2O2 resistance. The filamentous phenotype associated with DnaK deficiency was suppressed by cultivation in a defined glucose medium. When starved for glucose, the nonfilamentous and rod-shaped dnaK mutant strain failed to convert into the small spherical form typical of starving wild-type cells. The dnaK mutant retained the ability to develop adaptive H2O2 resistance during growth but not adaptive resistance to heat. Complementation of DnaK deficiency by using Ptac-regulated dnaK+ and dnaK+J+ expression plasmids confirmed a specific role for the DnaK molecular chaperone in these starvation-induced phenotypes.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazole- carboxamide by Escherichia coli in a Shaking Culture

Various attempts were made to accumulate 5 (4) -amino-4 (5) -imidazolecarboxamide (abreviated as AICA; in this paper, separate analyses for the riboside forms were not attempted, and hence AICA and AICA-riboside will be presented as total AICA) in a shaking culture medium by Escherichia coli strain B. The accumulation of non-acetylatable, diazotizable amines was accomplished by the addition of 0.01% of sulfadiazine, 0.2% of glucose, and 2% of peptone in the medium for sixteen hours at 30°C. E coli strain B was able to accumulate the amines in the pepton medium, even when glucose and the sulfonamide inhibtor were omitted. Although paper chromatographic and spectrophotometric analyses proved the accumulation of AICA and AICA-riboside by E. coli train B in the medium, another substance colored by the Bratton and Marshall method was also accumulated.     

DISTURBANCE OF STREAM PERIPHYTON BY PERTURBATIONS IN SHEAR STRESS: TIME TO STRUCTURAL FAILURE AND DIFFERENCES IN COMMUNITY RESISTANCE1

The resistance of stream periphyton to structural disturbance by increases in shear stress (simulating a spate) was investigated in a laboratory flow tank. We monitored loss of biomass from a filamentous community (dominated by Melosira varians) under four different levels of shear stress. In each case, any loss that was going to occur did so within 10 min for this community. In a second experiment, we tested the resistance of four different communities (two dominated by nonfilamentous diatoms and two dominated by filamentous green algae/diatoms) to increases in shear stress. Nine different levels of shear stress were used, ranging from 1- to 70-fold higher than the conditions to which the communities were acclimated. All communities were 14 days old, but some differences in initial biomass occurred that influenced the degree of resistance independently of species composition. Overall, the nonfilamentous diatom communities were the most resistant, and the filamentous communities were the least resistant. The kinetics of the sloughing process varied among community types, with a community dominated by Melosira varians/Gom-phonema parvulum losing 50% of its biomass with only a 3-fold increase in shear stress. In contrast, a community dominated by the nonfilamentous diatoms Fragilaria vaucheriae/Cymbella minuta lost <50% of its biomass after a 70-fold increase in shear stress. Shear stresses required for 50% loss of biomass for the different communities were as follows: 3.6 Newtons.m^?2 for the Melosira varians/Gomphonema parvulum community, 10.0 N.m^?2 for the Spirogyra sp./Gomphoneis her-culeana/Ulothrix zonata community, 50.6 N.m^?2 for the Fragilaria construens/Cymbella minuta/Ach-nanthes minutissima community, and >90.0 N.m^?2for the Fragilaria vaucheriae/Cymbella minuta community. These results show that spates without bedload movement can potentially have widely differing disturbance effects on periphyton loss among streams depending on the initial taxonomic composition of resident communities. These results have important implications for stream ecosystem analysis and modeling.     

Importance of some factors on the dimorphism of Candida albicans

The passage between the yeast and mycelial forms of Candida albicans B 311-10 was studied by using the minimal syntehtic medium of Shepherd et al. [19] modified without biotin and with low glucose concentrations. It was observed that biotin, aminoacids and particularly pH are not important factors in the dimorphism of C. albicans. The only factor of notable importance in the passage of yeast form to mycelial form in C. albicans was glucose concentration.     

Enhanced Formation of β-Glucosidase by Aspergillus niger VKMF-2092 in Fed-Batch Operation with Frequently Intermittent Glucose Addition

The wild strain Aspergillus niger VKMF-2092 forms β-glucosidase on the basis of glucose as the sole carbon source. The formation of β-glucosidase is initiated after the glucose concentration in the medium has reached a low level since the formation is subjected to catabolite repression. The β-glucosidase is mainly cell-bound and only released after a longer period. Under non-repressed conditions the total formation of β-glucosidase is not associated with growth. By supplying glucose in a fed-batch-technique, maintaining a low actual glucose concentration in the medium, the formation of β-glucosidase is enhanced in comparison to simple batch fermentation. Using a fed-batch-technique with a frequently intermittent addition of glucose it is possible to increase the formation of β-glucosidase with regard to both productivity and the activity related to the mycelium and the fermentation broth as well. The increase of productivity is about two to four times greater than at constant feed rate of the same overall amount of glucose. The reason for this increase will be discussed below. A method is presented which permits to investigate the influence of the substrate concentration and other parameters on the enzyme formation in short periods of one and the same fermentation run.     

Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates

Lactobacillus rhamnosus is a heterolactic acid bacterium, which can be used to produce flavour compounds like diacetyl and acetoin. Various startegies have been applied to improve the growth rate and diacetyl yield. The use of multiple substrates affected growth as well as the yield of diacetyl. Growth on a medium containing glucose demonstrated a diauxic growth profile, with the second phase of growth being on the product, lactic acid. L. rhamnosus also grew on a medium containing citrate. Growth on medium containing glucose+citrate demonstrated simultaneous utilization of carbon sources. L. rhamnosus did not grow in a medium containing acetate and also did not co-metabolize it with glucose. Maximum specific growth rate ( _max) was found to increase in the case of simultaneous utilization of glucose+citrate (0.38 h^–1) as compared to glucose as the sole carbon source (0.28 h^–1). The yields of diacetyl were also found to increase for glucose + pyruvate and glucose + citrate (0.10 and 0.05 g g^–1 of glucose, respectively) as compared to glucose alone (0.01 g g^–1 of glucose). The productivity of diacetyl on medium containing glucose and citrate was double that of a medium containing only citrate, although the yields were comparable.     

Induction of mycelial type of development inCandida albicans by the antibiotic monorden and N-acetyl-D-glucosamine

Two new effective methods for synchronous germ tube production inCandida albicans have been described. Both are based on the use of stationary grown cultures and their further incubation in an aerated simple mineral medium enriched with vitamins containing either high glucose concentration (100 mmol/l) and the antibiotic monorden being added, or N-acetyl-D-glucosamine (100 mmol/l) as the sole carbon source. On the other hand yeast morphology could be maintained in the medium with high glucose concentration.On the basis of the methods developed it was possible to compare respiration intensity, respiration quotients, and sensitivity against some metabolic inhibitors in both morphological forms. Labeling experiments showed slight differences in the time course of glycine incorporation. The mycelial cell walls contained more chitin than the yeastlike cells. Using light and electron microscopy the interrelationships between concentration of monorden, or N-acetyl-D-glucosamine, physiological state of inoculum and the germ tube frequency were determined.The results are discussed with regard to the induction of germ tubes by low glucose concentration inCandida albicans from the more general aspect of regulation of fungal morphogenesis.     

Ectomycorrhiza formation ofTricholoma matsutake isolates on seedlings ofPinus densiflora in vitro

Tricholoma matsutake forms ectomycorrhizas withPinus densiflora under field conditions. The present study aimed to test the ability ofT. matsutake isolates to form mycorrhizas with aseptic seedlings ofP. densiflora in vitro. Pine seeds were germinated aseptically on a nutrient agar medium, and pairs of 1-wk-old seedlings were transplanted into polymethylpentene bottles containing autoclaved sphagnum moss/vermiculite substrate. The substrate was saturated with nutrient medium containing glucose. At the same time, the bottles were inoculated with aT. matsutake isolate. Three mo after inoculation, the fungus formed a sheath and Hartig net on the pine lateral roots. Ectomycorrhizas were also confirmed on 4-6-mo-old seedlings which showed the same or slinghtly better growth than the control plants. These results indicate that culturedT. matsutake mycelium can form true ectomycorrhizas withP. densiflora seedlings in vitro.     

Fruiting ofLyophyllum tylicolor in plate culture on Soytoneglucose agar and urea-treated soil extract agar

Lyophyllum tylicolor, which forms mycelial basidia (and basidiospores), produced fruit-bodies when cultivated at 20°C under continuous illumination of 400–700 lux on agar plates containing Bacto-Soytone and glucose or an extract from urea-treated soil. Under these conditions, mycelial basidia were also observed on the Soytone-glucose agar, but not on the soil extract agar. In darkness, fruit-bodies and mycelial basidia were not observed on either medium. In culture on the soil extract agar, fruit-body primordia were produced at the position of the margin of the colony when it was transferred from darkness to continuous light; stipes did not elongate under illumination of ca. 2000 lux; and mycelial basidia and basidiospores, but not fruit-bodies, developed when glucose concentration in the medium was as high as 1% (w/v).     

Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation

Summary Mutants of Saccharomyces cerevisiae without detectable phosphofructokinase activity were isolated. They were partly recessive and belonged to two genes called PFK1 and PFK2. Mutants with a defect in only one of the two genes could not grow when they were transferred from a medium with a nonfermentable carbon source to a medium with glucose and antimycin A, an inhibitor of respiration. However, the same mutants could grow when antimycin A was added to such mutants after they had been adapted to the utilization of glucose. Double mutants with defects in both genes could not grow at all on glucose as the sole carbon source. Mutants with a single defect in gene PFK1 or PFK2 could form ethanol on a glucose medium. However, in contrast to wild-type cells, there was a lag period of about 2 h before ethanol could be formed after transfer from a medium with only nonfermentable carbon sources to a glucose medium. Wild-type cells under the same conditions started to produce ethanol immediately. Mutants with defects in both PFK genes could not form ethanol at all. Mutants without phosphoglucose isomerase or triosephosphate isomerase did not form ethanol either. Double mutants without phosphofructokinase and phosphoglucose isomerase accumulated large amounts of glucose-6-phosphate on a glucose medium. This suggested that the direct oxidation of glucose-6-phosphate could not provide a bypass around the phosphofructokinase reaction. On the other hand, the triosephosphate isomerase reaction was required for ethanol production. Experiments with uniformly labeled glucose and glucose labeled in positions 3 and 4 were used to determine the contribution of the different carbon atoms of glucose to the fermentative production of CO₂. With only fermentation operating, only carbon atoms 3 and 4 should contribute to CO₂ production. However, wild-type cells produced significant amounts of radioactivity from other carbon atoms and pfk mutants generated CO₂ almost equally well from all six carbon atoms of glucose. This suggested that phosphofructokinase is a dispensable enzyme in yeast glycolysis catalyzing only part of the glycolytic flux.     

Production of Inosine from Hydrocarbons by Corynebacterinm petrophilum

Using the adenine auxotroph of hydrocarbonoclastic microorganism, Corynebacterium petrophilum, the effects of glucose on the inosine productivity were investigated. The mutant did not produce inosine from glucose as the sole source of carbon. Production of inosine in n-C₁₆ medium was found to be inhibited by the addition of glucose. To obtain information on such effect of glucose, several characters were compared between the cells grown in glucose medium and those grown in n-C₁₆ medium. Intracellular content of UV-absorbing materials of the glucose-cells was higher than that of hydrocarbon-cells. The glucose-celle could not grow in media containing adenosine or 5′-AMP. On the other hand, hydrocarbon-cells were able to achieve growth, with adenine, adenosine and 5′-AMP contained in the hydrocarbon medium, but, in the case of glucose medium, the cells could grow only in the presence of adenine. Furthermore, the growth of this mutant in n-C₁₆ medium was found to be inhibited by a larger amount of adenine than that required for the maximum growth, and this inhibition was overcome by the addition of guanine. The significance of the effect of guanine was discussed.     

Expression of capsule-associated genes of Cryptococcus neoformans

Cryptococcus neoformans produces an extracellular polysaccharide capsule that is related to its virulence. The production of capsular components was reported to be accelerated when cultured on media with lower amount of glucose. In this study, relationship between capsule synthesis and expression of capsule-associated genes (CAP genes) was investigated by quantitative real-time PCR analysis. Normally encapsulated strains and a stable acapsular strain were cultured in 1% polypepton medium with 0.1% or 15% glucose. The results of assessment of the capsule size showed that the capsule of yeast cells cultured in the medium with low amount of glucose was thicker than that with high amount of glucose. The CAP gene expressions of normally encapsulated strains were higher in the medium with 0.1% glucose than in the medium with 15% glucose. Furthermore, CAP10, CAP59 and CAP60 genes were expressed very low in a stable acapsular strain, and CAP64 gene was not expressed. Results of assessment of capsule size and CAP gene expressions by quantitative real-time PCR analysis indicated that CAP gene expressions might be related to the production of capsule, and that glucose concentration in culture media might be related to the expression of CAP genes.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Biology of a problematic marine fungus,dermocystidium sp.

Summary The life cycle of a nonfilamentous marine phycomycete, Dermocystidium sp., is described from its growth in axenic culture under controlled conditions. Reproduction occurs by vegetative means involving a process of cell enlargement and ecdysis modifiable by the methods of cultivation. Zoospores and sexuality were not observed. The results of inoculation studies with algae, phanerogam debris, and molluscs suggest that the fungus may be a free-living saprophyte in coastal waters.     

Acanthamoeba profilin affects the mechanical properties of nonfilamentous actin

We investigated the mechanical properties of two abundant, cytoplasmic proteins from Acanthamoeba, profilin and actin, and found that while both profilin and nonfilamentous actin alone behaved as solids, mixtures of the two proteins were viscoelastic liquids. When allowed to equilibrate, profilin formed a viscoelastic solid with mechanical properties similar to filamentous and nonfilamentous actin. Consequently, profilin itself may contribute significantly to the elasticity and viscosity of cytoplasm. The addition of profilin to nonfilamentous actin caused a phase transition from gel (viscoelastic solid) to sol (viscoelastic liquid) when the concentration of free actin became too low to form a gel. In contrast, profilin had little effect on the rigidity and viscosity of actin filaments. We speculate that nonfilamentous actin and profilin, both of which form shear-sensitive structures, can be modeled as flocculant materials. We conclude that profilin may regulate the rigidity (elasticity) of the cytoplasm not only by inhibiting polymerization of actin, but also by modulating the mechanical properties of nonfilamentous actin.