An analysis of the Impact of <Emphasis Type="Italic">NRG1</Emphasis> Overexpression on the <Emphasis Type="Italic">Candida albicans</Emphasis> Response to Specific Environmental Stimuli

The ability of the opportunistic fungal pathogen Candida albicans to form filaments has been strongly linked to its capacity to cause disease in humans. We previously described the construction of a strain in which filamentation can be modulated both in vitro and in vivo by placing one copy of the NRG1 gene under the control of a tetracycline-regulatable promoter. To further characterize the role of NRG1 in controlling filamentous growth, and in an attempt to determine whether NRG1 downregulation is a requirement for filamentation per se, or is only necessary under certain environmental conditions, we have conducted an analysis of the growth of the tet-NRG1 strain under a variety of in vitro conditions. Through overexpression of NRG1, we were able to block filamentation of C. albicans in both liquid media and on solid media. Filamentation in response to the low-oxygen environment of embedded growth was also inhibited. In all of these conditions, normal filamentation could be restored by down regulating expression from the tet-NRG1 allele. Interestingly, although elevated NRG1 levels were able to inhibit the formation of true hyphae in response to a wide range of environmental stimuli, elevated NRG1 expression did not affect the formation of pseudohyphae on nitrogen-limiting synthetic low ammonia dextrose (SLAD) medium. This work further illustrates the key role played by NRG1 in the control of filamentation and suggests that, although NRG1 repression plays a key role in regulating true hyphal growth, it apparently does not regulate pseudohyphal growth in the same fashion.     

<Emphasis Type="Italic">MPK1</Emphasis> gene is required for filamentous growth induced by isoamyl alcohol in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from the alcoholic fermentation

The aim of this study was to evaluate the MPK1 (SLT2) gene deletion upon filamentous growth induced by isoamyl alcohol (IAA) in two haploid industrial strains of Saccharomyces cerevisiae using oligonucleotides especially designed for a laboratory S. cerevisiae strain. The gene deletion was performed by replacing part of the open reading frames from the target gene with the KanMX gene. The recombinant strains were selected by their resistance to G418, and after deletion confirmation by polymerase chain reaction, they were cultivated in a yeast extract peptone dextrose medium + 0.5% IAA to evaluate the filamentous growth in comparison to wild strains. Mpk1 derivatives were obtained for both industrial yeasts showing the feasibility of the oligonucleotides especially designed for a laboratory strain (Σ1278b) by Martinez-Anaya et al. (In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint. J Cell Sci 116:3423–3431, 2003). The filamentation rate in these derivatives was significantly lower for both strains, as induced by IAA. This drastic reduction in the filamentation ability in the deleted strains suggests that the gene MPK1 is required for IAA-induced filamentation response. The growth curves of wild and derivative strains did not differ substantially. It is not known yet whether the switch to filamentous growth affects the fermentative characteristics of the yeast or other physiological traits. A genetically modified strain for nonfilamentous growth would be useful for these studies, and the gene MPK1 could be a target gene. The feasibility of designed oligonucleotides for this deletion in industrial yeast strains is shown.     

Glucose fermentation to acetate,CO2 and H2 in the anaerobic hyperthermophilic eubacterium Thermotoga maritima: involvement of the Embden-Meyerhof pathway

The hyperthermophilic anaerobic eubacterium Thermotoga maritima was grown on glucose as carbon and energy source. During growth 1 mol glucose was fermented to 2 mol acetate, 2 mol CO₂ and 4 mol H₂. The molar growth yicld on glucose (Y_glucose) was about 45 g cell dry mass/mol glucose. In the presence of elemental sulfur growing cultures of T. maritima converted 1 mol glucose to 2 mol acetate, 2 mol CO₂ about 0.5 mol H₂ and about 3.5 mol H₂S. Y_glucose was about 45 g/mol. Cell extracts contained all enzymes of the Embden-Meyerhof pathway: hexokinase (0.29 U/mg, 50°C), glucose-6-phosphate isomerase (0.56 U/mg, 50°C), phosphofructokinase (0.19 U/mg, 50° C), fructose-1,6-bisphosphate aldolase (0.033 U/mg, 50°C), triosephosphate isomerase (6.3 U/mg, 50°C), glyceraldehyde-3-phosphate dehydrogenase (NAD⁺ reducing: 0.63 U/mg, 50°C), phosphoglycerate kinase (3.7 U/mg, 50°C), phosphoglycerate mutase (0.4 U/mg, 50°C); enolase (4 U/mg, 80°C), pyruvate kinase (0.05 U/mg, 50°C). Furthermore, cell extracts contained pyruvate: ferredoxin oxidoreductasee (0.43 U/mg, 60°C); NADH: ferredoxin oxidoreductase (benzylviologen reduction: 0.46 U/mg, 80°C); hydrogenase (benzylviologen reduction: 15 U/mg, 80°C), phosphate acetyltransferase (0.13 U/mg, 80°C), acetate kinase (1.2 U/mg, 55°C), lactate dehydrogenase (0.16 U/mg, 80°C) and pyruvate carboxylase (0.02 U/mg, 50°C). The findings indicate that the hyperthermophilic eubacterium T. maritima ferments sugars (glucose) to acetate, CO₂ and H₂ involving the Embden-Meyerhof pathway, phosphate acetyltransferase and acetate kinase. Thus, the organism differs from the hyperthermophilic archaeon Pyrococcus furiosus which ferments sugars to acetate, CO₂ and H₂ involving a modified non-phosphorylated Entner-Doudoroff pathway and acetyl-CoA synthetase (ADP forming).     

Utilization of UF-permeate for production of beta-galactosidase by lactic acid bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.     

Starch conversion by amylases fromAureobasidium pullulans

Summary A color variant strain (NRRL Y-12974) ofAureobasidium pullulans produced a saccharifying -amylase and two forms of glucoamylase extracellularly when grown on starch at 28°C for 4 days. A sugar syrup containing DP1 (degree of polymerization) and DP2 (31) was made from maltodextrin DE (dextrose equivalent) 10 (35%, w/w) at 55°C and pH 4.5 using the amylase preparation (40 U g^–1 DS (dry substance). The syrup composition was highly dependent upon substrate concentration but nearly independent of enzyme dose. Glucose syrup containing 93% glucose was made from maltodextrin DE 10 (35%, w/w) at 65°C and pH 4.5 using the same enzyme preparation at 100 U g^–1 DS. The enzyme preparation (100 U g^–1 DS) produced 98–100% glucose from raw corn starch at pH 4.5 and 50°C.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned. Abbreviations: DE, dextrose equivalent (an indication of polymerization; reducing sugars as percentage glucose); DP, degree of polymerization; DP1, glucose; DP2, disaccharide; DP3, trisaccharide; DP4, tetrasaccharide; DS, dry substance.     

Composition and morphology of Candida utilis grown in continuous culture with decreasing concentrations of phosphate

Candida utilis was grown in continuous culture with decreasing concentrations of phosphate. At a constant dllution rate three successlve growth patterns were observed: carbon-limited growth, carbon and phosphate dual-limited growth, and phosphatelimited growth. Phosphate deficlency as well as phosphate limitation produced significant modifications in cell composition and morphology, including cell size. At the lowest phosphate concentration (6 mg/P/I), the cell size was the largest and the relative contents of phosphorus and RNA in the cells was very low, with 96% of the total phosphorus being located in the RNA fraction.M.E. Lucca and M.E. Romero are with the Cátedra de Microbiología Industrial, Fac. Bioq. y Farmacia (U.N.T.) C.C. 90, suc. 2 (4.000) S.M. Tucumán, Argentina, J.C. Díaz Ricci, O.A. Garro and D.A.S. Callieri are with PROIMI, Avda Belgrano y Pje Caseros (4000) S.M. Tucumán, Argentina.     

Conditional filamentation as an adaptive trait of bacteria and its ecological significance in soils

Bacteria can regulate cell morphology in response to environmental conditions, altering their physiological and metabolic characteristics to improve survival. Conditional filamentation, in which cells suspend division while continuing lateral growth, is a strategy with a range of adaptive benefits. Here, we review the causes and consequences of conditional filamentation with respect to bacterial physiology, ecology and evolution. We describe four major benefits from conditional filamentation: stress tolerance, surface colonization, gradient spanning and the facilitation of biotic interactions. Adopting a filamentous growth habit involves fitness trade-offs which are also examined. We focus on the role of conditional filamentation in soil habitats, where filamentous morphotypes are highly prevalent and where environmental heterogeneity can benefit a conditional response. To illustrate the use of information presented in our review, we tested the conditions regulating filamentation by the forest soil isolate Paraburkholderia elongata 5N^T. Filamentation by P. elongata was induced at elevated phosphate concentrations, and was associated with the accumulation of intracellular polyphosphate, highlighting the role of filamentation in a phosphate-solubilizing bacterium. Conditional filamentation enables bacteria to optimize their growth and metabolism in environments that are highly variable, a trait that can impact succession, symbioses, and biogeochemistry in soil environments.     

Characterization of industrial strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> exhibiting filamentous growth induced by alcohols and nutrient deprivation

Summary The use of microorganisms in biotechnology is an important economic area of interest in Brazil, especially the use of Saccharomyces cerevisiae in the baking and alcohol fermentation industries. Dimorphism in S. cerevisiae (cell morphology alterations from budding cells to filamentous structures) has been observed in conditions of nitrogen and carbon deprivation and in the presence of fusel alcohols. This can be described as a defense mechanism that allows the yeast to forage for nutrients through cell elongation, hyphal formation and invasive growth. In this work fifteen industrial strains of S. cerevisiae (including haploid and diploid strains) isolated from the fermentative process for alcohol production were characterized for filamentation on solid culture media under growth conditions of carbon- and nitrogen-deprivation and in the presence of fusel alcohols. The majority of strains showed filamentation induced by isoamyl alcohol, butanol, isopropanol and isobutanol, but not by methanol. In rich medium (YEPD), both haploid and diploid strains showed invasive growth, although this kind of filamentous growth was more common in haploid strains. Similar results were observed when fructose or mannose was used as the sole carbon source. In nitrogen-deficient medium (SLAD) the strains did not filament. The results obtained indicate that the filamentation induced by higher alcohols and carbon deprivation (specially carbon) is a common process in industrial strains of S. cerevisiae contributing towards their maintenance/survival in adverse conditions.     

Rhizosphere mediated nutrient management in Allium hookeri Thwaites by using phosphate solubilizing rhizobacteria and tricalcium phosphate amended soil

This work describes integrated nutrient management for cultivation of Allium hookeri by using phosphate solubilizing bacteria (PSB) applied in rhizosphere, along with tricalcium phosphate (TCP). Arthrobacter luteolus S4C7, Enterobacter asburiae S5C7, Klebsiella pneumoniae S4C9, S4C10 and S6C1, and K. quasipneumoniae S6C2, were isolated from rhizosphere of Allium hookeri Thwaites, and were found to release substantial amount of soluble phosphate (124.8–266.4?μg/mL) from TCP in vitro conditions. These isolates were experimented for plant growth promoting attributes, including IAA, siderophore, and nitrogen-fixation. Treatment with PSB resulted in enhanced growth of A. hookeri Th., which was even better with TCP amendment with PSB. K.quasipneumoniae  S6C2 resulted in 39.1% and 533.3% increase (p?≤?0.05) of root length and weight respectively. The treatment with these isolates, in TCP amended soil also resulted in 200–250% increase in available P in soil, which was maximum for K. quasipneumoniae (1.866?mg/g).     

PERCEPTION OF PORK PATTIES WITH SHIITAKE (LENTINUS EDODE P.) MUSHROOM POWDER AND SODIUM TRIPOLYPHOSPHATE AS MEASURED BY KOREAN AND UNITED STATES CONSUMERS

This study explored consumer liking and perception of pork patties with an added functional health ingredient: shiitake (Lentinus edodes P.) mushroom powder. In addition, patties with and without sodium tripolyphosphate were tested. Both native Korean and U.S. consumers evaluated the products. Pork patties made with both phosphate and mushroom powder were found to be acceptable by Korean consumers, but increases in mushroom powder in patties with phosphate decreased acceptability for U.S. consumers. However, addition of mushroom powder to patties without phosphate increased acceptability for U.S. consumers, perhaps because it tended to increase texture acceptance and juiciness. Thus, addition of mushroom powder as a functional ingredient in pork patties, while possible, would require different formula modifications to appeal to consumers of differing nationalities.     

ELEMENTAL AND BIOCHEMICAL COMPOSITION OF RHINOMONAS RETICULATA (CRYPTOPHYTA) IN RELATION TO LIGHT AND NITRATE‐TO‐PHOSPHATE SUPPLY RATIOS1

The biochemical basis for variations in the critical nitrogen‐to‐phosphorus (N:P) ratio, which defines the transition between N‐ and P‐limitation of growth rate, is currently not well understood. To assess this issue, we cultured the cryptophyte Rhinomonas reticulata NOVARINO in chemostats with inflow nitrate‐to‐phosphate ratios ranging from 5 to 60 mol N·(mol P)^?1 at two light intensities. The nitrate‐to‐phosphate ratio marking the transition between N‐ and P‐limitation was independent of light intensity and was between 30 and 45 mol N/mol P. In N‐limited cells, the particulate N:P ratio was stable at around 23 mol N/mol P over a range of inflow nitrate‐to‐phosphate from 5 to 30, whereas in P‐limited cells this ratio was around 90 mol N/mol P at inflow nitrate‐to‐phosphate ratios of 45 and 60. Cell phosphorus decreased with increasing nitrate‐to‐phosphate ratio up to the critical nitrate‐to‐phosphate ratio for each light intensity, above which they remained stable. The C:P of R. reticulata cells increased with increasing inflow nitrate‐to‐phosphate from around the Redfield value (106 mol C/mol P) to around 700. There was a significant effect of light on C:P in the N‐ limited cells, with higher C:P under high light conditions that was not observed in the P‐limited chemostats. Cellular RNA was not influenced by light but was greatly influenced by the type of nutrient limitation. In contrast, chl a, C, N, and protein were not influenced by the nitrate‐to‐phosphate in the inflow medium. Total protein per RNA was independent of light intensity but exhibited a maximum at inflow nitrate‐to‐phosphate of 30. Our results suggest a strong “two‐level” homeostatic mechanism of cellular N and P content in R. reticulata with two distinct states that are determined by the type of nutrient limitation and not by light.     

Dimorphism and virulence in Candida albicans

Two regulatory pathways govern filamentation in the pathogenic fungus Candida albicans. Recent virulence studies of filamentation regulatory mutants argue that both yeast and filamentous forms have roles in infection. Filamentation control pathways seem closely related in C. albicans and in Saccharomyces cerevisiae, thus permitting speculation about C. albicans filamentation genes not yet discovered.     

Factors determining soil microbial biomass and nutrient immobilization in desert soils

We examined the 10-day response of soil microbial biomass-N to additions of carbon (dextrose) and nitrogen (NH₄NO₃) to water-amended soils in a factorial experiment in four plant communities of the Chihuahuan desert of New Mexico (U.S.A.). In each site, microbial biomass-N and soil carbohydrates increased and extractable soil N decreased in response to watering alone. Fertilization with N increased microbial biomass-N in grassland soils; whereas, fertilization with C increased microbial biomass-N and decreased extractable N and P in all communities dominated by shrubs, which have invaded large areas of grassland in the Chihuahuan desert during the last 100 years. Our results support the hypothesis that the control of soil microbial biomass shifts from N to C when the ratio of C to N decreases during desertification.     

Phosphate excretion rate ofSalpa fusiformis Cuvier (Tunicata : Thaliacea)

The rate of phosphate excretion bySalpa fusiformis was measured for animals of various weights at temperatures of 12.5 to 25 °C. Blastozooids excreted only small, often undetectable quantities of phosphate, in contrast to oozooids, in which the effects of individual weight and temperature were mainly studied. Excretion rate and specific excretion rates were independent of body weight (as total protein). The specific excretion rate increased exponentially with temperature, giving a Q10 of 4.54 (3.17 to 6.43) over the whole temperature range. Using the measurements of Andersen & Nival (1986) on the ammonia excretion rate ofS. fusiformis, we calculated the atomic N/P ratio, which appeared, for oozooids, similar to N/P ratios reported for other zooplanktonic species.     

GROWTH DYNAMICS OF ULVA ROTUNDATA (CHLOROPHYTA) IN A FISH FARM: IMPLICATIONS FOR BIOMITIGATION AT A LARGE SCALE1

Changes in biomass of several macroalgae [Ulva rotundata Bliding; Gracilariopsis longissima (S. G. Gmel.) Steentoft, L. M. Irvine et Farnham; Ulva intestinalis L.; and Cladophora sp.] and marine plants (Zostera noltii and Ruppia cirrhosa) growing naturally in earthen ponds of a fish farm (Acuinova, San Fernando, Southern Spain) were recorded during a year. The farm is mainly devoted to the culture of gilthered seabream (Sparus aurata). The most conspicuous algal species thriving in the ponds was U. rotundata, which reached densities up to 600 g dry mass · m⁻² and produced up to 20.45 g C · m⁻² · d⁻¹. Dissolved nutrients (phosphate and ammonium), tissue nutrient content, and growth rates of this species were estimated during 2001 and 2002. Evidence of natural biomitigation by U. rotundata when water circulates throughout the fish farm is presented. Due to the fish cultivation, both phosphate and ammonium increased as water circulated from the preculture ponds to the postculture ponds. As a consequence, U. rotundata tissue nitrogen (N) and phosphorus (P) increased from algae growing in preculture ponds to algae growing in the outflow channel, so that mean C:N:P ratio varied from 773:57:1 in preculture ponds to 567:64:1 in the outflow channel. Phosphorus limited growth of U. rotundata during the spring. As growth rates increased as a function of tissue P, data were fitted to the Droop equation. From this equation, the estimated maximal growth rate was 0.295 ± 0.041 d⁻¹, the subsistence quota was 0.05 ± 0.01% P of dry mass, and the critical quota was 0.215% P of dry mass. The results suggest that management of the fish farm based on a large-scale integrated mariculture system of fish and macroalgae may increase the total ecological and economic benefits, both for the farm and for the environment.     

Immobilization of cyclodextrin glucanotransferase on amberlite IRA-900 for biosynthesis of transglycosylated xylitol

Cyclodextrin glucanotransferase (CGTase) fromThermoanaerobacter sp. was adsorbed on the ion exchange resin Amberlite IRA-900. The optimum conditions for the immobilization of the CGTase were pH 6.0 and 600 U CGTase/g resin, and the maximum yield of immobilization was around 63% on the basis of the amount ratio of the adsorbed enzyme to the initial amount in the solution. Immobilization of CGTase shifted the optimum temperature for the enzyme to produce transglycosylated xylitol from 70°C to 90°C and improved the thermal stability of immobilized CGTase, especially after the addition of soluble starch and calcium ions. Transglycosylated xylitol was continuously produced using immobilized CGTase in the column type packed bed reactor, and the operating conditions for maximum yield were 10% (w/v) dextrin (13 of the dextrose equivalent) as the glycosyl donor, 10% (w/v) xylitol as the glycosyl acceptor, 20 mL/h of medium flow rate, and 60°C. The maximum yield of transglycosylated xylitol and productivity were 25% and 7.82 g·L⁻¹·h⁻¹, respectively. The half-life of the immobilized CGTase in a column type packed bed reactor was longer than 30 days.     

Inhibition of growth of Sclerotinia minor and other pathogens by citrinin in the filtrate of Penicillium citrinum

Penicillium citrinum Thom, isolated from the sclerotia of Sderotinia minor, was cultured in a broth of Czapek-Dox for 4 to 8 weeks. The filtrate obtained was incorporated into potato dextrose agar or Czapek-Dox agar at different concentrations (v/v). The amended media were tested for mycelial growth of S. minor and other pathogens. Mycelial growth of S. minor was completely inhibited on media amended with 20% (v/v) filtrate of P. citrinum, and considerable inhibition of S. minor occurred at 10 and 15% concentrations. Mycelial growth of S. major, Sclerotium rolfsii, Rhizoctonia solani (AG-4) was inhibited by similar concentrations of filtrate of P. citrinum. Inhibitor(s) in the filtrate were extracted with ethyl acetate and tentatively identified as citrinin. Citrinin was shown to be an active component in the filtrate against mycelial growth of S. minor, S. major and Sclerotium rolfsii.Cooperative investigation of U.S. Department of Agriculture, Agriculture Research Service and Oklahoma State University. Journal Article No. 4989, Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by USDA or by Oklahoma State University, or imply their approval to the exclusion of other products or vendors that may also be suitable.