Cytokinesis-Based Constraints on Polarized Cell Growth in Fission Yeast

The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at new ends of fic1Δ cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division. Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data highlight a role for previous cell cycles in defining a cell''s capacity to polarize at specific sites, and they additionally provide insight into how a unicellular yeast can transition into a quasi-multicellular state.     

Adhesive specificity of developing cerebellar cells on lectin substrata

Ten lectins, each with a different carbohydrate-binding specificity, have been coupled to tissue culture substrata with carbodiimide [1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate] and assayed for their efficacy as substrates for the carbohydrate-specific adhesion of cells dissociated from mouse cerebellum at embryonic Day 13 and postnatal Days 0 and 7. On surfaces treated with concanavalin A, succinyl-concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin, both embryonic and early postnatal cerebellar cells formed a monolayer. On surfaces coupled with Ricinus communis_I agglutinin (120,000 daltons) both embryonic and postnatal cells formed cellular aggregates with extensive fiber outgrowth. On surfaces treated with peanut agglutinin, Dolichos bifloris agglutinin, Wistaria floribunda agglutinin, soybean agglutinin, or Ulex europaeus_I agglutinin, embryonic cerebellar cells formed cellular aggregates with a cell viability of 25–35% and little or no fiber outgrowth. Postnatal cerebellar cells, in contrast, formed cellular aggregates with a cell viability of 60–70% and extensive fiber outgrowth. On surfaces treated with Ulex europaeus_I agglutinin, cells from postnatal Day 7 formed limited areas of monolayer in addition to cellular aggregates. After 12 hr in vitro the specific attachment of cerebellar cells to lectin-derivatized substrata was inhibited 60–80% by the inclusion of free hapten carbohydrate (50–100 mM) in the growth medium. The addition of soluble concanavalin A or Ricinus communis_I agglutinin (100 μg/ml) was toxic. These studies suggest the presence of glycoconjugate-binding sites for concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin which promote cerebellar cellular adhesion.     

Hydrostatic Pressure Shows That Lamellipodial Motility in Ascaris Sperm Requires Membrane-associated Major Sperm Protein Filament Nucleation and Elongation

Sperm from nematodes use a major sperm protein (MSP) cytoskeleton in place of an actin cytoskeleton to drive their ameboid locomotion. Motility is coupled to the assembly of MSP fibers near the leading edge of the pseudopod plasma membrane. This unique motility system has been reconstituted in vitro in cell-free extracts of sperm from Ascaris suum: inside-out vesicles derived from the plasma membrane trigger assembly of meshworks of MSP filaments, called fibers, that push the vesicle forward as they grow (Italiano, J.E., Jr., T.M. Roberts, M. Stewart, and C.A. Fontana. 1996. Cell. 84:105–114). We used changes in hydrostatic pressure within a microscope optical chamber to investigate the mechanism of assembly of the motile apparatus. The effects of pressure on the MSP cytoskeleton in vivo and in vitro were similar: pressures >50 atm slowed and >300 atm stopped fiber growth. We focused on the in vitro system to show that filament assembly occurs in the immediate vicinity of the vesicle. At 300 atm, fibers were stable, but vesicles often detached from the ends of fibers. When the pressure was dropped, normal fiber growth occurred from detached vesicles but the ends of fibers without vesicles did not grow. Below 300 atm, pressure modulates both the number of filaments assembled at the vesicle (proportional to fiber optical density and filament nucleation rate), and their rate of assembly (proportional to the rates of fiber growth and filament elongation). Thus, fiber growth is not simply because of the addition of subunits onto the ends of existing filaments, but rather is regulated by pressure-sensitive factors at or near the vesicle surface. Once a filament is incorporated into a fiber, its rates of addition and loss of subunits are very slow and disassembly occurs by pathways distinct from assembly. The effects of pressure on fiber assembly are sensitive to dilution of the extract but largely independent of MSP concentration, indicating that a cytosolic component other than MSP is required for vesicle-association filament nucleation and elongation. Based on these data we present a model for the mechanism of locomotion-associated MSP polymerization the principles of which may apply generally to the way cells assemble filaments locally to drive protrusion of the leading edge.     

A Helper-Independent Adenovirus Vector with E1, E3, and Fiber Deleted: Structure and Infectivity of Fiberless Particles

The adenovirus (Ad) fiber protein largely determines viral tropism through interaction with specific cell surface receptors. This molecule may also be involved in virion assembly or maturation, as some previously characterized fiber mutants were defective for processing of viral structural proteins. We previously described packaging cell lines that express Ad type 5 (Ad5) fiber and can complement the temperature-sensitive Ad fiber mutant H5ts142. We have now used these packaging cells to construct a new adenoviral vector (Ad5.βgal.ΔF) with E1, E3, and L5 (fiber) deleted and analyzed the fiber null phenotype. Ad5.βgal.ΔF growth was completely helper independent, and fiberless particles were produced by a single final round of growth in 293 cells. Cryoelectron microscopic studies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the structure and composition of these particles was nearly identical to those of first-generation Ad vectors. As expected, fiberless particles had reduced infectivity on epithelial cells, but they retained the ability to infect monocytic cells via an integrin-dependent pathway. These studies provide a novel approach to developing retargeted Ad gene therapy vectors.     

Anaerobic Degradation of Ethylbenzene by a New Type of Marine Sulfate-Reducing Bacterium

Anaerobic degradation of the aromatic hydrocarbon ethylbenzene was studied with sulfate as the electron acceptor. Enrichment cultures prepared with marine sediment samples from different locations showed ethylbenzene-dependent reduction of sulfate to sulfide and always contained a characteristic cell type that formed gas vesicles towards the end of growth. A pure culture of this cell type, strain EbS7, was isolated from sediment from Guaymas Basin (Gulf of California). Complete mineralization of ethylbenzene coupled to sulfate reduction was demonstrated in growth experiments with strain EbS7. Sequence analysis of the 16S rRNA gene revealed a close relationship between strain EbS7 and the previously described marine sulfate-reducing strains NaphS2 and mXyS1 (similarity values, 97.6 and 96.2%, respectively), which grow anaerobically with naphthalene and m-xylene, respectively. However, strain EbS7 did not oxidize naphthalene, m-xylene, or toluene. Other compounds utilized by strain EbS7 were phenylacetate, 3-phenylpropionate, formate, n-hexanoate, lactate, and pyruvate. 1-Phenylethanol and acetophenone, the characteristic intermediates in anaerobic ethylbenzene degradation by denitrifying bacteria, neither served as growth substrates nor were detectable as metabolites by gas chromatography-mass spectrometry in ethylbenzene-grown cultures of strain EbS7. Rather, (1-phenylethyl)succinate and 4-phenylpentanoate were detected as specific metabolites in such cultures. Formation of these intermediates can be explained by a reaction sequence involving addition of the benzyl carbon atom of ethylbenzene to fumarate, carbon skeleton rearrangement of the succinate moiety (as a thioester), and loss of one carboxyl group. Such reactions are analogous to those suggested for anaerobic n-alkane degradation and thus differ from the initial reactions in anaerobic ethylbenzene degradation by denitrifying bacteria which employ dehydrogenations.     

Vanadium Respiration by Geobacter metallireducens: Novel Strategy for In Situ Removal of Vanadium from Groundwater

Vanadium can be an important contaminant in groundwaters impacted by mining activities. In order to determine if microorganisms of the Geobacteraceae, the predominant dissimilatory metal reducers in many subsurface environments, were capable of reducing vanadium(V), Geobacter metallireducens was inoculated into a medium in which acetate was the electron donor and vanadium(V) was the sole electron acceptor. Reduction of vanadium(V) resulted in the production of vanadium(IV), which subsequently precipitated. Reduction of vanadium(V) was associated with cell growth with a generation time of 15 h. No vanadium(V) was reduced and no precipitate was formed in heat-killed or abiotic controls. Acetate was the most effective of all the electron donors evaluated. When acetate was injected into the subsurface to enhance the growth and activity of Geobacteraceae in an aquifer contaminated with uranium and vanadium, vanadium was removed from the groundwater even more effectively than uranium. These studies demonstrate that G. metallireducens can grow via vanadium(V) respiration and that stimulating the activity of Geobacteraceae, and hence vanadium(V) reduction, can be an effective strategy for in situ immobilization of vanadium in contaminated subsurface environments.     

Effect of the addition of nitrogen sources to cassava fiber and carbon-to-nitrogen ratios on Agaricus brasiliensis growth

The same substratum formulation to grow Agaricus bisporus has been used to grow Agaricus brasiliensis since its culture started in Brazil. Despite being different species, many of the same rules have been used for composting or axenic cultivation when it comes to nitrogen content and source in the substrate. The aim of this study was to verify the mycelial growth of A. brasiliensis in different ammonium sulfate and (or) urea concentrations added to cassava fiber and different carbon-to-nitrogen (C:N) ratios to increase the efficiency of axenic cultivation. Two nitrogen sources (urea and (or) ammonium sulfate) added to cassava fiber were tested for the in vitro mycelial growth in different C:N ratios (ranging from 2.5:l to 50:l) in the dark at 28 degrees C. The radial mycelial growth was measured after 8 days of growth and recorded photographically at the end of the experiment. Nitrogen from urea enhanced fungal growth better than ammonium sulfate or any mixture of nitrogen. The best C:N ratios for fungal growth were from 10:l to 50:l; C:N ratios below 10:l inhibited fungal growth.     

Copper-Induced Inhibition of Growth of Desulfovibrio desulfuricans G20: Assessment of Its Toxicity and Correlation with Those of Zinc and Lead

The toxicity of copper [Cu(II)] to sulfate-reducing bacteria (SRB) was studied by using Desulfovibrio desulfuricans G20 in a medium (MTM) developed specifically to test metal toxicity to SRB (R. K. Sani, G. Geesey, and B. M. Peyton, Adv. Environ. Res. 5:269–276, 2001). The effects of Cu(II) toxicity were observed in terms of inhibition in total cell protein, longer lag times, lower specific growth rates, and in some cases no measurable growth. At only 6 μM, Cu(II) reduced the maximum specific growth rate by 25% and the final cell protein concentration by 18% compared to the copper-free control. Inhibition by Cu(II) of cell yield and maximum specific growth rate increased with increasing concentrations. The Cu(II) concentration causing 50% inhibition in final cell protein was evaluated to be 16 μM. A Cu(II) concentration of 13.3 μM showed 50% inhibition in maximum specific growth rate. These results clearly show significant Cu(II) toxicity to SRB at concentrations that are 100 times lower than previously reported. No measurable growth was observed at 30 μM Cu(II) even after a prolonged incubation of 384 h. In contrast, Zn(II) and Pb(II), at 16 and 5 μM, increased lag times by 48 and 72 h, respectively, but yielded final cell protein concentrations equivalent to those of the zinc- and lead-free controls. Live/dead staining, based on membrane integrity, indicated that while Cu(II), Zn(II), and Pb(II) inhibited growth, these metals did not cause a loss of D. desulfuricans membrane integrity. The results show that D. desulfuricans in the presence of Cu(II) follows a growth pattern clearly different from the pattern followed in the presence of Zn(II) or Pb(II). It is therefore likely that Cu(II) toxicity proceeds by a mechanism different from that of Zn(II) or Pb(II) toxicity.     

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

Background

Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.

Results

The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation. All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.

Conclusions

Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.     

Skeletal Muscle Characterization of Japanese Quail Line Selectively Bred for Lower Body Weight as an Avian Model of Delayed Muscle Growth with Hypoplasia

This study was designed to extensively characterize the skeletal muscle development in the low weight (LW) quail selected from random bred control (RBC) Japanese quail in order to provide a new avian model of impaired and delayed growth in physically normal animals. The LW line had smaller embryo and body weights than the RBC line in all age groups (P<0.05). During 3 to 42 d post-hatch, the LW line exhibited approximately 60% smaller weight of pectoralis major muscle (PM), mainly resulting from lower fiber numbers compared to the RBC line (P<0.05). During early post-hatch period when myotubes are still actively forming, the LW line showed impaired PM growth with prolonged expression of Pax7 and lower expression levels of MyoD, Myf-5, and myogenin (P<0.05), likely leading to impairment of myogenic differentiation and consequently, reduced muscle fiber formation. Additionally, the LW line had delayed transition of neonatal to adult myosin heavy chain isoform, suggesting delayed muscle maturation. This is further supported by the finding that the LW line continued to grow unlike the RBC line; difference in the percentages of PMW to body weights between both quail lines diminished with increasing age from 42 to 75 d post-hatch. This delayed muscle growth in the LW line is accompanied by higher levels of myogenin expression at 42 d (P<0.05), higher percentage of centered nuclei at 42 d (P<0.01), and greater rate of increase in fiber size between 42 and 75 d post-hatch (P<0.001) compared to the RBC line. Analysis of physiological, morphological, and developmental parameters during muscle development of the LW quail line provided a well-characterized avian model for future identification of the responsible genes and for studying mechanisms of hypoplasia and delayed muscle growth.     

Growth of Methanogenic Bacteria in Pure Culture with 2-Propanol and Other Alcohols as Hydrogen Donors

Two types of mesophilic, methanogenic bacteria were isolated in pure culture from anaerobic freshwater and marine mud with 2-propanol as the hydrogen donor. The freshwater strain (SK) was a Methanospirillum species, the marine, salt-requiring strain (CV), which had irregular coccoid cells, resembled Methanogenium sp. Stoichiometric measurements revealed formation of 1 mol of CH₄ by CO₂ reduction, with 4 mol of 2-propanol being converted to acetone. In addition to 2-propanol, the isolates used 2-butanol, H₂, or formate but not methanol or polyols. Acetate did not serve as an energy substrate but was necessary as a carbon source. Strain CV also oxidized ethanol or 1-propanol to acetate or propionate, respectively; growth on the latter alcohols was slower, but final cell densities were about threefold higher than on 2-propanol. Both strains grew well in defined, bicarbonate-buffered, sulfide-reduced media. For cultivation of strain CV, additions of biotin, vitamin B₁₂, and tungstate were necessary. The newly isolated strains are the first methanogens that were shown to grow in pure culture with alcohols other than methanol. Bioenergetic aspects of secondary and primary alcohol utilization by methanogens are discussed.     

Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.)

Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and induced somatic embryogenesis across a wide range of concentrations (1–50 μm). However, somatic embryogenesis was accompanied by callus formation at concentrations of 20 μm and above. Cell suspension cultures were established with the TDZ-induced callus and groups of large cell clumps were formed within 2–3 weeks. Plants were regenerated from both directly formed somatic embryos and somatic embryos derived from cell suspensions plated on semisolid medium devoid of growth regulators. Regenerated plantlets continued to grow after transfer to a greenhouse environment and were similar phenotypically to zygotic seedlings. This simple regeneration system may be beneficial for mass propagation of selected elite clones of neem. Received: 13 May 1997 / Revision received: 13 November 1997 / Accepted: 2 December 1997     

Growth of Thiobacillus ferrooxidans on Formic Acid

A variety of acidophilic microorganisms were shown to be capable of oxidizing formate. These included Thiobacillus ferrooxidans ATCC 21834, which, however, could not grow on formate in normal batch cultures. However, the organism could be grown on formate when the substrate supply was growth limiting, e.g., in formate-limited chemostat cultures. The cell densities achieved by the use of the latter cultivation method were higher than cell densities reported for growth of T. ferrooxidans on ferrous iron or reduced sulfur compounds. Inhibition of formate oxidation by cell suspensions, but not cell extracts, of formate-grown T. ferrooxidans occurred at formate concentrations above 100 μM. This observation explains the inability of the organism to grow on formate in batch cultures. Cells grown in formate-limited chemostat cultures retained the ability to oxidize ferrous iron at high rates. Ribulose 1,5-bisphosphate carboxylase activities in cell extracts indicated that T. ferrooxidans employs the Calvin cycle for carbon assimilation during growth on formate. Oxidation of formate by cell extracts was NAD(P) independent.     

In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system

1. Cultured neurons from embryonic chick sympathetic ganglia or dorsal root ganglia grow nerve fibers extensively on simple substrata containing fibronectin, collagens (types I, III, IV), and especially laminin. 2. The same neurons cultured on substrata containing glycosaminoglycans grow poorly. Glycosaminoglycans (heparin) inhibit nerve fiber growth on fibronectin substrata. 3. Proteolytic fragments of fibronectin support nerve fiber growth only when the cell attachment region is intact. For example, a 105 kD fragment, encompassing the cell attachment region, supports growth when immobilized in a substratum, but a 93 kD subfragment, lacking the cell attachment region, is unable to support fiber growth. When it is added to the culture medium, the 105 kD fragment inhibits fiber growth on substrata containing native fibronectin. 4. In culture medium lacking NGF, DRG neurons extend nerve fibers only on laminin and not on fibronectin, collagen or polylysine. Studies with radioiodinated laminin indicate that laminin binds with a relatively high affinity (kd approximately equal to 10(-9) M) to DRG neurons, and to a variety of other neural cells (NG108 cells, PC12 cells, rat astrocytes, chick optic lobe cells). We have isolated a membrane protein (67 kD) by affinity chromatography on laminin columns and are characterizing this putative laminin receptor. 5. Dissociated DRG neurons or ganglionic explants cultured on complex substrata consisting of tissue sections of CNS or PNS tissues extend nerve fibers onto the PNS (adult rat sciatic nerve) but not CNS (adult rat optic nerve) substrata. Other tissue substrata which support fiber growth in vivo (embryonic rat spinal cord, goldfish optic nerve) support growth in culture. While substrata from adult CNS, which support meager regeneration in vivo (adult rat spinal cord) support little fiber growth in culture. 6. Ganglionic explants cultured in a narrow space between a section of rat sciatic nerve and optic nerve grow preferentially onto the sciatic nerve suggesting that diffusible growth factors are not responsible for the differential growth on the two types of tissues. 7. Dissociated neurons adhere better to sections of sciatic nerve than optic nerve. Laminin, rather than fibronectin or heparan sulfate proteoglycan, is most consistently identifiable by immunocytochemistry in tissues (sciatic nerve, embryonic spinal cord, goldfish optic nerve) which support nerve fiber growth. Taken together, these data suggest that ECM adhesive proteins are important determinants of nerve regeneration.     

Ultraviolet radiation-induced neoplastic transformation of normal human cells,in vitro

Human foreskin cell cultures in scheduled DNA synthesis (S phase) of the cell cycle were exposed to UV irradiation at a dose of 10 J · m^?2 in the presence of insulin. These treated cell populations, when selectively passaged in a high amino acid supplemented complete growth medium (CM) after 20 Dulbecco's phosphate buffered saline (pH 6.8) (PDL), were able to be grown in soft agar. These treated cell populations were also grown in 1% serum supplemented growth medium and at 41°C in 10% serum supplemented growth medium. Cell populations 4–5 PDL after treatment exhibited altered colony morphology and altered lectin agglutination profiles but would not grow in soft agar. These events appeared to be associated with the early stages in the expression phase of the transformed phenotype. After 20 PDL, we observed that these cells would grow in soft agar at a frequency of 20 colonies/10⁵ cells seeded in soft agar. The cell populations derived from these colonies, when propagated and injected into the nude mice, formed myxofibromas at the injection sites rather than the type of tumor (fibrosarcoma) previously described for chemical carcinogen-induced neoplasms.     

Evaluation of storage conditions for refrigerated rabbit skin

An evaluation of various refrigerated (4 °C) storage solutions and conditions was conducted using rabbit skin. Two in vitro methods to assay skin viability are presented: one which directly measures basal cell viability and one which assesses the skin's ability to grow in culture following storage. The superiority of storage in nutrient medium supplemented with fetal bovine serum over conventional storage in saline is clearly demonstrated. Storage in nutrient medium with 10% fetal calf serum resulted in basal cell viabilities which were over 30% higher than viabilities of skin stored by conventional methods in saline. Skin stored in saline failed to grow in culture, while 100% of the cultures of skin stored in medium plus fetal calf serum grew. Although addition of fetal calf serum to the saline improved the basal cell viability, growth in culture occurred only when the skin was stored in a capped tube. Skin stored in medium without serum gave viability results which were not significantly different from the unstored control, but growth rates in culture did differ significantly from the control values. Our study shows that the viability of rabbit skin and its ability to grow in vitro are depressed when the tissue is maintained at 4 °C in saline or in petri dishes, and optimal when refrigerated in nutrient medium supplemented with FBS in a sealed tube.     

Isolation of a wheat cell line with altered membrane properties

A spontaneous dimethylsulfoxide (DMSO)-tolerant cell line was isolated from a cell culture of wheat (Triticum monococcum L.). The tolerant cells were able to grow in the presence of 4% DMSO. Cells formed from protoplasts of the tolerant line required DMSO for division in culture medium of high osmotic value.     

Identification and Characterization of the Myxococcus xanthus argE Gene

The chromosomal acetylornithine deacetylase (argE) gene of Myxococcus xanthus was identified via homology to acetylornithine deacetylases from other bacterial species. A mutant carrying a disruption in argE was unable to grow on minimal media lacking supplemental arginine and formed fruiting bodies and spores in response to arginine starvation at high cell density.     

Determinants in Microbial Colonization of the Murine Gastrointestinal Tract: pH, Temperature, and Energy-Yielding Metabolism of Torulopsis pintolopesii

Torulopsis pintolopesii is an indigenous yeast that colonizes the secreting epithelia in the stomachs of mice and rats. A wild-type strain of this microbe was isolated and identified. To attempt to learn characteristics of the yeast that are advantageous to it in colonizing its natural habitat in vivo, we examined some aspects of its nutrition and energy-yielding metabolism and some environmental conditions that influence its growth in vitro. The yeast appeared to be limited in the compounds it can utilize as carbon and nitrogen sources. It grew best at 37°C and did not grow at 23 or 43°C. It grew optimally at neutral pH but could grow aerobically at pH values as low as 2.0 and anaerobically at pH values as low as 3.4. As assessed by measurements of growth rates and yield coefficients, it grew better aerobically than anaerobically. When grown aerobically, it had a cyanide-sensitive system for taking up O₂ and tested positively for cytochrome c oxidase activity. A petite mutant strain isolated from the wild-type strain had a growth rate and yield coefficient when incubated aerobically that were essentially the same as those of the wild-type parent grown anaerobically. Likewise similar to the wild-type parent grown anaerobically, the petite strain, though incubated aerobically, did not take up O₂. Yeast-free mice associated with either the wild-type or the petite mutant strain were colonized at essentially the same rates and to similar final population levels by both strains. The yeast's capacity to respire may be of little advantage to it in its natural environment. By contrast, its abilities to grow best at 37°C and to grow at low pH values are undoubtedly advantageous characteristics in this respect. The limitations in its carbon and nitrogen nutrition are difficult to evaluate as ecological factors in its colonization of the natural habitat.