Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks. Received 20 January 1999/ Accepted in revised form 30 April 1999     

An Escherichia coli Mutant Having Altered D-Xylose Uptake Activity and Cloning of a Gene for D-Xylose Uptake

An Escherichia coli C600 mutant having an altered D-xylose uptake activity was isolated. The growth rate and D-xylose uptake activity of the mutant grown on the minimal medium with D-xylose at 25°C were much lower than those of the parental strain grown under the same conditions, although the activities of D-xylose-binding proteins and the enzymes involved in D-xylose metabolism were almost the same for the two strains. An uptake study on sugars at the low temperature (25°C) indicated that the mutant was deficient in D-xylose uptake activity. A gene responsible for the D-xylose uptake activity at the low temperature was isolated and cloned onto vector plasmid pBR322. The gene specifically improved the D-xylose uptake activity of the mutant at the low temperature when it was introduced into the mutant cells. Based on these results, it was suggested that another D-xylose transport system other than the D-xylose-binding protein mediated system might be functioning in E. coli cells.     

Dimorphic and yeast-like mutants of the genusCephalosporium Cda

A series of mutants, in which the mycelial type of growth gradually changes to the dimorphic and permanent yeast-like forms, were isolated from cultures ofCephalosporium sp. subjected to UV radiation. The intermediate stage between the mycelial and dimorphic strains (mutants 2/29 and 2/R) is characterized by the absence of aerial hyphae, ability to form conidiophores inside agar and by polymorphism of conidia. The Y-M transformation of two dimorphic mutants obtained from the 2/R mutant depends on temperature. Another mutant isolated from the 2/29 strain was found to form the mycelial phase only when osmolarity of the medium increased. At 22°C the transformation of all three dimorphic strains was influenced by the carbon source: the Y phase predominated in glucose-containing media, the M phase predominated in media with amino acids or citrate serving as carbon sources. Another mutant (2/7R) was found to grow permanently in the Y phase and was not influenced by temperature, osmolarity of the medium and by the carbon source. It is assumed that the dimorphism of the mutants is caused by a conformational mutation inhibiting the apical growth. This mutation can be phenotypically reversed by some factors of the environment.     

Some required events in conidial germination of Neurospora crassa.

A temperature-sensitive mutant of Neurospora crassa, with reduced levels of protein synthesis at 37°C, was used to identify some essential events in conidial germination. Conidia of mutant strain psi-1 were incubated for 2 hr at 37°C and then shifted to 20°C. Germination was inhibited at 37°C, but commenced after 1.5 hr at 20°C. Increases in aspartate transcarbamylase activity, cell wall synthesis, and nuclear number preceded germination. However, increases in glutamate dehydrogenase activity, amino acid uptake, and DNA synthesis were inhibited prior to germination. Although all of these events were correlated with germination in control cultures of the mutant at 20°C and of its parent strain at 20 and 37°C, some events were apparently not essential for germination. The requirement for aspartate transcarbamylase activity was demonstrated independently by the failure of strain pyr-3d (lacking the activity) to germinate in the absence of uridine. The dispensability of glutamate dehydrogenase activity and DNA synthesis for the germination of some conidia was verified by the germination of strain am-1 (lacking glutamate dehydrogenase activity) in the absence of glutamate and by the germination of the parent strain in the presence of hydroxyurea (an inhibitor of DNA synthesis). These findings identify some landmarks in germination which may be useful in further studies of the regulation of a developmental program. They also provide preliminary evidence that the resting conidia may contain nuclei arrested at different stages of their division cycle.     

Isolation and growth characteristics of a nystatin- resistant mutant of a thermotolerant yeast Hansenula polymorpha

A nystatin-resistant mutant (NR-21) of a thermotolerant yeast, Hansenula polymorpha CK-1, was isolated by mutagenesis with ethyl methanesulfonate, followed by selection for resistance to nystatin (50 units/ml). The mutant was defective in ergosterol biosynthesis. Specific growth rates (h⁻¹ of the mutant were reduced to 0.35 at 40°C and 0.16 at 50°C as compared with the wild type (0.53 at 40°C and 0.28 at 50°C). The mutant grown with ergosterol-phosphatidylcholine emulsion at 50°C incorporated ergosterol and its specific growth rate was increased to 0.41, which was comparable to that of the wild type grown under the same conditions.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Temperature-sensitive flocculation during growth ofSaccharomyces cerevisiae

Summary Cell wall surface proteins were extracted from a temperature-sensitive flocculent strain ofSaccharomyces cerevisiae. Electrophoretic analysis identified two protein bands (28 and 43 kDa) present when grown at 21°C. These proteins were initially absent when the strain was grown at 37°C, but intensified after 6 days of growth concomitant with the onset of flocculation.     

Dimorphism and hydrocarbon metabolism in Yarrowia lipolytica var. indica

Yarrowia lipolytica is able to metabolize high Mr hydrophobic natural compounds such as fatty acids and hydrocarbons. Characteristically, strains of Y. lipolytica can grow as populations with variable proportions of yeast and filamentous forms. In the present study, we describe the dimorphic characteristics of a variant designated as Y. lipolytica var. indica isolated from petroleum contaminated sea water and the effect of cell morphology on hydrocarbon metabolism. The variant behaved as a yeast monomorphic strain, under conditions at which terrestrial Y. lipolytica strain W29 and its derived strains, grow as almost uniform populations of mycelial cells. Using organic nitrogen sources and N-acetylglucosamine as carbon source, var. indica was able to form mycelial cells, the proportion of which increased when incubated under semi-anaerobic conditions. The cell surface characteristics of var. indica and W29 were found to be different with respect to contact angle and percent hydrophobicity. For instance, percent hydrophobicity of var. indica was 89.93 ± 1.95 while that of W29 was 70.78 ± 1.1. Furthermore, while all tested strains metabolize hydrocarbons, only var. indica was able to use it as a carbon source. Yeast cells of var. indica metabolized hexadecane with higher efficiency than the mycelial form, whereas the mycelial form of the terrestrial strain metabolized the hydrocarbon more efficiently, as occurred with the mycelial monomorphic mutant AC11, compared to the yeast monomorphic mutant AC1.     

Single cell protein production from yacon extract using a highly thermosensitive and permeable mutant of the marine yeast Cryptococcus aureus G7a and its nutritive analysis

The intracellular protein in the highly thermosensitive and permeable mutant can be easily released when they are incubated both in the low-osmolarity water and at the non-permissive temperature (usually 37 °C). After the mutant was grown in the yacon extract for 45 h, the crude protein content in the highly thermosensitive and permeable mutant Z114 was 59.1% and over 61% of the total protein could be released from the cells treated at 37 °C. The mutant cells grown in the yacon extract still contained high level of essential amino acids and other nutrients. This means that the yacon extract could be used as the medium for growth of the highly thermosensitive and permeable mutant which contained high content of crude protein.     

Cloning and expression of the gene for an insect haemocyte anti‐aggregation protein (VPr3), from the venom of the endoparasitic wasp,Pimpla hypochondriaca

A venom protein from the endoparasitic wasp, Pimpla hypochondriaca, was recently biochemically isolated. This protein possessed haemocyte anti‐aggregation activity in vitro and shares the same N‐terminal amino acid sequence as that deduced from a gene termed vpr3. The vpr3 gene was identified by sequence analysis of randomly isolated cDNAs from a P. hypochondriaca venom gland library. Presently, the gene for the full‐length sequence of mature VPr3 protein was amplified from the P. hypochondriaca venom gland cDNA library by PCR. The amplicon was directionally cloned into a pET expression vector so that recombinant VPr3 (rVPr3) would have an N‐terminal polyhistidine (His) tag. High levels of target protein expression were obtained following addition of IPTG (1 mM) and growth of the bacteria at 37°C for 5 h, or at 24°C for 20 h. Following lysis of bacteria grown at 37°C, the target protein partitioned into the insoluble fraction. However, at 24°C, a small amount of soluble protein was consistently detected. The amount of soluble rVPr3 was subsequently increased when the transformed bacteria were grown in Overnight Express Instant TB medium at 24°C. Soluble rVPr3 was purified utilizing the MagneHis Protein Purification System. Recombinant VPr3 was determined to have adverse effects on the cytoskeleton of Lacanobia oleracea haemocytes and to inhibit the ability of these cells to form aggregates in vitro. © 2009 Wiley Periodicals, Inc.     

Isolation, and morphological and chemical properties of an autolysis-deficient mutant of Clostridium botulinum type A.

An autolysis-deficient mutant was isolated from Clostridium botulinum type A 190L by treatment with ethyl methanesulfonate. The cell wall prepared from the mutant autolyzed at much slower rate than that from the parent strain, accompanying with much less liberation of both amino terminals and reducing groups. Electron microscopic observation revealed that the mutant strain was converted to short rod or curved spherical form with thickened cell walls when the growth temperature was shifted from 37 to 45 C. The mutant had a significantly larger amount of non-peptidoglycan-carbohydrate complexes than did the parent strain and became markedly resistant to the autolysin partially purified from the parent, compared with the parent strain. Furthermore, the mutant was fairly tolerant to killing by penicillin. These results suggest that the autolysis deficiency of the mutant was due not only to the deficient production of autolysin but also to the excess accumulation of carbohydrate in the cell wall.     

Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15°C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37°C membranes, while 15°C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15°C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15°C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37°C, but only 50% at 15°C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15°C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Fucose-specific adhesins on germ tubes of Candida albicans

Lectin-like adhesins of hyphal-form Candida albicans were investigated by conventional fluorescence microscopy, fluorescence microscopy with image analysis, spectrofluorimetry and flow cytometry. Labelling was done with neoglycoprotein probes consisting of sugars (fucose, mannose, glucose, galactose, lactose) covalently linked to bovine serum albumin (BSA), which itself was labelled with fluorescein. The fucose probe bound to both the yeast and germ-tube portions of hyphal-form cells, not especially at the tip, but in the adjacent region of the germ-tube portion. Probes with the other sugars did not label the hyphal-form cells. Fucose-probe binding to the cells was optimal at pH 5.0 in citrate buffer, and was a time-dependent reaction requiring 30–60 min and reaching saturation concentration at 100 μg ml⁻¹. Each hyphal-form cell of C. albicans grown in 199 medium was calculated to have about 2×10⁷ fucose probe-binding sites. There appeared to be no requirement for Ca²⁺ or Mg²⁺ in binding. Binding of the fucose probe to the hyphal-form cells was higher at 37°C than at 22°C or 4°C. Fluorescence intensity of the fucose-labelled yeast forms was not increased over the hyphal-form cells. A germ-tube-deficient mutant when exposed to hyphal-form growth conditions for 2 h showed much less binding of the fucose probe than the wild-type which produced germ tubes. Confirmation of specificity and the need for a carrier molecule was obtained by showing that Fuc-BSA (without fluorescein) effectively inhibited the binding of the fucose probe, although l-fucose itself was inactive, as was Gal-BSA.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

Ornithine decarboxylase in Paracoccidioides brasiliensis

Ornithine decarboxylase in Paracoccidioides brasiliensis, a dimorphic human pathogenic fungus, was more active at 37° C in the yeast phase and at 30° C in the mycelial phase. In contrast to other fungal systems, yeast growth and mycelium-to-yeast transition in P. brasiliensis were accompanied by a high activity of ornithine decarboxylase at the onset of the budding process, the activity of which was inhibited by 1,4-diamino-2-butanone. The activity of ornithine decarboxylase remained at a basal level during vegetative growth of both the mycelial phase and the late stage of yeast phase, and also through the yeast-to-mycelium transition. Received: 18 December 1995 / Accepted: 8 March 1996     

A yeast lysis mutant: potential biotechnological applications

The conditional yeast lysis mutant cly8 was studied for potential biotechnological applications. The strain stops to grow immediately after a shift to elevated temperatures ( > 30°C). Cell viability (colony forming capacity) decreases at 37°C at a rate depending on the composition of the medium. However, at the elevated temperature cells still consume glucose and incorporate [¹⁴C]leucine into cell material. With decreasing viability the mutant cells become leaky for small, predominantly cytoplasmic components such as leucine or uridine but not for vacuolar storage products like arginine. No trichloroacetic acid-precipitable material could be detected in the medium after the shift to the elevated temperature indicating that leakiness was restricted to low molecular weight compounds. On acetate medium mutant cells became permeable only after prolonged incubation at 37°C but could be used for the oxidation of exogenous NADH. In comparison to the wild type the mutant also produced more glycerol. When the mutant cells were immobilized, glycerol production was in the same range at room temperature and at 28°C and could be maintained for several days.     

Characterization of the carbohydrate component of fraction I in the Neurospora crassa cell wall.

The carbohydrate portion of fraction I of the Neurospora crassa cell wall has been analyzed for sugar composition by gas-liquid chromatography and colorimetric methods. The analysis was performed comparatively in a wild-type strain (RL 3-8A) and three morphological mutants: scumbo (FGSC 49), peak-2a (a mutant known to be allelic to biscuit), and ragged (FGSC 296). Fraction I of all strains studied contains glucose, mannose, and galactose as the main sugars. Uronic acids and amino sugars are also present in small amounts. The glycosidic linkages binding the neutral sugars were analyzed by Lindberg's combined gas chromatography-mass spectrometry techniques for identification of the partially methylated alditol acitate sugar derivatives. The main polymeric portion of fraction I seems to be a linear glucan with the glucose residues linked by 1 leads to 3 and 1 leads to 4 bonds. A mannan portion with a branched configuration is also present, with galactose as the sugar residue which serves as branches in the molecule(s). The branched mannan portion appears to increase in amount in correlation with more drastic morphological changes of the mycelia. In this respect, the mutant ragged has the lowest mycelial growth rate and the largest amount of mannan. The importance of the polysaccharide structure of fraction I on the colonial morphology of the mycelia is discussed.     

Application of temperature-sensitive mutants for single-cell protein production

Cell-division-cycle, temperature-sensitive mutants of Saccharomyces cerevisiae were investigated as a means of altering the morphological characteristics and subsequent physical properties of single-cell protein (SCP). Strain 4471, harboring mutation cdc 4, formed a visible complex mass at the nonpermissive temperature, after being grown at 30°C and then transferred to 37°C for 8 hr. Microscopic observation showed that the mother cell was unable to complete the budding process at the nonpermissive temperature, which caused the cells to enlarge. Viscosity measurements were used to establish and characterize optimum morphological changes in the yeast. The Maximum increase in viscosity occurred when cells were incubated at 30°C and then shifted to 37°C for 8 hr. Strain 4471 exhibited yield stress, whereas A364A did not. Maximum change in yield stress occurred when cells were shifted from 30 to 37°C for 8 hr. No significant loss of protein or RNA occurred in strain 4471, as compared to strain A364A, when incubated at the nonpermissive temperature.     

Effect of Temperature on the Contents of the Two Energy-Reserve Substances,Paramylon and Wax Esters,in Euglena gracilis1

ABSTRACT. When a streptomycin-bleached mutant of Euglena gracilis strain Z was cultured in the dark at 33, 26, or 15°C, the content of paramylon was higher at lower growing temperature while that of wax esters was higher at higher temperature. Transfer of the cells grown at 33°C–15°C decreased the wax ester content while increasing the paramylon content; transfer in the reverse direction caused reverse changes. On incubation with labeled acetate, the cells grown at 33°C showed more distribution of radioactivity in wax esters than the cells grown at lower temperatures. Apparently the two energy-reserve substances have different physiological functions.     

Mutants of Streptococcus pneumoniae that contain a temperature-sensitive autolysin

Two mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-L-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 degrees C or at 30 degrees C, although only the cell extracts obtained at 30 degrees C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 degrees C, mutant cultures grown at 30 degrees C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 degrees C.