Candida Maltosa Research Articles

Effect of lactic acid on the growth dynamics of Candida maltosa YP1

The growth dynamics of the oxidative imperfect yeast strain Candida maltosa YP1 isolated from the surface of fruit yoghurt was studied in relation to the lactic acid concentration ranging from 0 to 1.6% (w/v). The maximal specific growth rate of 0.36 h<sup>–1</sup> and minimal lag-phase duration of 2.9 h were found in the glucose solution without lactic acid at 25°C. The decrease of the natural logarithm of both the specific growth rate (ln µ) and the lag-phase prolongation (ln ) in the dependence on the increase of lactic acid concentration (0–1.59%) was significantly linear (ln µ = –1.1458 – 0.6056 c; R<sup>2</sup><sub>(µ) </sub>= 0.9526; ln l = 1.0141 + 1.9766 c; R<sup>2</sup><sub>() </sub>= 0.9577). Based on these equations, the prediction of the time necessary for C. maltosa YP1 to reach 1 × 10<sup>6</sup> CFU/ml in the dependance on lactic acid concentration and, the initial density of the yeast culture was calculated. For example, C. maltosa YP1 was able to reach the level of 1 × 10<sup>6</sup> CFU/ml in a model glucose solution at the initial concentration N<sub>0</sub> = 1 CFU/ml, 0.9% lactic acid and 25°C within 2 d. The growth predictions presented indicate a considerable resistance of C. maltosa YP1 to lactic acid in the concentration of up to 1.3% (w/v).  

Conservation of a portion of the S. cerevisiae Ure2p prion domain that interacts with the full-length protein.

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating inactive amyloid form of the Ure2 protein. Ure2p residues 1-65 constitute the prion domain, and the remaining C-terminal portion regulates nitrogen catabolism. We have examined the URE2 genes of wild-type isolates of S. cerevisiae and those of several pathogenic yeasts and a filamentous fungus. We find that the normal function of the S. cerevisiae Ure2p in nitrogen regulation is fully complemented by the Ure2p of Candida albicans, Candida glabrata, Candida kefyr, Candida maltosa, Saccharomyces bayanus, and Saccharomyces paradoxus, all of which have high homology in the C-terminal nitrogen regulation domain. However, there is considerable divergence of their N-terminal domains from that of Ure2p of S. cerevisiae. [URE3(Sc)] showed efficient transmission into S. cerevisiae ure2Delta cells if expressing a Ure2p of species within Saccharomyces. However, [URE3(Sc)] did not seed self-propagating inactivation of the Ure2p's from the other yeasts. When overexpressed as a fusion with green fluorescent protein, residues 5-47 of the S. cerevisiae prion domain are necessary for curing the [URE3] prion. Residues 11-39 are necessary for an inactivating interaction with the full-length Ure2p. A nearly identical region is highly conserved among many of the yeasts examined in this study, despite the wide divergence of sequences found in other parts of the N-terminal domains.

Enantioselective microbial reduction of 2-oxo-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′-naphthalenyl)acetic acid and its ethyl ester

The chiral ester ethyl (2 R)-hydroxy-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′-naphthalenyl) acetate 2 and the corresponding acid 4 were prepared as intermediates in the synthesis of the retinoic acid receptor gamma-specific agonist ( R)-3-fluoro-4-[[hydroxy(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)acetyl]amino]benzoic acid 7. Enantioselective reduction of ethyl 2-oxo-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′naphthalenyl)acetate 1 to alcohol 2 was carried out using Aureobasidium pullulans SC 13849 in 98% yield and with an enantiomeric excess (e.e.) of 96%. Among microorganisms screened for the reduction of 2-oxo-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′-naphthalenyl)acetic acid 3 to hydroxy acid 4, Candida maltosa SC 16112 and two strains of Candida utilis (SC 13983, SC 13984) gave reaction yields of >53% with e.e.s of >96%.

Selenium tolerance of yeasts

Selenium tolerance of yeasts widely varies: the growth of some yeasts can be inhibited by a selenium concentration as low as 10(-4) M, whereas others can grow in the presence of 10(-1) M selenium. Homogeneous yeast taxa are characterized by a certain level of selenium tolerance, and heterogeneous taxa show a variable level of tolerance to selenium. In general, ascomycetous yeasts are more tolerant to selenium than basidiomycetous yeasts. Among the ascomycetous yeasts, the genera Dekkera and Schizosaccharomyces exhibited the lowest and the species Candida maltosa, Hanseniaspora valbyensis, Kluyveromyces marxianus, and Yarrowia lipolytica the highest tolerance to selenium. Among the basidiomycetous yeasts, the genera Bullera, Cryptococcus, and Holtermannia showed the lowest and the species Cryptococcus curvatus, Cr. humicola, and Trichosporon spp. the highest tolerance to selenium. The selenium tolerance of yeasts depends on the composition of the growth medium, in particular, on the presence of sulfate, sulfur-containing amino acids, and glutamine in the medium.

Pseudohyphal growth in a dimorphic yeast, Candida maltosa, after disruption of the C-GCN4 gene, a homolog of Saccharomyces cerevisiae GCN4.

The transcriptional activator protein Gcn4p increases the transcription of many genes that code for amino acid synthesis genes during amino acid starvation in Saccharomyces cerevisiae. Here we showed that after the disruption of C-GCN4, a homolog in Candida maltosa of GCN4 in S. cerevisiae, formed pseudohyphae in minimal medium. This is the first report that a GCN4 homolog is involved in the control of morphological transition.

Large-scale screening of selected Candida maltosa, Debaryomyces hansenii and Pichia anomala killer toxin activity against pathogenic yeasts

Large-scale screening of selected Candida maltosa, Debaryomyces hansenii and Pichia anomala killer toxin activity against pathogenic yeasts

Ray38p, a Homolog of a Purine Motif Triple-Helical DNA-Binding Protein, Stm1p, Is a Ribosome-Associated Protein and Dissociated from Ribosomes prior to the Induction of Cycloheximide Resistance in Candida maltosa

Cycloheximide (CYH) resistance in Candida maltosa is dependent on the induction of a ribosomal protein, Q-type L41, the 56th residue of which is glutamine, not proline as in ordinary P-type L41. We found that a 38-kDa protein in a wild-type C. maltosa ribosomal fraction became undetectable upon CYH treatment but detectable again with the establishment of CYH resistance by the induction of Q-type L41. We cloned a gene coding for this protein and named it RAY38 (ribosome-associated protein of yeast). Ray38p is a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, and has a putative RNA-binding motif RGG. The ribosome-associated Ray38p was phosphorylated at serine and threonine residues, and Ray38p that was dissociated from ribosome by CYH treatment was highly phosphorylated in threonine residues. A ray38 null mutant recovered faster from CYH-caused growth stasis than the wild-type strain, suggesting that the dissociation of Ray38p from ribosome facilitates the induction of CYH resistance in C. maltosa.

Microbiology and physiology of Cachaça (Aguardente) fermentations.

Cachaça (aguardente) is a rum-style spirit made from sugar cane juice by artisanal methods in Brazil. A study was made of the production, biochemistry and microbiology of the process in fifteen distilleries in Sul de Minas. Identification of 443 yeasts showed Saccharomyces cerevisiae to be the predominant yeast but Rhodotorula glutinis and Candida maltosa were predominant in three cases. Bacterial infection is a potential problem, particularly in older wooden vats, when the ratio of yeasts:bacteria can be 10:1 or less. A study of daily batch fermentations in one distillery over one season in which 739 yeasts were identified revealed that S. cerevisiae was the predominant yeast. Six other yeast species showed a daily succession: Kluyveromyces marxianus, Pichia heimii and Hanseniaspora uvarum were present only at the beginning, Pichia subpelliculosa and Debaryomyces hansenii were detected from mid to the end of fermentation, and Pichia methanolica appeared briefly after the cessation of fermentation. Despite a steady influx of yeasts from nature, the species population in the fermenter was stable for at least four months suggesting strong physiological and ecological pressure for its maintenance. Cell densities during the fermentation were: yeasts - 4 x 108/ml; lactic acid bacteria -4 x 10(5)/ml; and bacilli - 5 x 10(4)/ml. Some acetic acid bacteria and enterobacteriaceae appeared at the end. Sucrose was immediately hydrolysed to fructose and glucose. The main fermentation was complete after 12 hours but not all fructose was utilised when harvesting after 24 hours.

Large-scale screening of selected Candida maltosa, Debaryomyces hansenii and Pichia anomala killer toxin activity against pathogenic yeasts.

The killer activity of selected crude toxins produced by nine Candida maltosa, Debaryomyces hansenii and Pichia anomala strains was tested at 37 degrees C against 383 strains belonging to 19 pathogenic species of 10 genera (Candida, Clavispora, Cryptococcus, Filobasidiella, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Stephanoascus and Trichosporon). The broad killer spectra exhibited by all crude toxins may lead to the development of new antimycotic agents against medically important yeasts.

Significance of physical attachment of fungi for bio-treatment of water

The inhibitory effect of xenobiotics known to damage cell surface structures was studied. The sensitivity of suspended cells of the two fungi Candida maltosa and Fusarium proliferatum was compared with that of artificial or natural biofilms of these fungi. The results obtained indicate that the resistance of attached cell populations to model xenobiotics is increased compared with suspended cells. Only the attached fungal cells had the capacity to degrade acetone and phenol and to adapt to increasing concentrations of these substances, so they seem ideally suited for bioremediation of waste water.

Competition between n-alkane-assimilating yeasts and bacteria during colonization of sandy soil microcosms.

An n-alkane-assimilating strain of Candida tropicalis was selected in sandy soil inoculated with microorganisms from contaminated sites. Competition experiments with n-alkane utilizers from different strain collections confirmed that yeasts overgrow bacteria in sandy soil. Acidification of the soil is one of the colonization factors useful for the yeasts. It can be counteracted by addition of bentonite, a clay mineral with high ion exchange capacity, but not, however, by kaolin. Strains of different yeast species showed different levels of competitiveness. Strains of Arxula adeninivorans, Candida maltosa, and Yarrowia lipolytica overgrew strains of C. tropicalis, C. shehatae or Pichia stipitis. Two strains of C. maltosa and Y. lipolytica coexisted during several serial transfers under microcosm conditions.

Exploring redundancy in the yeast genome: an improved strategy for use of the cre– loxP system

Gene families having more than three members are a common phenomenon in the Saccharomyces cerevisiae genome. As yeast research enters the post-genome era, the development of existing deletion strategies is crucial for tackling this apparent redundancy, hence a method for performing rapid multiple gene disruptions in this organism has been developed. We constructed three replacement cassettes in which different selectable markers were placed between two loxP loci. Multiple deletions (of members of a gene family) were generated, in one strain, using sequential integration of different replacement markers ( kanMX, LYS2, KlURA3 and SpHIS5). Their excision from the genome was performed simultaneously, as the final step, using a new cre recombinase vector, which carries the cycloheximide-resistance gene from Candida maltosa as a selectable marker. Our multiple gene deletion system significantly accelerates and facilitates the functional analysis process and is particularly useful for studying gene families in either laboratory or industrial yeast strains.

Biodiversity of killer activity in yeasts isolated from the Brazilian rain forest.

The occurrence of killer activity against a panel composed of 22 industrially and (or) medically important yeasts was investigated in 438 yeast and yeast-like cultures belonging to 96 species, isolated from different environments of the Brazilian rain forest. Altogether, 26% of ascomycetes, 56% of basidiomycetes, and 42% of yeast-like cultures exhibited killer activity against at least one of the panel yeasts. More than 15 species never reported before as toxin producers were found, with Pseudozyma antarctica, Trichosporon asteroides, and Geotrichum klebahnii, showing the broader activity spectra. Plasmid curing did not cure the killer phenotypes of Candida maltosa, Debaryomyces hansenii, G. klebahnii, Tr. asteroides, Cryptococcus laurentii, and Ps. antarctica.

Glycosylphosphatidylinositol-anchored Glucanosyltransferases Play an Active Role in the Biosynthesis of the Fungal Cell Wall

A novel 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus was recently characterized. This enzyme splits internally a 1,3-beta-glucan molecule and transfers the newly generated reducing end to the non-reducing end of another 1, 3-beta-glucan molecule forming a 1,3-beta linkage, resulting in the elongation of 1,3-beta-glucan chains. The GEL1 gene encoding this enzyme was cloned and sequenced. The predicted amino acid sequence of Gel1p was homologous to several yeast protein families encoded by GAS of Saccharomyces cerevisiae, PHR of Candida albicans, and EPD of Candida maltosa. Although the expression of these genes is required for correct morphogenesis in yeast, the biochemical function of the encoded proteins was unknown. The biochemical assays performed on purified recombinant Gas1p, Phr1p, and Phr2p showed that these proteins have a 1,3-beta-glucanosyltransferase activity similar to that of Gel1p. Biochemical data and sequence analysis have shown that Gel1p is attached to the membrane through a glycosylphosphatidylinositol in a similar manner as the yeast homologous proteins. The activity has been also detected in membrane preparations, showing that this 1,3-beta-glucanosyltransferase is indeed active in vivo. Our results show that transglycosidases anchored to the plasma membrane via glycosylphosphatidylinositols can play an active role in fungal cell wall synthesis.

Cloning and Characterization ofEPD2, a Gene Required for Efficient Pseudohyphal Formation of a Dimorphic Yeast,Candida maltosa

Candida maltosa is a dimorphic fungus and its pseudohyphal growth is partly induced by additional copies of the centromeric DNA (CEN) region1) or n-alkane. In the course of analyzing the induction mechanism of pseudohyphal growth, we isolated EPD1, which is similar in sequence to PHR1 and PHR2 of Candida albicans. Epdlp could be involved in cell wall maintenance and is essential for pseudohyphal growth induced by CEN and n-hexadecane at pH 4 and by n-hexadecane at pH 7.) In this paper, we cloned EPD2 of C. maltosa, which is highly similar to EPD1, PHR1, and PHR2. The transcription of EPD2 is induced strongly when cells are grown in SD medium of higher pH (pH 7), but not in SD medium of lower pH (pH 4). This pattern of expression was an inverse of that of EPD1. This alternate expression is similar to that between PHR1 and PHR2. The expression of EPD2 was much higher when C. maltosa was grown on the n-hexadecane solid medium than grown in the n-hexadecane liquid medium. The efficiency of pseudohyphal formation of an epd2 null mutant on n-hexadecane medium at pH 7 or 7.5 was lower than that of the wild-type strain. These results suggest that Epd2p is required for efficient pseudohyphal formation induced by n-hexadecane in the medium at pH 7.

A Gene Coding for a Ribosomal Protein L41 in Cycloheximide-Resistant Ribosomes Has a Promoter Which Is Upregulated under the Growth-Inhibitory Conditions in Yeast,Candida maltosa

We previously found by using yeast,Candida maltosa,that cycloheximide (CYH) sensitivity of ribosomes is dependent on the 56th amino acid residues of a ribosomal protein, L4133L41 ofSaccharoymces cerevisiaehas been renamed L42A/B in the recent paper by R. J. Planta and W. H. Mager (Yeast14,471–477, 1998).(proline in sensitive and glutamine in resistant ribosomes). We also revealed that in this yeast, which has bothL41-Ptype andL41-Qtype genes, the expression of the latter type genes is induced by the addition of CYH in the medium to make the cells inducibly resistant to CYH. In this paper, we analyzed the promoter region ofL41-Q2a,one of the CYH-inducibleL41-Qtype genes and found two elements required for the induction of expression: one was a GCRE (Gcn4p-responsive element ofSaccharomyces cerevisiae)-like element and the other was a GT-rich element. This promoter region was also required for its expression under some other growth inhibitory conditions. Furthermore, it was suggested that Q-type ribosomes synthesized under these conditions are more resistant to these inhibitory conditions.

Cyanobacteria—a potential source of new biologically active substances

Hydrophilic and lipophilic extracts of twelve cyanobacterial strains, isolated from fresh and brackish water, and two waterblooms, collected during the summer from the Baltic Sea, were investigated for their antibiotic activities against seven microorganisms. No inhibitory effects were found against the three Gram-negative bacteria Escherichia coli, Proteus mirabilis and Serratia marcescens and the yeast Candida maltosa. Of all cyanobacterial samples, extracts from seven species inhibited the growth of at least one of the Gram-positive bacteria Micrococcus flavus, Staphylococcus aureus and Bacillus subtilis. M. flavus proved to be the most sensitive bacterium in the agar diffusion test system. In particular, the hexane and dichlormethane extracts showed antimicrobial effects. But only one water extract, prepared from material of a natural waterbloom, was found to be active.

Null mutation in IRE1 gene inhibits overproduction of microsomal cytochrome P450Alk1 (CYP 52A3) and proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae.

Overproduction of microsomal cytochrome P450Alk1 (P450Alk1) of Candida maltosa in Saccharomyces cerevisiae resulted in an extensive proliferation of endoplasmic reticulum (ER) and induction of Kar2p and Pdi1p. The ire1 null mutation severely suppressed ER proliferation, reduced the level of functional P450Alk1, and showed no induction of these ER chaperones, suggesting that the function of Ire1p is required for ER proliferation upon the overproduction of P450Alk1. Cerulenin, a potent inhibitor of lipid biosynthesis, also induced these chaperones in an Ire1p-dependent manner and limited the production of functional P450Alk1. These results imply that Ire1p may function to restore the balance between membrane proteins and lipids of the ER when the ER is relatively overcrowded by membrane proteins.

Candida maltosa NADPH-cytochrome P450 reductase: cloning of a full-length cDNA, heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum.

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resulted in an approximately 70-fold increase in NADPH-cytochrome c reductase activity in the microsomal fraction. The functional integrity of the heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified state. The signal-anchor sequence of the reductase was identified within the N-terminal region of the protein by means of constructing and expressing fusion proteins with the cytosolic form of yeast invertase. The first 33 amino acids turned out to be sufficient for stable membrane insertion, wild-type membrane orientation and retention in the endoplasmic reticulum. As shown by immunoelectron microscopy, the heterologously expressed reductase was integrated into the endoplasmic reticulum of the host organism. It triggered a strong proliferation of the membrane system. This membrane-inducing property of the reductase was transferable to the cytosolic reporter protein with the same N-terminal sequences that confer membrane insertion.

Oxygenation Cascade in Conversion of n-Alkanes to α,ω-Dioic Acids Catalyzed by Cytochrome P450 52A3

Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system. Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1, 16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1, 16-hexadecanedioic acid. As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity. Kinetic studies of the individual sequential reactions revealed high Vmax values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min-1, respectively), whereas the oxidation of hexadecanoic acid, 1, 16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min-1, respectively). 1-Hexadecanol was found to be the main branch point between mono- and diterminal oxidation. Taken together with data on the incorporation of 18O2-derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono- and diterminal monooxygenation reactions from n-alkanes to alpha, omega-dioic acids with high regioselectivity.