表型相似的热带假丝酵母和麦芽糖假丝酵母在脉冲电泳核型上的差异

热带假丝酵母Ｃａｎｄｉｄａ ｔｒｏｐｉｃａｌｉｓ （Ｃａｓｔｅｌｌａｎｉ ）Ｂｅｒｋｈｏｕｔ和麦芽糖假丝酵母Ｃ． ｍａｌｔｏｓａＫｏｍａｇａｔａ, Ｎａｋａｓｅ ＆ Ｋａｔｓｕｙａ是两种可利用烃类作为碳和能量来源的酵母菌,前者还是一种条件致病菌,可引起系统感染。这两种假丝酵母菌在形态和生理生化性状上非常相似,用常规分类方法不易准确地鉴别。本研究对Ｃ． Ｔｒｏｐｉｃａｌｉｓ和Ｃ ｍａｌｔｏｓａ的模式菌株以及中国普通微生物菌种保藏中心（ＣＧＭＣＣ）保藏的归于这两个种名下的其它菌株进行了脉冲电泳核型比较分析。发现这两个表型相似的种具有明显不同的染色体ＤＮＡ分子带型,而同一种内的不同菌株却具有相同或相似的分子核型。Ｃ．Ｔｒｏｐｉｃａｌｉｓ的特异染色体ＤＮＡ分子带谱为２条８．５—１．２ Ｍｂ的带, ４条２．３－３．４ Ｍｂ的带。 Ｃ ｍａｌｔｏｓａ的特异带谱为： ３～４条分子量在１．１－１．３Ｍｂ范围内的带, １条约为２．２Ｍｂ的带以及２－３条大小为３．２－３．５Ｍｂ的带。 Ｃ ｔｒｏｐｉｃａｌｉｓ与Ｃ ｍａｌｔｏｓａ在染色体ＤＮＡ分子带型上的差异与二者在可溶性淀粉的同化能力和４０℃下的生长能力上的差异具有明显的相关性…     

Physiological and DNA characterization of Candida maltosa,a hydrocarbon-utilizing yeast

Selected yeasts classified as Candida sake van Uden et Buckley were examined for their physiological, morphological and immunological properties and their DNA relatedness. Candida maltosa Komagata, Nakase et Katsuya is herein recognized as a species separate from C. sake. Candida maltosa was distinguished from C. sake and from C. tropicalis by insignificant DNA reassociation. In addition, C. maltosa was distinguished from C. sake by its higher maximal growth temperature and lower guanine plus cytosine content of its DNA and from C. tropicalis by its failure to utilize soluble starch for growth and its resistance to cycloheximide. The species C. cloacae and C. subtropicalis are placed in synonymy with C. maltosa.     

Phylogenetic identification of n-alkane assimilating Candida yeasts based on nucleotide divergence in the 59 end of LSU rDNA gene

Phylogenetic relationships of several species within the n-alkane assimilating Candida yeasts were investigated by using characters from the nucleotide sequence of the variable D1/D2 region at the 5' end of a large-subunit (26S) ribosomal DNA (rDNA) gene. First the nucleotide sequences of D1/D2 domain of Candida sp. 1098 (formerly identified as C. tropicalis 1098) and its dicarboxylic acid-producing-mutant strain M1210 were investigated. These two nucleotide sequences were identical and lacked only one base pair compared with that of C. maltosa CBS 5611 (type strain), and they were identified as C. maltosa. We then showed that C. maltosa IFO 1978 (formerly identified as C. cloacae) and C. maltosa IFO 1975 (formerly identified as C. subtropicalis) had the same nucleotide sequence and had only one base pair substitution compared with C. maltosa CBS 5611 (type strain), which is consistent with conventional classification. We also found that another widely studied n-alkane assimilating Candida yeast, C. tropicalis pk233, to be C. viswanathii.     

Evaluation of industrial yeasts for pathogenicity.

Eleven yeasts representative of species of industrial interest were compared with Candida albicans for their potential pathogenicity for untreated and cortisone-treated mice. Only C. tropicalis produced a progressive infection similar to that produced by C. albicans. Candida lipolytica, Torulopsis spp., and Hansenula polymorpha were not recovered from mice 6 days after inoculation. Kluyveromyces fragilis, C. pseudotropicalis, C. utilis, C. guilliermondii and C. maltosa were recovered from mice but did not produce evidence of infection.     

Microorganisms form exocellular structures, trophosomes, to facilitate biodegradation of oil in aqueous media

Cytochemical staining and microscopy were used to study the trophic structures and cellular morphotypes that are produced during the colonization of oil-water interfaces by oil-degrading yeasts and bacteria. Among the microorganisms studied here, the yeasts (Schwanniomyces occidentalis, Torulopsis candida, Candida tropicalis, Candida lipolytica, Candida maltosa, Candida paralipolytica) and two representative bacteria (Rhodococcus sp. and Pseudomonas putida) produced exocellular structures composed of biopolymers during growth on petroleum hydrocarbons. Four of the yeasts including S. occidentalis, T. candida, C. tropicalis and C. maltosa excreted polymers through modified sites in their cell wall ('canals'), whereas C. lipolytica and C. paralipolytica and the two bacterial species secreted polymers over the entire cell surface. These polymers took the form of fibrils and films that clogged pores and cavities on the surfaces of the oil droplets. A three-dimensional reconstruction of the cavities using serial thin sections showed that the exopolymer films isolated the ambient aqueous medium together with microbial cells and oil to form both closed and open granules that contained pools of oxidative enzymes utilized for the degradation of the oil hydrocarbons. The formation of such granules, or 'trophosomes,' appears to be a fundamental process that facilitates the efficient degradation of oil in aqueous media.     

A mutated hygromycin resistance gene is functional in the n-alkane-assimilating yeast Candida tropicalis

Development of a transformation system in the n-alkane-assimilating diploid yeast Candida tropicalis requires an antibiotic resistance gene in order to establish a selectable marker. The resistance gene for hygromycin B has often been used as a selectable marker in yeast transformation. However, C. tropicalis harboring the hygromycin resistance gene (HYG) was as sensitive to hygromycin B as the wild-type strain. Nine CTG codons were found in the ORF of the HYG gene. This codon has been reported to be translated as serine rather than leucine in Candida species. Analysis of the tRNA gene in C. tropicalis with the anticodon CAG [tRNA(CAG) gene], which is complementary to the codon CTG, showed that the sequence was highly similar to that of the C. maltosa tRNA(CAG) gene. In C. maltosa, the codon CTG is read as serine and not leucine. These results suggested that the HYG gene was not functional due to the nonuniversal usage of the CTG codon. Each of the nine CTG codons in the ORF of the HYG gene was changed to a CTC codon, which is read as leucine, by site-directed mutagenesis. When a plasmid containing the mutated HYG gene (HYG#) was constructed and introduced into C. tropicalis, hygromycin-resistant transformants were successfully obtained. This mutated hygromycin resistance gene may be useful for direct selection of C. tropicalis transformants.     

Characterization of mitochondrial DNA in various Candida species: isolation, restriction endonuclease analysis, size, and base composition.

A practical and effective method for the extraction of mitochondrial DNA from Candida species was developed. Zymolyase was used to induce yeast protoplasts, and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Restriction endonuclease analyses of mitochondrial DNAs from 19 isolates representing seven species of Candida (C. albicans, C. kefyr, C. lusitaniae, C. maltosa, C. parapsilosis, C. shehatae, and C. tropicalis) and Lodderomyces elongisporus revealed different cleavage patterns that appeared to be specific for the species. Few common restriction fragments were evident. The genome sizes of the mitochondrial DNAs ranged from 26.4 to 51.4 kilobase pairs, and the guanine-plus-cytosine contents ranged from 20.7 to 36.8 mol%. There was no correlation between the base compositions of nuclear and mitochondrial DNAs. Eight isolates of C. parapsilosis, including the type culture, and an ascosporogenous strain of L. elongisporus, which was once proposed as the teleomorph of C. parapsilosis, had similar mitochondrial DNA molecular sizes (30.2 and 28.8 kilobase pairs); however, restriction endonuclease patterns of these organisms were distinct. These data provide additional support for discrimination of these two species. The results of our experiments demonstrate that mitochondrial DNA analyses may provide useful criteria for the differentiation of yeast species.     

Application of Agglutinins for the Rapid and Accurate Identification of Medically Important Candida Species

Agglutinins have been prepared against the medically important Candida species. Crude antisera to the various species demonstrated intense cross-reactions with heterologous yeastlike fungi as well as with many true yeasts. However, carefully monitored adsorptions of selected antisera allowed the production of six factor sera that proved useful in a slide agglutination test. These six sera permitted the rapid and specific identification of C. guilliermondii, C. krusei, C. parapsilosis, and C. pseudotropicalis. They also allowed the delineation of two groups: (i) C. albicans (type A)-C. tropicalis and (ii) C. albicans (type B)-C. stellatoidea. C. albicans type A could be readily distinguished from C. tropicalis by its ability to form germ tubes in serum. C. stellatoidea could be distinguished from C. albicans type B by its predominantly filamentous growth on a nutritionally deficient medium. The medically important Candida species could be identified within 24 hr by the combined use of serological and morphological procedures.     

Yeasts isolated from plant-associated beetles and other insects: seven novel Candida species near Candida albicans

Yeasts related to Candida albicans were isolated from the digestive tracts of beetles in eight families and various orders of insects such as earwigs, crickets, and roaches, most of which were caught at light traps or in a few cases directly from plant materials. Based on comparisons of DNA sequences and other taxonomic characteristics, a total of 41 isolates were identified as Candida orthopsilosis, Candida pseudorhagii, Candida maltosa, Candida parapsilosis, Candida tropicalis, Candida neerlandica, Lodderomyces elongisporus, and seven new Candida species. The new species and type strains are designated as Candida gigantensis NRRL Y-27736T, Candida bohiensis NRRL Y-27737T, Candida alai NRRL Y-27739T, Candida buenavistaensis NRRL Y-27734T, Candida frijolesensis NRRL Y-48060T, Candida labiduridarum NRRL Y-27940T, and Candida tetrigidarum NRRL Y-48142T. A phylogeny based on SSU and LSU rRNA gene sequences indicated that most of the new species were closely related to members of the C. albicans/L. elongisporus clade, such as C. albicans, Candida dulbliniensis, C. neerlandica, Candida chauliodes, and Candida corydali. Candida alai was placed near this clade, but no closely related sister taxon was identified. The ecology of the insect-associated yeasts is discussed and compared with the results from other studies.     

Sequences of two tandem genes regulated by carbon sources, one being essential for n-alkane assimilation in Candida maltosa

Several n-alkane-inducible clones were isolated from the genomic library of an n-alkane-assimilation yeast, Candida maltosa, by the differential hybridization method. Among these, one of the most predominantly expressed clones was analyzed. The nucleotide sequence of the cloned DNA fragment showed that it contained two open reading frames, one encoding a protein of 127 amino acids (aa) and the other a protein of 276 aa. The former was named POX18Cm, because the sequence was highly homologous to that of the Candida tropicalis gene, POX18, which already had been identified as encoding a small oleate-inducible peroxisomal protein. The latter, named ALI1, had no homologous sequences in the EMBL database (1990 release). Northern-blot hybridization indicated that the expression of these two genes was regulated by carbon sources in the media. From gene-disruption experiments, it was concluded that ALI1 was essential for assimilation of n-alkane by C. maltosa.     

Detection of specificity of a new antigen in Candida tropicalis and its evaluation by taxonomic DNA analyses

Monospecific factor serum for identifying Candida tropicalis was obtained either from rabbit antiserum to heated cells of C. tropicalis M 1519 (S 96) or from antiserum to C. tropicalis IFO 1400, by adsorption with heated cells of Candida albicans serotype A, or C. albicans (A) and Candida krusei, respectively. We designated this adsorbed serum factor t serum. The monospecific factor serum reacted with 31 out of 32 strains of C. tropicalis, only when tested on heat-treated cell antigens, whereas it did not react with any of 72 strains of the six other medically important species of Candida. The morphological and physiological characteristics of the one strain of C. tropicalis that did not react with the factor t serum, designated the t- -strain, were shown to be similar to those of the type strain of C. tropicalis by most of the methods employed for identifying Candida. Therefore, cell wall mannan from the t- -strain was compared with that from several typical strains of C. tropicalis for its specificity by the precipitation reaction and also for its 1H-nuclear magnetic resonance spectrum. The results showed that these mannans are similar to each other serologically and physicochemically, suggesting that the new antigen t is not mannan. Taxonomic characterization of the t-- and several typical strains of C. tropicalis was carried out by determining the mol% G+C of their DNA and also their DNA homology. Although the mol% G+C values of four typical strains of C. tropicalis were fairly similar (35.2 to 36.2 mol% by the Tm method and 35.5 to 36.4 mol% by the HPLC method), the t- -strain had a G+C content of 44.1 (Tm) and 43.3 (HPLC) mol%. Furthermore, the DNAs of the t- -strain and the type strain of C. tropicalis showed only 18.2% relatedness. These results suggest that the antigen corresponding to serum factor t exists only in the cell wall of C. tropicalis strains, not in those of the other medically important Candida, and that the t- -strain should not be classified as C. tropicalis. In conclusion, the taxonomic value and usefulness of factor t serum is primarily for differentiating C. tropicalis from C. albicans serotype A serologically.     

Evolution of codon usage patterns: the extent and nature of divergence between Candida albicans and Saccharomyces cerevisiae.

Codon usage in a sample of 28 genes from the pathogenic yeast Candida albicans has been analysed using multivariate statistical analysis. A major trend among genes, correlated with gene expression level, was identified. We have focussed on the extent and nature of divergence between C.albicans and the closely related yeast Saccharomyces cerevisiae. It was recently suggested that significant differences exist between the subsets of preferred codons in these two species [Brown et al. (1991) Nucleic Acids Res. 19, 4293]. Overall, the genes of C.albicans are more A + T-rich, reflecting the lower genomic G + C content of that species, and presumably resulting from a different pattern of mutational bias. However, in both species highly expressed genes preferentially use the same subset of 'optimal' codons. A suggestion that the low frequency of NCG codons in both yeast species results from selection against the presence of codons that are potentially highly mutable is discounted. Codon usage in C.albicans, as in other unicellular species, can be interpreted as the result of a balance between the processes of mutational bias and translational selection. Codon usage in two related Candida species, C.maltosa and C.tropicalis, is briefly discussed.     

Molecular typing of Candida spp. by random amplification of polymorphic DNA and analysis of restriction fragment length polymorphism of ribosomal DNA repeats

Random amplification of polymorphic DNA (RAPD) using an arbitrary oligonucleotide primer (5'-CGGTGCGACG) and analysis of restriction fragment length polymorphism of ribosomal DNA (rDNA-RFLP) after digestion of genomic DNA with restriction endonuclease EcoRI were investigated as tools for genotypic delineation beyond the species level of 91 Candida clinical isolates and four reference strains including 33 Candida albicans, 19 Candida tropicalis, 22 Candida krusei and 21 Candida (Torulopsis) glabrata. Results indicated that both techniques can be useful for typing isolates of the above species, although showing a variable discriminative potential with different species. As compared to RAPD fingerprinting, the discriminative potential of rDNA-RFLP appeared to be highest for C. albicans and lowest for C. glabrata, being overall similar for C. krusei and identical for C. tropicalis. A comparative analysis of the results obtained with the two typing techniques showed that, except for C. tropicalis, they were able to provide non-redundant information, and that their use in combination could enhance the discriminative potential for delineation among C. glabrata and C. krusei isolates.     

Cloning and molecular characterization of a gene coding D-xylulokinase (CmXYL3) from Candida maltosa

AIMS: To clone and identify a gene (CmXYL3) coding D-xylulokinase from Candida maltosa Xu316 and understand its physiological function. METHODS AND RESULTS: Based on the conserved regions of the known D-xylulokinase-encoding genes, a pair of degenerate primers was designed to clone the CmXYL3 gene from C. maltosa Xu316. The coding region and sequences flanking the CmXYL3 gene were obtained by PCR-based DNA walking method. Southern blotting analysis suggested that there is a single copy of the CmXYL3 gene in the genome. The open reading frame starting from ATG and ending with TAG stop codon encoded 616 amino acids with a calculated molecular mass of 68889.743 Da. The CmXYL3 gene under the control of the GPD1 promoter was heterologously expressed in Saccharomyces cerevisiae deficient in D-xylulokinase (deltaScXKS1::LEU2) activity, and restored growth on D-xylulose. The specific activity of D-xylulokinase varied during xylose fermentation and was correlated with aeration level. After growth on different pentoses and pentitols as sole carbon sources, the highest specific activity of D-xylulokinase was observed on D-xylose. CONCLUSIONS: The CmXYL3 gene isolated from C. maltosa Xu316 encodes a novel D-xylulokinase that plays a pivotal role in xylulose metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes the isolation and cloning of D-xylulokinase gene (CmXYL3) from C. maltosa Xu316. D-xylulokinase is pivotal for growth and product formation during xylose metabolism. Better understanding of the biochemical properties and the physiological function of D-xylulokinase will contribute to optimizing fermentation conditions and determining the strategies for metabolic engineering of C. maltosa Xu316 for further improvement of xylitol yield and productivity.     

CYP52 (cytochrome P450alk) multigene family in Candida maltosa: molecular cloning and nucleotide sequence of the two tandemly arranged genes

Southern blot analysis under low-stringency conditions using a previously isolated n-alkane-inducible cytochrome P450 (P450alk) gene as a probe revealed the presence of multiple P450alk-related genes in the genome of Candida maltosa. Nine P450alk-related genes (one reported previously and eight in the present report) were isolated from a genomic library constructed from this strain, and these were classified on the basis of sequence similarities into three pairs of putative allelic genes and three nonallelic genes. Two pairs of these alleles were tandemly arranged in the genome. The complete nucleotide sequences of one of these pairs were determined and compared to other members of this P450 family (CYP52) in C. maltosa and C. tropicalis. Northern blot analysis further showed that these genes were regulated by carbon sources. These results provide evidence for a P450alk (CYP52) multigene family in C. maltosa.     

Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome.

To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae.     

A PCR-based approach to sequence the Candida tropicalis HSP90 gene

The heat shock protein 90 (hsp90) gene sequence is known to be highly conserved across the species barrier. A PCR-based method was thus utilised in an attempt to sequence the Candida tropicalis hsp90 gene. Primers for PCR were designed from conserved regions of the gene, which were identified by comparing the Saccharomyces cerevisiae and Candida albicans hsp90 gene sequences. Different sets of primers were designed to amplify and obtain overlapping DNA sequences of the C tropicalis gene. PCR was carried out on genomic DNA of Candidca tropicalis and the PCR products were cloned into suitable vector molecules for sequencing. In this way, a 2,070-basepair sequence of the C. tropicalis hsp90 gene was obtained. The PCR-based approach proved to be an easier method of obtain the sequence of a highly conserved gene, as compared to more conventional methods.     

New yeast vectors containing the autonomously replicating sequences from Candida maltosa genome

Two different DNA sequences from the yeast Candida maltosa confer the ability to replicate autonomously to the yeast integrative vector pLD700 on which they are cloned. The recombinant plasmids pLD701 and pLD702 with autonomously replicating sequences (ARS) from Candida maltosa and LEU2 gene from Saccharomyces cerevisiae transform the auxotrophic strain S. cerevisiae DC5 with the efficiency 3-5 x 10(3) per microgram of DNA. Like other yeast vectors harbouring ARS, these plasmids are not stable in yeast cells. Restriction and hybridization analyses have revealed the pLD701 plasmid to contain ARS from chromosomal DNA of C. maltosa. Plasmid pLD701 appears to be a useful vector for yeast transformation.     

26S rDNA单链构象多态性分析在临床酵母菌菌种鉴定中的应用

摘要： 【目的】探讨酵母菌临床分离株26S rDNA D1/D2区序列种内相似性和种间差异性的快速检测方法,为临床酵母菌菌种鉴定方法的改进奠定基础。调查北京地区临床酵母菌的种群多样性,为国内酵母菌感染的流行病学研究提供新的基础数据。【方法】用5种常见临床酵母菌种的模式和权威菌株作为标准参考菌株,从北京四家综合性医院收集临床酵母菌260余株,PCR扩增其26S rDNA D1/D2区,对扩增产物进行单链构象多态性（Single-Strand Conformation Polymorphism,SSCP）分析和序列测定分析。【结果】常见病原酵母菌26S rDNA D1/D2区的SSCP图谱具有明显的种间差异性和种内相似性,可以通过该方法对菌株进行初步的菌种鉴定。D1/D2-SSCP和序列分析相结合,对260余株临床酵母菌进行了菌种鉴定,共鉴定有10个属20个种,优势属为念珠菌属(Candida),优势种及其所占比例分别是：C. albicans (57.7%), C. parapsilosis (10.0%), C. tropicalis (9.2%), C. glabrata (6.7%)和C. krusei (5.8%),并发现过去从未或很少报道致病的酵母菌种,愈来愈多地出现在临床分离菌株中。【结论】 26S rDNA D1/D2区的SSCP图谱分析为临床酵母菌株的快速鉴定提供了新的方法;北京地区酵母菌临床分离株呈种群多样性分布,C. albicans虽然仍占优势,但其它念珠菌种的比例已达42%。