利用可重复使用的URA3标记基因建立热带假丝酵母基因敲除系统

热带假丝酵母(Candida tropicalis)在发酵工业中具有重要的应用潜力,但二倍体遗传结构和较低的遗传转化效率限制了其代谢工程育种技术的应用。建立可靠的遗传转化技术并高效的删除目的基因是代谢工程改造热带假丝酵母的重要前提。文章以C. tropicalis ATCC 20336为出发菌株,通过化学诱变筛选获得了尿嘧啶缺陷型突变株C. tropicalis XZX(ura3/ura3)。以丙酮酸脱羧酶(Pyruvate decarboxylase,PDC)基因作为靶基因构建了两端包含同源臂并在选择性标记C. tropicalis URA3(Orotidine-5′-phosphate decarboxylase,乳清酸核苷-5-磷酸脱羧酶)基因两侧同向插入源于沙门氏菌(Salmonella typhimurium)的hisG序列的基因敲除盒PDC1-hisG-URA3-hisG- PDC1(PHUHP),并转化宿主菌株C. tropicalis XZX,筛选获得PHUHP片段正确整合到染色体的PDC基因位点的转化子XZX02。在此基础上,将转化子XZX02涂布于5-FOA(5-氟乳清酸)选择培养基上,筛选得到URA3基因从PHUHP片段中丢失的营养缺陷型菌株XZX03。进一步构建了第2个PDC等位基因的删除表达盒PDCm- URA3-PDCm,并转化C. tropicalis XZX03菌株,获得转化子C. tropicalis XZX04。经PCR和DNA测序确认转化子C. tropicalis XZX04细胞染色体上的两个PDC等位基因被成功敲除。文章建立了一种营养缺陷型标记可重复使用的热带假丝酵母遗传转化技术,利用该技术成功敲除了细胞的PDC基因,为进一步利用代谢工程改造热带假丝酵母奠定了基础。     

热带假丝酵母唑类耐药相关基因的研究进展

临床上热带假丝酵母(又称热带念珠菌)的分离率越来越高,唑类抗真菌药物因较低的细胞毒性且大多可口服给药,是治疗热带念珠菌感染的常用药物。我国耐唑类药物热带念珠菌的分离率较高,因此有必要了解其具体机制,为寻求新的药物作用靶点提供依据。目前认为,与热带念珠菌唑类耐药有关的主要机制有靶基因ERG11过度表达和突变、编码转录因子的upc2基因过度表达和突变、外排泵基因过度表达及其他相关基因过度表达等。本文就目前热带念珠菌唑类耐药机制的基因水平研究进展进行综述。     

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.     

Development of an integrative DNA transformation system for the yeast Candida tropicalis.

We developed the alkane and fatty-acid utilizing yeast Candida tropicalis as a host for DNA transformations. The system is based on an auxotrophic mutant host of C. tropicalis which is defective in orotidine monophosphate decarboxylase (ura3). The ura3 host was isolated by mutagenesis and a double-selection procedure that combined nystatin enrichment selection and 5-fluoro-orotic acid resistance selection. As a selectable marker, we isolated and characterized the C. tropicalis URA3 gene. Plasmid vectors that contained the C. tropicalis URA3 gene transformed the C. tropicalis mutant host at a frequency of 10(3) to 10(4) transformants per micrograms of plasmid DNA. Vectors that contained the Saccharomyces cerevisiae URA3 gene could not transform C. tropicalis. DNA transfer was accomplished by modified versions of either spheroplast generation (CaCl2-polyethylene glycol)-fusion or cation (LiCl) procedures developed for S. cerevisiae. Plasmid vectors that had been cut within the C. tropicalis URA3 fragment integrated by homologous recombination at the URA3 locus.     

A Xenopus tropicalis oligonucleotide microarray works across species using RNA from Xenopus laevis

Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.     

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.     

Interspecific complementation analysis by protoplast fusion of Candida tropicalis and Candida albicans adenine auxotrophs.

A protocol employing inositol starvation was used to isolate proline and adenine auxotrophs of Candida tropicalis. Interspecific hybrids between red adenine auxotrophs of C. tropicalis and Candida albicans were formed by protoplast fusion. These C. tropicalis red adenine auxotrophs were shown to fall into two complementation groups by crossing them with a known C. albicans ade1 tester strain. It is suggested that these two groups correspond to the ade1 and ade2 mutants of Saccharomyces cerevisiae and C. albicans and that these defined mutants may be useful in attempts to develop transformation systems for C. tropicalis.     

Hydroxylation of phenol to catechol by Candida tropicalis: involvement of cytochrome P450

Microsomal preparations isolated from yeast Candida tropicalis (C. tropicalis) grown on three different media with or without phenol were isolated and characterized for the content of cytochrome P450 (CYP) (EC 1.14.15.1). While no CYP was detected in microsomes of C. tropicalis grown on glucose as the carbon source, evidence was obtained for the presence of the enzyme in the microsomes of C. tropicalis grown on media containing phenol. Furthermore, the activity of NADPH: CYP reductase, another enzyme of the microsomal CYP-dependent system, was markedly higher in cells grown on phenol. Microsomes of these cells oxidized phenol. The major metabolite formed from phenol by microsomes of C. tropicalis was characterized by UV/vis absorbance and mass spectroscopy as well as by the chromatographic properties on HPLC. The characteristics are identical to those of catechol. The formation of catechol was inhibited by CO, the inhibitor of CYP, and correlated with the content of cytochrome P450 in microsomes. These results, the first report showing the ring hydroxylation of phenol to catechol with the microsomal enzyme system of C. tropicalis, strongly suggest that CYP-catalyzed reactions are responsible for this hydroxylation. The data demonstrate the progress in resolving the enzymes responsible for the first step of phenol degradation by the C. tropicalis strain.     

侵袭性热带念珠菌感染流行病学及药物敏感性

全球范围内,随着抗肿瘤药物、免疫抑制剂和广谱抗菌药物的使用,真菌感染的发病率显著提高,其中念珠菌感染占到绝大多数。目前热带念珠菌已经成为非白念珠菌中最常见的病原菌。我国热带念珠菌的临床分离率及对氟康唑及伏立康唑的耐药率都明显高于世界平均水平。但是,相比于白念珠菌,关于热带念珠菌的研究及相关临床信息相对较少。该文就侵袭性热带念珠菌感染危险因素、流行病学以及药物敏感性进行全面的综述。     

Comparison of human and soil Candida tropicalis isolates with reduced susceptibility to fluconazole

Infections caused by treatment-resistant non-albicans Candida species, such as C. tropicalis, has increased, which is an emerging challenge in the management of fungal infections. Genetically related diploid sequence type (DST) strains of C. tropicalis exhibiting reduced susceptibility to fluconazole circulated widely in Taiwan. To identify the potential source of these wildly distributed DST strains, we investigated the possibility of the presence in soil of such C. tropicalis strains by pulsed field gel electrophoresis (PFGE) and DST typing methods. A total of 56 C. tropicalis isolates were recovered from 26 out of 477 soil samples. Among the 18 isolates with reduced susceptibility to fluconazole, 9 belonged to DST149 and 3 belonged to DST140. Both DSTs have been recovered from our previous studies on clinical isolates from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) program. Furthermore, these isolates were more resistant to agricultural azoles. We have found genetically related C. tropicalis exhibiting reduced susceptibility to fluconazole from the human hosts and environmental samples. Therefore, to prevent patients from acquiring C. tropicalis with reduced susceptibility to azoles, prudent use of azoles in both clinical and agricultural settings is advocated.     

Tol2 transposon-mediated transgenesis in Xenopus tropicalis

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.     

N-acetylglucosamine induces white-to-opaque switching and mating in Candida tropicalis, providing new insights into adaptation and fungal sexual evolution

Pathogenic fungi are capable of switching between different phenotypes, each of which has a different biological advantage. In the most prevalent human fungal pathogen, Candida albicans, phenotypic transitions not only improve its adaptation to a continuously changing host microenvironment but also regulate sexual mating. In this report, we show that Candida tropicalis, another important human opportunistic pathogen, undergoes reversible and heritable phenotypic switching, referred to as the "white-opaque" transition. Here we show that N-acetylglucosamine (GlcNAc), an inducer of white-to-opaque switching in C. albicans, promotes opaque-cell formation and mating and also inhibits filamentation in a number of natural C. tropicalis strains. Our results suggest that host chemical signals may facilitate this phenotypic switching and mating of C. tropicalis, which had been previously thought to reproduce asexually. Overexpression of the C. tropicalis WOR1 gene in C. albicans induces opaque-cell formation. Additionally, an intermediate phase between white and opaque was observed in C. tropicalis, indicating that the switching could be tristable.     

Construction of BAC library for the amphibian Xenopus tropicalis

Xenopus tropicalis has become an alternative model to the amphibian Xenopus laevis because it is better suited for genetic and genomic studies. We have constructed a genomic BAC library consisting of over 100,000 clones from sperm of Xenopus tropicalis. Analysis by pulsed field gel electrophoresis of representative BAC clones indicated the average size of insert DNA to be 100 kb, and we estimated the library covers 6 times the Xenopus tropicalis genome of 1.7 x 10(9) base pairs. To evaluate the BAC library, we attempted to isolate BAC clones which contain a protocadherin gamma (Pcdh gamma) gene and found that the isolated BAC clones are assembled as two separate contigs. This result suggests the presence of at least two clusters for the Pcdh gamma gene in the genome of X. tropicalis.     

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.     

Phosphorus-31 and carbon-13 nuclear magnetic resonance study of glucose and xylose metabolism in agarose-immobilized Candida tropicalis.

Candida tropicalis can ferment both hexose and pentose sugars. Here, we have used 31P and 13C nuclear magnetic resonance spectroscopy to study the capacity of this yeast species to metabolize glucose or xylose when immobilized in small (< 1-mm-diameter) agarose beads. Immobilized C. tropicalis metabolizing glucose showed rapid initial growth within the beads. A corresponding drop in the intracellular pH (from 7.8 to 7.25) and hydrolysis of intracellular polyphosphate stores were observed. Although the initial rate of glucose metabolism with immobilized C. tropicalis was similar to the rate observed previously in cell suspensions, a decrease by a factor of 2.5 occurred over 24 h. In addition to ethanol, a significant amount of glycerol was also produced. When immobilized C. tropicalis consumed xylose, cell growth within the beads was minimal. The intracellular pH dropped rapidly by 1.05 pH units to 6.4. Intracellular ATP levels were lower and intracellular Pi levels were higher than observed with glucose-perfused cells. Consumption of xylose by immobilized C. tropicalis was slower than was previously observed for oxygen-limited cell suspensions, and xylitol was the only fermentation product.     

Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle

The African clawed frog Xenopus laevis has been instrumental to investigations of both development and cell biology, but the utility of this model organism for genetic and proteomic studies is limited by its long generation time and unsequenced pseudotetraploid genome. Xenopus tropicalis, which is a small, faster-breeding relative of X. laevis, has recently been adopted for research in developmental genetics and functional genomics, and has been chosen for genome sequencing. We show that X. tropicalis egg extracts reconstitute the fundamental cell cycle events of nuclear formation and bipolar spindle assembly around exogenously added sperm nuclei. Interestingly, X. tropicalis spindles were approximately 30% shorter than X. laevis spindles, and mixing experiments revealed a dynamic, dose-dependent regulation of spindle size by cytoplasmic factors. Measurements of microtubule dynamics revealed that microtubules polymerized slower in X. tropicalis extracts compared to X. laevis, but that this difference is unlikely to account for differences in spindle size. Thus, in addition to expanding the range of developmental and cell biological experiments, the use of X. tropicalis provides novel insight into the complex mechanisms that govern spindle morphogenesis.     

Phosphatase Activity Among Candida Species and Other Yeasts Isolated from Clinical Material

A group of 277 yeasts isolated from burned children and 14 reference strains were tested for phosphatase activity by using phenolphthalein phosphate substrates. Phosphatase activity was widely distributed among various species and strains representing seven genera. Candida albicans, which was the most common yeast isolated from clinical material, was notably absent in producing the enzyme, whereas Candida tropicalis was the most consistent, strong, and rapidly active phosphatase-producing organism. The characteristic enzyme activity of a selected isolate of C. tropicalis was demonstrated in the presence of concentrations of inorganic phosphate which inhibited enzyme activity of other species. The greater enzyme activity of C. tropicalis was not related to more rapid or greater cell growth or decrease in the pH of culture media. Extracellular constitutive heat-labile acid phosphatase was found in broth filtrates of C. tropicalis, C. krusei, and a strain of Staphylococcus aureus.     

热带假丝酵母1230细胞色素P450基因CYPA14与CYPA16的克隆和表达

细胞色素P450(CYP)是一种单加氧酶,在热带假丝酵母(Candidatropicalis)ω-氧化过程中发挥关键作用。通过对来源不同的P450基因进行同源性分析,首先克隆到热带假丝酵母1230中P450基因的部分序列,再利用基因组步行法克隆其未知序列,结果分别获得了两个P450同工酶基因CYPA14和CYPA16的完整序列。经PCR方法证实,二者在染色体上的位置相邻,其读码框分别编码522和540个氨基酸残基的肽链。经NCBIBLAST搜索比较后发现,二者与热带假丝酵母ATCC20336中的P450成员CYP52A14和CYP52A16分别编码的序列几乎完全一致,与热带假丝酵母ATCC750中的P450成员CYP52A2和CYP52A1也具有较高的相似性。同时,对经诱变后的几株二元酸生产菌株的CYPA14与CYPA16也进行了克隆和序列比较,发现部分序列中的个别氨基酸残基发生了突变。CYPA14和CYPA16均在酿酒酵母中获得了有效表达,其中CYPA16的P450表达含量高于CYPA14,后者有部分表达产物发生了变性。