微紫青霉菌(Penicillium janthinellum)P-type ATPase及金属硫蛋白相关基因的克隆

本研究以高抗多种重金属盐的微紫青霉菌(Penicillium janthinellum)菌株GXCR为材料构建基因组fosmid文库。其插入片段集中在36～50kb,含13348个克隆,重组率为100%,大约覆盖了GXCR基因组的14.83倍。基于序列特异性和简并引物,利用PCR扩增分析了与酿酒酵母(Saccharomyces cerevisiae)重金属盐抗性相关的CRS5和CUP2基因;基于兼并引物和序列特异性引物,利用PCR扩增分析了GXCR的P-type ATPase基因。通过菌落原位杂交和Southern blot鉴定了一个含铜转运P-type ATPase基因的阳性fosmid克隆,经亚克隆测序分析表明该基因与棒曲霉(Aspergillus clavatus菌株)NRRL1的P-type copper ATPase相似性达97%。没有筛选到与CRS5和CUP2基因同源的克隆,说明GXCR中可能不存在与酿酒酵母CUP2和CRS5高度同源的MT基因,同时也暗示酵母与丝状真菌的重金属盐的抗性机制有本质上的差异或者独特性。     

Molecular characterization of the food-borne fungus Neosartorya fischeri (Malloch and Cain).

The food-borne fungus Neosartorya fischeri, which is phenotypically related to the human opportunistic pathogen Aspergillus fumigatus, causes spoilage of heat-processed fruit products. Genomic methods were used to type N. fischeri strains and identify the genomic relationship between A. fumigatus and N. fischeri and between the different varieties of N. fischeri. EcoRI restriction fragment length polymorphism (RFLP) patterns obtained after ethidium bromide staining could differentiate most of N. fischeri var. glabra and N. fischeri var. spinosa strains. On the contrary, all N. fischeri var. fischeri strains tested exhibit the same RFLP pattern, which was similar to the A. fumigatus pattern. Similarly, Southern hybridization with a ribosomal probe showed some polymorphism between N. fischeri var. glabra and N. fischeri var. spinosa strains but could not distinguish between N. fischeri var. fischeri and A. fumigatus strains. By using the endonucleases EcoRI, HindIII, and BglII to generate Southern blot patterns with a fragment of the A. fumigatus gene coding for a 33-kDa protease, it was possible to differentiate N. fischeri var. fischeri from A. fumigatus. The difference between N. fischeri and A. fumigatus was confirmed by the use of moderately repetitive nonribosomal A. fumigatus sequences. These results are in agreement with previous studies that showed important infraspecific polymorphism within N. fischeri var. glabra and N. fischeri var. spinosa and, in contrast, the homogeneity of N. fischeri var. fischeri strains. A unique Southern blot pattern was seen for each strain of N. fischeri fingerprinted with the A. fumigatus repetitive sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incidence of Heat-Resistant Molds in Eastern Orchards and Vineyards

Over 70% of the samples of fruit, vegetation, and soil obtained in surveys of New York orchards and vineyards were contaminated with heat-resistant molds. The counts generally were low, under one per gram. Byssochlamys fulva was the most common isolate. Other isolates were identified as B. nivea, Paecilomyces varioti, Aspergillus fischeri, A. fischeri var. spinosus, A. fumigatus, Penicillium vermiculatum, and P. ochro-chloron.     

Antigenic profile of some typical and septate phialide-strains of Aspergillus fumigatus

Antigens from typical and septate phialide-strains of Aspergillus fumigatus, A. fumigatus var. ellipticus, A. fischeri, A. flavus and A. niger were analysed by fused rocket and two-dimensional immunoelectrophoresis. Depsite strain-to-strain variations, isolates of the two morphological forms of A. fumigatus showed a close similarity in their antigenic profile; the percentage of qualitative sharing ranged between 80% and 100%. The number of antigens shared by the isolates of A. fumigatus var. ellipticus and A. fischeri as compared with a typical A. fumigatus isolate was relatively high. By contrast isolates of A. flavus and A. niger, which are taxonomically not so closely related to A. fumigatus, revealed a qualitative sharing of only 16% and 8%, respectively. The number of common antigenically active components identified in culture filtrate antigens from the typical and septate phialide-strains of A. fumigatus was around 29. Two-dimensional electrophoresis with tandem and intermediate gel proved further that the two types of A. fumigatus isolates are antigenically homologous. Present findings also support the view that the antigenically active components of this pathogen are predominantly protein-glycoprotein in nature.     

Genomic characteristics comparisons of 12 food-related filamentous fungi in tRNA gene set, codon usage and amino acid composition

Filamentous fungi are widely exploited in food industry due to their abilities to secrete large amounts of enzymes and metabolites. The recent availability of fungal genome sequences has provided an opportunity to explore the genomic characteristics of these food-related filamentous fungi. In this paper, we selected 12 representative filamentous fungi in the areas of food processing and safety, which were Aspergillus clavatus, A. flavus, A. fumigatus, A. nidulans, A. niger, A. oryzae, A. terreus, Monascus ruber, Neurospora crassa, Penicillium chrysogenum, Rhizopus oryzae and Trichoderma reesei, and did the comparative studies of their genomic characteristics of tRNA gene distribution, codon usage pattern and amino acid composition. The results showed that the copy numbers greatly differed among isoaccepting tRNA genes and the distribution seemed to be related with translation process. The results also revealed that genome compositional variation probably constrained the base choice at the third codon, and affected the overall amino acid composition but seemed to have little effect on the integrated physicochemical characteristics of overall amino acids. The further analysis suggested that the wobble pairing and base modification were the important mechanisms in codon-anticodon interaction. In the scope of authors' knowledge, it is the first report about the genomic characteristics analysis of food-related filamentous fungi, which would be informative for the analysis of filamentous fungal genome evolution and their practical application in food industry.     

Isolation and Characterization of Antigens of Aspergillus Fischeri

Dilute acid extraction of Aspergillus fischeri mycelia contained two distinct antigens that were purified and separated by ethanol fractionation, concanavalin A precipitation, and chromatography on a Sephadex G-100 column. One antigen which emerges from the column among the early fractions contained 66% hexose determined as galactose and mannose but no demonstrable protein. The second antigen contained mannose as the sole hexose, was inactivated by pronase, and had a molecular weight of approximately 8000.

Fungi of the genus Aspergillus are ubiquitous saprophytes capable of causing debilitating disease under certain conditions. Although aspergilli can attack any part of the body, they are most often encountered as respiratory pathogens. Aspergillus fumigatus is the species most frequently responsible for infection.

Aspergillus fischeri is an ascosporic species within the aspergilli group, closely related to A. fumigatus This organism grows on Czapek's agar at 25 and 37°C and at 37°C it produces abundant conidia indistinguishable from A. fumigatus. Aspergillus fischeri is worldwide in distribution and can be readily isolated from the soil. Only rarely has it been associated with disease in man and it is generally considered a nonpathogen. However, A. fischeri has been shown by Biguet et al. to have at least eight antigens in common with A_. fumigatus. Aspergillus fischeri has been neglected in serological studies of the aspergilli, probably because of its noted nonpathogenicity. However, the obvious morphological similarity of these two aspergilli led us to study the antigens shared by the two species.     

Tools to study molecular mechanisms of Aspergillus pathogenicity

The unique nature of Aspergillus fungi represents a challenge for scrutinizing the attributes that render these saprophytic microorganisms pathogenic or allergenic under certain environmental circumstances. Recent publication of the genomic sequence from an isolate of the major pathogen Aspergillus fumigatus denotes enormous progress in aiming at cellular features and gene products that contribute to its pathogenicity. Latest developments to study virulence-related characteristics comprise profiling techniques, conditional gene inactivation and precise manipulation of the genome by means of gene targeting. Advances in assessing the virulence potential of particular mutant strains in alternative test systems complement these approaches.     

Unique biosynthesis of dehydroquinic acid?

A search of the genomic sequences of the thermophilic microorganisms Aquifex aeolicus, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii for the first seven enzymes (aroG, B, D, E, K, A, and C ) involved in the shikimic acid biosynthetic pathway reveal two key enzymes are missing. The first enzyme in the pathway, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase (aroG) and the second enzyme in the pathway, 5-dehydroquinic acid synthase (aroB) are "missing." The remaining five genes for the shikimate pathway in these organism are present and are similar to the corresponding Escherichia coli genes. The genomic sequences of the thermophiles Pyrococcus abyssi and Thermotoga maritima contain the aroG and aroB genes. Several fungi such as Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pneumocystis carinii f. sp. carinii, and Neurospora crassa contain the gene aroM, a pentafunctional enzyme whose overall activity is equivalent to the combined catalytic activities of proteins expressed by aroB, D, E, K, and A genes. Two of these fungi also lack an aroG gene. A discussion of potential reasons for these missing enzymes is presented.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 October 2009-30 November 2009

This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.     

DNA sequence characterization and molecular evolution of MAT1 and MAT2 mating-type loci of the self-compatible ascomycete mold Neosartorya fischeri

Degenerate PCR and chromosome-walking approaches were used to identify mating-type (MAT) genes and flanking regions from the homothallic (sexually self-fertile) euascomycete fungus Neosartorya fischeri, a close relative of the opportunistic human pathogen Aspergillus fumigatus. Both putative alpha- and high-mobility-group-domain MAT genes were found within the same genome, providing a functional explanation for self-fertility. However, unlike those in many homothallic euascomycetes (Pezizomycotina), the genes were not found adjacent to each other and were termed MAT1 and MAT2 to recognize the presence of distinct loci. Complete copies of putative APN1 (DNA lyase) and SLA2 (cytoskeleton assembly control) genes were found bordering the MAT1 locus. Partial copies of APN1 and SLA2 were also found bordering the MAT2 locus, but these copies bore the genetic hallmarks of pseudogenes. Genome comparisons revealed synteny over at least 23,300 bp between the N. fischeri MAT1 region and the A. fumigatus MAT locus region, but no such long-range conservation in the N. fischeri MAT2 region was evident. The sequence upstream of MAT2 contained numerous candidate transposase genes. These results demonstrate a novel means involving the segmental translocation of a chromosomal region by which the ability to undergo self-fertilization may be acquired. The results are also discussed in relation to their significance in indicating that heterothallism may be ancestral within the Aspergillus section Fumigati.     

Insight into the genome of Aspergillus fumigatus: analysis of a 922 kb region encompassing the nitrate assimilation gene cluster

Aspergillus fumigatus is the most ubiquitous opportunistic filamentous fungal pathogen of human. As an initial step toward sequencing the entire genome of A. fumigatus, which is estimated to be approximately 30 Mb in size, we have sequenced a 922 kb region, contained within 16 overlapping bacterial artificial chromosome (BAC) clones. Fifty-four percent of the DNA is predicted to be coding with 341 putative protein coding genes. Functional classification of the proteins showed the presence of a higher proportion of enzymes and membrane transporters when compared to those of Saccharomyces cerevisiae. In addition to the nitrate assimilation gene cluster, the quinate utilisation gene cluster is also present on this 922 kb genomic sequence. We observed large scale synteny between A. fumigatus and Aspergillus nidulans by comparing this sequence to the A. nidulans genetic map of linkage group VIII.     

简并PCR结合RACE技术克隆申克孢子丝菌未知过氧化氢酶基因

目的 克隆孢子丝菌未知过氧化氢酶基因,命名为Sscat基因.方法 根据生物信息库中7种已知真菌过氧化氢酶氨基酸序列的高度保守区域设计简并引物,PCR扩增获得部分Sscat基因cDNA片段,随后应用RACE技术分别扩增其3’端和5’端未知序列.结果 Sscat基因cDNA序列全长1746 bp,其中包括5’端121 b...     

Detecting anomalous gene clusters and pathogenicity islands in diverse bacterial genomes.

A gene in a genome is defined as putative alien (pA) if its codon usage difference from the average gene exceeds a high threshold and codon usage differences from ribosomal protein genes, chaperone genes and protein-synthesis-processing factors are also high. pA gene clusters in bacterial genomes are relevant for detecting genomic islands (GIs), including pathogenicity islands (PAIs). Four other analyses appropriate to this task are G+C genome variation (the standard method); genomic signature divergences (dinucleotide bias); extremes of codon bias; and anomalies of amino acid usage. For example, the cagA domain of Helicobacter pylori is highly deviant in its genome signature and codon bias from the rest of the genome. Using these methods we can detect two potential PAIs in the Neisseria meningitidis genome, which contain hemagglutinin and/or hemolysin-related genes. Additionally, G+C variation and genome signature differences of the Mycobacterium tuberculosis genome indicate two pA gene clusters.     

Hop,an active Mutator-like element in the genome of the fungus Fusarium oxysporum

A new type of active DNA transposon has been identified in the genome of Fusarium oxysporum by its transposition into the niaD target gene. Two insertions within the final exon, in opposite orientations at the same nucleotide site, have been characterized. These elements, called Hop, are 3,299 bp long, with perfect terminal inverted repeats (TIRs) of 99 bp. The sequencing of genomic copies reveals a 9-bp target site duplication and no apparent sequence specificity at the insertion sites. The sequencing of a cDNA indicates that Hop does not contain an intron and encodes a putative transposase of 836 amino acids. The structural features (length, TIRs size, and 9-bp duplication), together with the presence of conserved domains in the transposase, strongly suggest that Hop is a Mutator-like element (MULE). Hop is thus the first active member of this family found beyond plants. The high rate of excision observed indicates that Hop is very active and thus represents a promising efficient tagging system for the isolation of fungal genes. The distribution of Hop elements within the Fusarium genus revealed that they are present in different species, suggesting that related elements could be present in other fungal genomes. In fact, Hop-related sequences have been identified in the survey of the entire genome sequence of three other ascomycetes, Magnaporthe grisea, Neurospora crassa, and Aspergillus fumigatus.     

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.     

Occurrence of indole alkaloids among secondary metabolites of soil Aspergillus

The occurrence of indole alkaloids among secondary fungal metabolites was studied in species of the genus Aspergillus, isolated from soils that were sampled in various regions of Russia (a total of 102 isolates of the species A. niger, A. phoenicis, A. fumigatus, A. flavus, A. versicolor, A. ustus, A. clavatus, and A. ochraceus). Clavine alkaloids were represented by fumigaclavine, which was formed by A. fumigatus. alpha-Cyclopiazonic acid was formed by isolates of A. fumigatus, A. flavus, A. versicolor, A. phoenicis, and A. clavatus. The occurrence of indole-containing diketopiperazine alkaloids was documented for isolates of A. flavus, A. fumigatus, A. clavatus, and A. ochraceus. No indole-containing metabolites were found among the metabolites of A. ustus or A. niger.     

Genomic Conflict Settled in Favour of the Species Rather Than the Gene at Extreme GC Percentage Values

Wada and colleagues have shown that, whether prokaryotic or eukaryotic, each gene has a "homostabilising propensity" to adopt a relatively uniform GC percentage (GC%). Accordingly, each gene can be viewed as a "microisochore" occupying a discrete GC% niche of relatively uniform base composition amongst its fellow genes. Although first, second and third codon positions usually differ in GC%, each position tends to maintain a uniform, gene-specific GC% value. Thus, within a genome, genic GC% values can cover a wide range. This is most evident at third codon positions, which are least constrained by amino acid encoding needs. In 1991, Wada and colleagues further noted that, within a phylogenetic group, genomic GC% values can also cover a wide range. This is again most evident at third codon positions. Thus, the dispersion of GC% values among genes within a genome matches the dispersion of GC% values among genomes within a phylogenetic group. Wada described the context-independence of plots of different codon position GC% values against total GC% as a "universal" characteristic. Several studies relate this to recombination. We have confirmed that third codon positions usually relate more to the genes that contain them than to the species. However, in genomes with extreme GC% values (low or high), third codon positions tend to maintain a constant GC%, thus relating more to the species than to the genes that contain them. Genes in an extreme-GC% genome collectively span a smaller GC% range, and mainly rely on first and second codon positions for differentiation as "microisochores". Our results are consistent with the view that differences in GC% serve to recombinationally isolate both genome sectors (facilitating gene duplication) and genomes (facilitating genome duplication, e.g. speciation). In intermediate-GC% genomes, conflict between the needs of the species and the needs of individual genes within that species is minimal. However, in extreme-GC% genomes there is a conflict, which is settled in favour of the species (i.e. group selection) rather than in favour of the gene (genic selection).