Phylogenetic relation of Epidermophyton floccosum to the species of Microsporum and Trichophyton in chitin synthase 1 (CHS1) gene sequences

The Nucleotide sequence of the chitin synthase1 (CHS1) gene of Epidermophyton floccosum, an anthrophophilic dermatophyte which is the type species of the genus Epidermophyton was analyzed to determine its phylogenetic relation to eight other dermatophyte species belonging to the genera Microsporum and Trichophyton, which were sequenced in our previous studies. A genomic DNA fragment about 620 bp in length of the CHS1 genewas amplified from E. floccosum by polymerasechain reaction (PCR) and was sequenced. The CHS1 nucleotide sequence showed more than 85% similarity with sequences derived from the other dermatophytes. Phylogenetic analyses of the sequences from E. floccosum revealed that the genus Epidermophyton may be genetically distinct from Microsporum and Trichophyton. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phytogeny of Epidermophyton floccosum and other dermatophytes

Eleven strains of Epidermophyton floccosum were compared with 5 Microsporum and 5 Trichophyton species with respect to the restriction fragment length polymorphism (RFLP) of the mitochondrial DNA to reveal their phylogenetic relationships. The phylogeny of 11 species showed that the three dermatophyte genera could not be separated from each other and could be considered to be congeneric. This result is not inconsistent with the results from ribosomal RNA sequences.     

Molecular Analysis of Chitin Synthase 1 (CHS1) Gene Sequences of Trichophyton mentagrophytes Complex and T. rubrum

Nucleotide sequences of chitin synthase 1 (CHS1) gene of dermatophytes, Arthroderma benhamiae, A. simii, A. vanbreuseghemii, Trichophyton mentagrophytes var. interdigitale (T. interdigitale), and T. rubrum were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS1 gene were amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these five dermatophytes showed more than 90% similarity between the species. The phylogenetic analysis of their sequences revealed that A. benhamiae, A. simii, A. vanbreuseghemii, and T. rubrum were genetically distinct from one another, but T. interdigitale was genetically very close to A. vanbreuseghemii. On the other hand, a specific restriction endonuclease site of HinfI was present in the CHS1 gene fragment of T. rubrum but not in those of A. benhamiae, A. simii, A. vanbreuseghemii and T. interdigitale. The molecular analysis of CHS1 genes will provide useful information for the identification of these Trichophyton species and the understanding of their evolution. Received: 6 March 1998 / Accepted: 4 May 1998     

Characterization of siderophores produced by different species of the dermatophytic fungiMicrosporum andTrichophyton

The dermatophytic fungiTrichophyton spp andMicrosporum spp secrete ferrichrome type siderophores under low iron conditions. Three different species ofMicrosporum, i.e.M. qypseum, M. canis andM. audouinii, as well asT. rubrum produce ferrichrome C and ferricrocin, whereasT. mentagrophytes andT. tonsurans produce only ferrichrome. The identification of the siderophores was established by means of thin layer chromatography, high performance liquid chromatography and mass spectroscopy.     

Molecular confirmation of a Trichophyton violaceum isolate from human black-dot ringworm

A clinical isolate from a black-dot ringworm lesion of a 28-year-old female Japanese was investigated by morphological and biochemical analyses as well as molecular analyses. The isolate grew well onthiamine enriched agar and did not produce violetpigment, macroconidia or microconidia on Sabouraud's dextrose agar. Approximately 620-bp genomic DNA fragments of the CHS1 gene were amplified from Trichophyton mentagrophytes, T. rubrum, T. tonsurans and T. violaceum by polymerase chain reaction (PCR) and sequenced. The chitin synthase 1 (CHS1) nucleotide sequences of the clinical isolate showed more than 97% similarity to that of T. violaceum and less than 96% similarity to that of T. mentagrophytes, T. rubrum and T. tonsurans. The phylogenetic analysis of their sequences revealed that the clinical isolate was genetically close to T. violaceum and distinct from T. mentagrophytes, T. rubrum and T. tonsurans. Therefore, the isolate was confirmed as T. violaceum by mycological examination and molecular analyses. This revised version was published online in June 2006 with corrections to the Cover Date.     

Incidence of dermatophytes and cyclohexamide resistantfungi on healthy children hairs and nails in nurseries

In order to estimate the prevalence of dermatophytes and other fungi on healthy children hairs and nails, 92 hair samples and 85 nail samples (groups of 10 finger nails from each child) were collected from 5 nurseries (children aged 9 months up to 4 years) in Assiut city. From hair samples 22 species were collected, Trichophyton (2 species) and Microsporum (2 species) were the only recovered dermatophytes in addition to well known keratinophilic genus Chrysosporium (4 species). From nail samples, 18 species were identified, Trichophyton was represented by 4 species, Microsporum, 2 species and Chrysosporium, 4 species. Also, several other saprophytes and cycloheximide resistant fungi were isolated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of keratinophilic fungi from floors in Roman kindergarten and secondary schools

We examined the dust collected from the floors of forty classrooms, twenty in kindergarten schools (children aged 2–5) and twenty in secondary schools (students aged 11–14) in order to determine the diffusion of keratinophilic fungi in respect to such different factors as human presence and children's age. In the kindergarten schools 268 colonies of keratinophilic fungi were isolated: 50 were Microsporum, 6 Trichophyton and 212 Chrysosporium species. Members of the Chrysosporium genus were found the widely diffused. It is interesting to note the isolation of M. gypseum in two schools. In the secondary schools 847 colonies of keratinophilic fungi were isolated: 727 were Chrysosporium, 81 Microsporum, 38 Trichophyton and 1 Epidermophyton species. Again the Chrysosporium species were the most widely diffused. It is remarkable to point out the isolation of pathogenic species such as Epidermophyton floccosum, Microsporum canis, Microsporum gypseum and the rather rare Microsporum vanbreuseghemii.     

Evidence of the Importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa Protein Cytolytic Activity in Escherichia coli

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix α C corresponding to a substitution of Met¹¹⁵ by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.     

Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover

Summary The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions.     

Taxonomic characterization of Shiraia-like fungi isolated from bamboos in Japan

The molecular phylogeny of nuclear LSU rDNA sequences (D1/D2 domain), ITS regions, and beta-tubulin gene (tub2) showed that the seven strains of Shiraia-like fungi obtained from fresh bamboo tissues as endophytes were closely related to Shiraia bambusicola and had three distinctive lineages (groups A-C). The closest group (group A) to S. bambusicola produced distinctive prawn-shaped conidioma-like structures that differed from conidiomata in the anamorph of S. bambusicola. Currently, none of the morphological structures and molecular database records were compatible with our Shiraia-like fungi. These results reveal that Shiraia-like fungi group A is supposed to be a new species that should be assigned into a novel genus/species related to S. bambusicola.     

The sequence of the isoepoxydon dehydrogenase gene of the patulin biosynthetic pathway in <Emphasis Type="Italic">Penicillium</Emphasis> species

Interest in species of the genus Penicillium is related to their ability to produce the mycotoxin patulin and to cause spoilage of fruit products worldwide. The sequence of the isoepoxydon dehydrogenase (idh) gene, a gene in the patulin biosynthetic pathway, was determined for 28 strains representing 12 different Penicillium species known to produce the mycotoxin patulin. Isolates of Penicillium carneum, Penicillium clavigerum, Penicillium concentricum, Penicillium coprobium, Penicillium dipodomyicola, Penicillium expansum, Penicillium gladioli, Penicillium glandicola, Penicillium griseofulvum, Penicillium paneum, Penicillium sclerotigenum and Penicillium vulpinum were compared. Primer pairs for DNA amplification and sequencing were designed from the P. griseofulvum idh gene (GenBank AF006680). The two introns present were removed from the nucleotide sequences, which were translated to produce the IDH sequences of the 12 species for comparison. Phylogenetic relationships among the species were determined from rDNA (ITS1, 5.8 S, ITS2 and partial sequence of 28S rDNA) and from the idh nucleotide sequences minus the two introns. Maximum parsimony analysis showed trees based on rDNA and idh sequences to be congruent. It is anticipated that the genetic information obtained in the present study will aid in the design of probes, specific for patulin biosynthetic pathway genes, to identify the presence of these mycotoxigenic fungi. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Factors affecting growth and urease production by Trichophyton spp.

Among Trichophyton spp. examined for urease production, T. rubrum was negative, whereas T. mentagrophytes appeared to be the most active species. Urease was not detected in cell-free culture fluids of the tested fungi. The endocellular urease of the test fungi was essentially constitutive. Moreover, addition of urea to the growth medium of these organisms markedly inhibited their mycelial biomass and ureolytic yield. Environmental factors showed variable effects on the test fungi and there was no correlation between mycelial growth and urease activity of these fungi.     

Cloning of bovine LYST gene and identification of a missense mutation associated with Chediak-Higashi syndrome of cattle

An inheritable bleeding disorder with light coat color caused by an autosomal recessive gene has been reported in a population of Japanese black cattle. The disease has been diagnosed as Chediak-Higashi Syndrome (CHS) of cattle which correspond to a human inheritable disorder caused by mutation in LYST gene. To characterize the molecular lesion causing CHS in cattle, cDNAs encoding bovine LYST were isolated from a bovine brain cDNA library. The nucleotide and deduced amino acid sequences of bovine LYST had 89.6 and 90.2% identity with those of the human LYST gene, respectively. In order to identify the mutation within the LYST gene causing CHS in cattle, cDNA fragments of the LYST gene were amplified from an affected animal by RT-PCR and their nucleotide sequences were completely determined. Notably, a nucleotide substitution of A to G transition, resulting in an amino acid substitution of histidine to arginine (H2015R) was identified in the affected animal. The presence of the substitution was completely corresponding with the occurrence of the CHS phenotype among 105 members of pedigrees of the Japanese black cattle and no cattle of other populations had this substitution. These findings strongly suggested that H2015R is the causative mutation in CHS of Japanese black cattle. Received: 25 May 1999 / Accepted: 26 July 1999     

Sequence diversity of a domesticated transposase gene, <Emphasis Type="Italic">MUG1</Emphasis>, in <Emphasis Type="Italic">Oryza</Emphasis> species

MUG1 is a MULE transposon-related domesticated gene in plants. We assessed the sequence diversity, neutrality, expression, and phylogenetics of the MUG1 gene among Oryza ssp. We found MUG1 expression in all tissues analyzed, with different levels in O. sativa. There were 408 variation sites in the 3886 bp of MUG1 locus. The nucleotide diversity of the MUG1 was higher than functionally known genes in rice. The nucleotide diversity (π) in the domains was lower than the average nucleotide diversity in whole coding region. The π values in nonsynonymous sites were lower than those of synonymous sites. Tajima D and Fu and Li D* values were mostly negative values, suggesting purifying selection in MUG1 sequences of Oryza ssp. Genome-specific variation and phylogenetic analyses show a general grouping of MUG1 sequences congruent with Oryza ssp. biogeography; however, our MUG1 phylogenetic results, in combination with separate B and D genome studies, might suggest an early divergence of the Oryza ssp. by continental drift of Gondwanaland. O. longistaminata MUG1 divergence from other AA diploids suggests that it might not be a direct ancestor of the African rice species. These authors contributed equally to this work.     

A putative disease-associated haplotype within the SCN1A gene in Dravet syndrome

Dravet syndrome (DS), previously known as severe myoclonic epilepsy of infancy, is one of the most severe forms of childhood epilepsy. DS is caused by a mutation in the neuronal voltage-gated sodium-channel alpha-subunit gene (SCN1A). However, 25–30% of patients with DS are negative for the SCN1A mutation screening, suggesting that other molecular mechanisms may account for these disorders. Recently, the first case of DS caused by a mutation in the neuronal voltage-gated sodium-channel beta-subunit gene (SCN1B) was also reported. In this report we aim to make the molecular analysis of the SCN1A and SCN1B genes in two Tunisian patients affected with DS. The SCN1A and SCN1B genes were tested for mutations by direct sequencing. No mutation was revealed in the SCN1A and SCN1B genes by sequencing analyses. On the other hand, 11 known single nucleotide polymorphisms were identified in the SCN1A gene and composed a putative disease-associated haplotype in patients with DS phenotype. One of the two patients with putative disease-associated haplotype in SCN1A had also one known single nucleotide polymorphism in the SCN1B gene. The sequencing analyses of the SCN1A gene revealed the presence of a putative disease-associated haplotype in two patients affected with Dravet syndrome.     

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB088371–AB088374, AB089199, AB154393–AB154399, and AB162144.     

Flamingomyces and Parvulago, new genera of marine smut fungi (Ustilaginomycotina)

Teliospore walls, teliospore germinations, hyphal septations, cellular interactions, and nucleotide sequences from the D1/D2 region of the nuLSU rRNA gene of the marine smut fungi Melanotaenium ruppiae and Ustilago marina were examined and compared with findings in other Ustilaginomycotina. The data show that Melanotaenium ruppiae belongs to the Urocystaceae and Ustilago marina to the Ustilaginaceae. Within the Urocystaceae, Melanotaenium ruppiae is morphologically similar to Melanustilospora and Vankya. However, according to the molecular results Melanotaenium ruppiae can neither be ascribed to Melanustilospora nor to Vankya. Therefore, the new genus Flamingomyces is proposed for Melanotaenium ruppiae. Ustilago marina differs from the other Ustilaginaceae in the mode of sporulation, which exclusively occurs at the base of the host plant culms. Accordingly, the new genus Parvulago is proposed for Ustilago marina.     

芍药内生真菌的鉴定及产生活性次生代谢产物的评估

【目的】研究药用植物芍药(Paeonia lactiflora Pall.)内生真菌的种群多样性,同时对其可能存在的聚酮合酶(Polyketide synthase,PKS)和非核糖体多肽合成酶(Non-ribosomal peptide synthetase,NRPS)基因多样性进行评估,预测芍药内生真菌产生活性次生代谢产物的潜力。【方法】采用组织分离法获得芍药根部内生真菌菌株,结合形态学特征和ITS序列分析,进行鉴定;利用兼并性引物对内生真菌中存在的聚酮合酶(PKS)基因和非核糖体多肽合成酶(NRPS)基因进行PCR扩增及序列测定分析,构建系统发育树,明确芍药内真菌PKS基因序列和NRPS基因序列的系统进化地位。【结果】从芍药组织块中共分离得到105株内生分离物,去重复后获得52株内生真菌,菌株ITS基因序列信息显示,52株芍药内生真菌隶属于7目、13科、15属,其中小球腔菌属(Leptosphaeria)、土赤壳属(Ilyonectria)和镰孢属(Fusarium)为优势种群;从52株内生真菌中筛选获得13株含PKS基因片段的菌株,8株含NRPS基因片段的菌株,部分菌株功能基因的氨基酸序列与Gen Bank中已知化合物的合成序列具有一定的同源性,预示芍药根部内生真菌具有合成丰富多样的次生代谢产物的潜力。【结论】药用植物芍药根部具有丰富的内生真菌资源,且具有产生活性次生代谢产物的潜力,值得进一步开发研究和应用。     

Isolation and Sequencing of a New Glucoamylase Gene from an Aspergillus niger Aggregate Strain (DSM 823) Molecularly Classified as Aspergillus tubingensis

Based on morphological characteristics the taxa included in the Aspergillus aggregate can hardly be differentiated. For that reason the phylogeny of this genus was revised several times as different criteria, from morphological to later molecular, were used. We found, comparing nucleotide sequences of the ITS-region, that the strain Aspergillus niger (DSM 823) which is claimed to be identical to the strains ATCC 10577, IMI 027809, NCTC 7193 and NRRL 2322 can be molecularly classified as Aspergillus tubingensis, exhibiting 100% identity with the A. tubingensis CBS strains 643.92 and 127.49. We amplified, cloned and sequenced a new glucoamylase gene (glaA) from this strain of A. tubingensis (A. niger DSM 823) using primers derived from A. niger glucoamylase G1. The amplified cDNA fragment of 2013 bp contained an open reading frame encoding 648 amino acid residues. The calculated molecular mass of the glucoamylase, deduced from the amino acid sequence, was 68 kDa. The nucleotide sequence of glaA showed 99% similarity with glucoamylases from Aspergillus kawachii and Aspergillus shirousami, whereas the similarity with the glucoamylase G1 from A. niger was 92% An erratum to this article is available at .