Ribosomal DNA variation,recombination and inheritance in the basidiomycete Trichaptum abietinum: implications for reticulate evolution

Two divergent nuclear ribosomal DNA (nrDNA) types, designated alpha and beta, were found distributed in 11 North European populations of the basidiomycete Trichaptum abietinum. These types differed by a 220 bp indel in the internal transcribed spacer 1 (ITS1) sequence and a number of linked substitutions and small indel motives in the internal transcribed and intergenic spacers (ITS1, ITS2, IGS1 and IGS2). The alpha and beta haplotypes co-occurred in heterozygous somatic individuals (dikaryons) and segregated in a Mendelian fashion in monokaryotic single spore progenies. This result suggests that the haplotypes are encoded in different nuclei of field-collected dikaryons and inherited as a single locus. No meiotic recombinants were observed among the sequenced monokaryons. Population genetic analyses by PCR-RFLP revealed that a low frequency of evolutionary intermediate nrDNA types also existed in natural populations, presumably as a result of meiotic recombination of alpha and beta nrDNA. The existence of divergent nrDNA types in T. abietinum could be a result of a former independent evolution followed by a hybridization event. Phylogenetic analyses of ITS sequences suggest that the sister taxon T. fusco-violaceum has been involved in the evolutionary history of T. abietinum. Sequence polymorphisms observed in the translation elongation factor 1alpha (efa) and glyceraldehyde-3-phosphate dehydrogenase (gpd) genes, did not reveal two well-defined types of these genes. The results are discussed in the light of other evolutionary mechanisms as well.     

不同外源条件对4种白腐真菌溶藻效果的影响

【目的】评价白腐真菌Irpex lacteus XX-5、Trichaptum abietinum 1302BG、Ceriporia lacerata P2、Bjerkandera adusta XX-2处理铜绿微囊藻废水的应用潜力。【方法】采用分批次实验研究pH、温度、铜绿微囊藻浓度、金属离子、氮源、磷源对白腐真菌I. lacteus XX-5、T. abietinum 1302BG、C. lacerata P2、B. adusta XX-2溶解铜绿微囊藻的影响。【结果】在不同外源条件下,4种白腐真菌对铜绿微囊藻的抑制效果明显,均达60%以上。菌株C. lacerata P2和B. adusta XX-2受外源条件的影响很小,菌株C. lacerata P2的抑制率达70%以上,菌株B. adusta XX-2的抑制率达60%以上;菌株T. abietinum 1302BG、I. lacteus XX-5在不同外源条件下抑制率均会发生相应的变化,但抑藻率均可达60%以上。【结论】研究所使用的4种白腐真菌对抑制铜绿微囊藻具有较好的应用潜力,尤其是菌株C. lacerata P2和B. adusta XX-2。     

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.     

Regional and local population structure of the pioneer wood-decay fungus Trichaptum abietinum

The population structure of 11 Fennoscandian geographic populations of the pioneer wood-decay basidiomycete Trichaptum abietinum was assessed with PCR-RFLPs, intersequence simple repeats (ISSRs) and mating studies. The three codominant PCR-RFLP markers (1) internal transcribed spacer 2 (nrDNA), (2) glyceraldehyde-3-phosphate dehydrogenase and (3) translation elongation factor 1α showed that genotype distributions in most cases (94%) agreed with Hardy-Weinberg expectations and that random association of alleles occurred across loci. The molecular data suggest that T. abietinum is a highly outcrossing fungus that regularly proliferates and spreads by sexual spores. Interstock mating reactions suggest a high number of mating factors among individuals and that biological barriers to gene flow are nonexistent in the region. The three PCR-RFLP loci gave an overall F(ST) = 0.03, indicating a low level of genetic differentiation and presumably high gene flow among the geographic populations. The ISSR markers revealed no systematic substructuring and the among-population variance component was low (6.1%) in AMOVA. However, all PCR-RFLP and most ISSR markers (7/12) showed significant deviation from the null hypothesis of an even distribution of allele frequencies across the 11 geographic populations. Allele frequencies varied in an apparently random manner, suggesting that genetic drift might be an important structuring factor in T. abietinum. The spatial small-scale distribution of heterokaryons on three selected substrate units (logs) showed that most isolates represented discrete individuals and that a number of genets (19) may occupy a single log. The small-scale genotype distributions (within logs) were in agreement with panmictic Hardy-Weinberg expectations.     

多孔菌Trichaptum abietinum 1302BG自然条件下对合成染料刚果红和酸性品红的高效降解

选用杭州竹林土壤分离并筛选能够降解多种类型染料的真菌。经大量筛选发现一株编号为1302BG的真菌能够在固体培养基上分解所测试的全部9种染料(苯胺蓝、刚果红、橙黄G、甲基红、甲基橙、结晶紫、酸性品红、番红花红、碱性品红、甲基紫)。经形态学和分子生物学方法鉴定, 该菌1302BG为冷杉附毛孔菌(Trichaptum abietinum)。在液体培养基中研究了pH、温度、碳源、氮源、碳氮源组合、碳氮源浓度等参数对该菌脱色效果的影响, 以寻找最适最经济的脱色条件。在液体培养基中研究表明, 冷杉附毛孔菌1302BG既能在酸性又能在碱性条件下有效分解2种测试染料(酸性品红和刚果红)。该真菌能以仅含有0.5 g/L淀粉和0.05 g/L硫酸铵的经济、环境友好的培养基为底物, 能在灭菌和非灭菌(自然)的条件下高效脱色, 在24 h内对2种染料的脱色率均在90%以上。紫外/可见光谱及微核试验分析显示, 该菌脱色主要是以生物降解为主, 2种染料经该菌分解后的毒性也同时大大降低。这些优异特点显示了该菌具有非常广阔的工业染料废水处理应用潜力。     

Identification and typing of miso and soy sauce fermentation yeasts, Candida etchellsii and C. versatilis, based on sequence analyses of the D1D2 domain of the 26S ribosomal RNA gene, and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS sequence) of the miso and soy sauce fermentation yeasts, Candida etchellsii and Candida versatilis, in order to evaluate the usefulness of this sequence analysis for identification and typing of these two species. In the 26S rDNA sequence method, the numbers of base substitutions among C. etchellsii strains were up to 2 in 482 bp (99.6% similarity), and they were divided into three types (types A, B, and C). Those of C. versatilis strains were also up to 2 in 521 bp (99.6% similarity) and they were divided into three types (types 1, 2, and 3). In the ITS sequence method, those of C. etchellsii strains were zero in 433 bp (type a, 100% similarity). Those of C. versatilis were 5 in 409 bp (98.8% similarity), divided into 4 types (types I, II, III and IV). It was found that molecular methods based on the sequences of the 26S rDNA D1D2 domain and the ITS region were rapid and precise compared with the physiological method for the identification and typing of these two species.     

Pseudo-nitzschia pungens (Bacillariophyceae): A cosmopolitan diatom species?

Genetic, reproductive and morphological variation were studied in 193 global strains of the marine diatom species Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle to assess potential intraspecific variation and biogeographic distribution patterns. Genetic differentiation between allo- and sympatric strains was investigated using the ITS1–5.8S–ITS2 rDNA region. Three ITS clades were found. Clones of opposite mating type were sexually compatible within clades I or II, and viable F1 hybrid offspring were produced in crosses between them. The molecular differences between these clades were correlated with slight but consistent morphological differences. At present, nothing can be said about morphology and mating behavior for clade III clones because only ITS data were available. The three ITS clades showed different geographic distributions. Clade II was restricted to the NE Pacific, whereas clones belonging to clade III originated from geographically widely separated areas (Vietnam, China and Mexico). ITS clade I was recovered in all locations studied: the North Sea (Belgium, The Netherlands, France), the eastern and western N Atlantic (Spain, Canada), the NW and S Pacific (Japan, New Zealand) and the NE Pacific (Washington State). Clade I thus appears to be globally distributed in temperate coastal areas and provides the first strong evidence to date for the global distribution of a biologically, genetically and morphologically defined diatom species.     

Genetic characterisation of Toxoplasma gondii in wildlife from North America revealed widespread and high prevalence of the fourth clonal type

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study wild animals, from the USA were examined for T. gondii infection. Tissues of naturally exposed animals were bioassayed in mice for isolation of viable parasites. Viable T. gondii was isolated from 31 animals including, to our knowledge for the first time, from a bald eagle (Haliaeetus leucocephalus), five gray wolves (Canis lupus), a woodrat (Neotoma micropus), and five Arctic foxes (Alopex lagopus). Additionally, 66 T. gondii isolates obtained previously, but not genetically characterised, were revived in mice. Toxoplasma gondii DNA isolated from these 97 samples (31 + 66) was characterised using 11 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, 5′- and 3′-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22–8, c29–2, L358, PK1 and Apico). A total of 95 isolates were successfully genotyped. In addition to clonal Types II, and III, 12 different genotypes were found. These genotype data were combined with 74 T. gondii isolates previously characterised from wildlife from North America and a composite data set of 169 isolates comprised 22 genotypes, including clonal Types II, III and 20 atypical genotypes. Phylogenetic network analysis showed limited diversity with dominance of a recently designated fourth clonal type (Type 12) in North America, followed by the Type II and III lineages. These three major lineages together accounted for 85% of strains in North America. The Type 12 lineage includes previously identified Type A and X strains from sea otters. This study revealed that the Type 12 lineage accounts for 46.7% (79/169) of isolates and is dominant in wildlife of North America. No clonal Type I strain was identified among these wildlife isolates. These results suggest that T. gondii strains in wildlife from North America have limited diversity, with the occurrence of only a few major clonal types.     

IGS基因及RAPD标记分析在加特隐球酵母基因分类中的应用

根据ITS1-5.8S-ITS2区域的特异核酸序列变化,加特隐球酵母Cryptococcus gattii(≡新型隐球酵母加特变种Cryptococcu neoformans var.gattii)可分为6种基因型。本研究通过检测加特隐球酵母的IGS基因,发现其IGS序列有着更丰富的变异和信息位点。通过结合加特隐球酵母RAPD(随机扩增的多态性DNA)图谱比较研究,与IGS和ITS的序列分析结果大体一致,说明新近发现的加特隐球酵母ITS8型确实有别于以前报道过的其他加特隐球酵母ITS基因型。研究证明IGS1及IGS2基因片段分析可以作为加特隐球酵母基因分类鉴定中有效的辅助鉴别的分子生物学方法,联合多种基因分类鉴定的方法可以更有效地揭示新型隐球酵母加特变种种内不同基因亚型间的遗传进化关系。     

Separation of the major species of interstitial collagen by reverse-phase high-performance liquid chromatography

Results presented here demonstrate a further application of reverse-phase high-performance liquid chromatography to the separation of large proteins. At a pH near 4.5 with a high pyridine concentration, we have completely separated three major types of human collagen (Types I, II, and III) from mixtures. We illustrate the application of this technique to the preparation of Types I and II collagen from lathyritic chick cartilage extracts.     

Purification and characterization of heterologous component IIs of botulinum C2 toxin.

Botulinum C2 toxin (C2T) elaborated by certain strains of Clostridium botulinum types C and D is composed of separate and dissimilar two proteins, components I and II. Previous studies have shown that these two components of C2T produced by type C and D strains were immunologically heterologous and that C2T-producers were classified into three groups depending on the difference in molecular characteristics of the components I and II. In the present study, the heterologous component IIs of C2T were purified from three representative strains of the groups and the molecular characteristics of the components were compared. Immunological analyses by agar gel double immunodiffusion test showed that the component IIs purified from the three strains have the specific epitope(s) in addition to the common one(s). The biological activity of C2Ts reconstituted with component I purified from a fixed strain and component II each from the three strains differed depending on the source of the component II. These results indicate that the component II of C2T produced by C. botulinum types C and D differs in molecular structure, which reflects on the difference in the biological activity of the toxin. The present study suggests that the pathophysiological activity of C2T, which possibly causes a necrotic enteritis, is dependent on the C2T-producing bacteria infected.     

Cryptic species in the <Emphasis Type="Italic">Terfezia boudieri</Emphasis> complex

Phylogenetic analyses have corroborated the discovery of three internal transcribed spacer (ITS) Types in Terfezia boudieri isolates in the course of earlier studies and have emphasized the divergence of Type 2 from Types 1 and 3. The application of molecular and physiological tools described below, revealed the existence of cryptic species within T. boudieri. The markers used include sequences taken from the 5′ end of the ribosomal large subunit gene, a chitin synthase partial sequence, β-tubulin partial sequence and amplified fragment length polymorphism (AFLP)-based markers. Following initial sequencing of a single PCR amplified sample for each Type, mass analysis of specimens relied on RFLP differences between the Types. Over 100 fruit bodies, 30 or more specimens for each ITS Type, were tested with each of the markers. The markers analysis divided the isolates into three groups, each correlated to a specific ITS Type. Two of the physiological traits examined: mycelial proliferation and mycorrhiza formation, consistently showed responses paralleling the ITS Types; the data presented suggest that T. boudieri is comprised of three cryptic species.     

中国石蒜属种间亲缘关系ITS序列分析

本文利用核糖体DNA内转录间隔区(ITS)序列对石蒜属13个种(含变种)的亲缘关系进行分析。结果表明,各样品的ITS1长度为259~260 bp,ITS2为230 bp,分别有多个特异性信息位点。以ITS序列为依据对石蒜属植物亲缘关系进行分析,表明石蒜属13个种可分为三大类,其中类Ⅰ包括中国石蒜、地笑、安徽石蒜和长筒石蒜,核型为M+T型;类Ⅱ包括矮小石蒜、换锦花、玫瑰石蒜和红蓝石蒜,核型为ST型;类Ⅲ包括稻草石蒜、乳白石蒜、短蕊石蒜和两种人工杂交种,核型为ST+M+T。系统进化树与核型分析结果相似,第Ⅲ类可能为自然杂交种。     

临床分离耐甲氧西林溶血性葡萄球菌的SCCmec分型及同源性分析

目的了解临床分离耐甲氧西林溶血性葡萄球菌（MRSH）的SCCmec基因型别及相同SCCmec型别菌株的同源性。方法多重PCR进行SCCmec分型,ERIC-PCR法对相同SCCmec型别菌株进行同源性分析。结果83株临床分离MRSH菌株中,SCCmecI型有23株（27．7％）,SCCmecⅡ型有10株（12．1％）,SCCmecm型有24株（28．9％）,SCCmecIV型有1株（1．2％）,I、Ⅱ混合型有8株（9．6％）,I、Ⅲ混合型有6株（7．2％）,Ⅱ、11混合型有5株（6．0％）,I、Ⅱ、Ⅲ混合型有3株（3．6％）,未分型3株（3．6％）。ERIC—PCR结果显示,23株SCCmecI型分为11型,其中A型5株,B型5株,C型3株,其余8株各为1型,2株未分型;10株SCCmecⅡ型分为6型,其中D型4株,E型2株,3株各为1型,1株未分型;24株SCCmecm型分为9型,其中F型11株,G型2株,H型2株,I型2株,5株各为1型,2株未分型。结论临床分离MRSH中,SCCmecI、Ⅲ型为多,部分菌株呈混合型别;相同SCCmec型别的部分菌株之间可能存在克隆传播。     

Conservation of type III secretion system genes in Bradyrhizobium isolated from soybean

The distribution of rhcRST genes encoding the type III secretion system (T3SS) in a collection of Bradyrhizobium strains was characterized by PCR and Southern blot hybridization. The polymorphism of the corresponding sequences amplified by PCR was characterized by RFLP and sequencing together with those available in the databank. Genomic group I is characterized by the presence of Bradyrhizobium elkanii strains and group II by the presence of B. japonicum and B. liaoningense strains. Highly conserved T3SS-like genes were detected by PCR in all Bradyrhizobium strains isolated from soybean belonging to genomic group II, and in none of the strains belonging to genomic group I. These data were confirmed by Southern blot hybridization that further indicated the presence of sequences showing similarity to the rhcRST sequence in B. elkanii strains. The high level of conservation of rhcRST among Bradyrhizobia of genomic group II and sharing the same host-plant suggests that T3SS-like genes might have undergone horizontal genetic transfer within this genomic group. When considering the three Rhizobiaceae genera, a clear congruence was recorded between the rhcRST, rRNA gene and ITS sequences in bacteria harbouring sequences encoding T3SS, suggesting a relatively ancient emergence of the T3SS in these genera.     

Structural variation in the Waxy gene and differentiation in foxtail millet [Setaria italica (L.) P. Beauv.]: implications for multiple origins of the waxy phenotype

The origin and evolution of the waxy type of foxtail millet [Setaria italica (L.) P. Beauv] were studied by analyzing structural variation in the Waxy gene. Initially, the Waxy gene was amplified by RT-PCR, RACE and genomic PCR from a non-waxy strain to determine the structure of the wild-type gene. Secondly, we screened by PCR for polymorphisms at the Waxy locus in 79 strains with various waxy phenotypes. We then carried out genomic Southern analysis on 67 strains and identified seven RFLP classes which were designated as types I-VII. RFLP type was correlated with phenotype, such that types I and II corresponded to non-waxy, types III and VI to low-amylose, and types IV, V and VII to waxy phenotypes. The differences between RFLP types could be attributed to insertions in the Waxy gene. Types II and VI were caused by the insertion of a Tourist element into intron 1 and a SINE-like sequence into intron 12, respectively. Types III, IV, V and VII were characterized by the insertion of large sequences into the Waxy gene that may alter the expression of the gene. Thus, multiple, independent insertions in the Waxy gene appear to have caused the loss-of-function waxy phenotypes. Furthermore, the geographical distributions of the three RFLP types associated with the waxy phenotype (types IV, V and VII) were distinct, with type IV being found mainly in Taiwan and Japan, type V in Korea, and type VII in Myanmar. These results indicate a polyphyletic origin for the waxy phenotype in landraces of foxtail millet.     

Integron-bearing methicillin-resistant coagulase-negative staphylococci in South China, 2001–2004

A total of 53 methicillin-resistant coagulase-negative staphylococci strains isolated in a hospital in Guangzhou, China, were analyzed to detect class 1 integrons and SCC mec typing. Thirty strains had the class 1 integrase ( intI1 ) gene and 26 strains possessed the 3' conserved region of qacE Δ 1 - sul1 . Four different types of gene cassette arrays were found and a highly prevalent array of dfrA12-orfF-aadA2 gene cassettes was observed. Thirty class 1 integron-positive coagulase-negative staphylococci strains were subjected to Southern hybridization analysis; the result showed that class 1 integrons were located on chromosome, not plasmid. According to the results of SCC mec typing for 30 integron-bearing MRCNS strains, five, 15 and five strains belonged to type I, II and III SCC mec , respectively, and five strains were untypeable. For 23 non-integron-bearing methicillin-resistant coagulase-negative staphylococci strains, four, nine and seven strains belonged to type I, II and III SCC mec , respectively, and three strains were untypeable. None of the strains belonged to type IV or V. Twenty-three coagulase-negative staphylococci isolates of three Staphylococcal species that contained the dfrA12-orfF-aadA2 gene cassette array were phylogenetically unrelated to each other by randomly amplified polymorphic DNA, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer.     

Integron-bearing methicillin-resistant coagulase-negative staphylococci in South China, 2001-2004.

A total of 53 methicillin-resistant coagulase-negative staphylococci strains isolated in a hospital in Guangzhou, China, were analyzed to detect class 1 integrons and SCCmec typing. Thirty strains had the class 1 integrase (intI1) gene and 26 strains possessed the 3' conserved region of qacEDelta1-sul1. Four different types of gene cassette arrays were found and a highly prevalent array of dfrA12-orfF-aadA2 gene cassettes was observed. Thirty class 1 integron-positive coagulase-negative staphylococci strains were subjected to Southern hybridization analysis; the result showed that class 1 integrons were located on chromosome, not plasmid. According to the results of SCCmec typing for 30 integron-bearing MRCNS strains, five, 15 and five strains belonged to type I, II and III SCCmec, respectively, and five strains were untypeable. For 23 non-integron-bearing methicillin-resistant coagulase-negative staphylococci strains, four, nine and seven strains belonged to type I, II and III SCCmec, respectively, and three strains were untypeable. None of the strains belonged to type IV or V. Twenty-three coagulase-negative staphylococci isolates of three Staphylococcal species that contained the dfrA12-orfF-aadA2 gene cassette array were phylogenetically unrelated to each other by randomly amplified polymorphic DNA, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer.     

Nested allele-specific PCR primers distinguish genetic groups of Uncinula necator.

Isolates of the obligately biotrophic fungus Uncinula necator cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of U. necator. We used the nucleotide sequences of the gene that encodes eburicol 14alpha-demethylase (CYP51) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5. 8S regions. We identified four point mutations (three in CYP51 and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season.     

Host-controlled Restriction of T-even Bacteriophages: Relation of Four Bacterial Deoxyribonucleases to Restriction

Escherichia coli strains B and K-12, which restrict growth of nonglucosylated T- even phage (T(*) phage), and nonrestricting strains (Shigella sonnei and mutants of E. coli B) were tested for levels of endonuclease I and exonucleases I, II, and III, by means of in vitro assyas. Cell-free extracts freed from deoxyribonucleic acid (DNA) were examined with three substrates: E. coli DNA, T(*)2 DNA, and T2 DNA. Both restricting and nonrestricting strains had comparable levels of the four nuclease activities and had similar patterns of preference for the three substrates. In addition, mutants of E. coli B and K-12 that lack endonuclease I were as effective as their respective wild types in restricting T(*) phage.