A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron

In the present study, we determined the sequence of group I self-splicing introns found in the large ribosomal RNA subunit of Candida albicans, Candida stellatoidea and the recently-described species Candida dubliniensis. It was found that both the intron and ribosomal RNA nucleotide sequences are almost perfectly identical between different C. albicans strains as well as between C. albicans and C. stellatoidea strains. Comparisons of ribosomal RNA sequences suggest that local isolates of atypical C. albicans from individuals infected with human immunodeficiency virus can be assigned to the C. dubliniensis species. C. dubliniensis strains also harbor a group I intron in their ribosomal RNA, as observed in about 40% of C. albicans strains and all C. stellatoidea strains. This novel C. dubliniensis group I intron is identical to the C. albicans and C. stellatoidea intron, except for two widely divergent stem-loop regions. Despite these differences, the C. dubliniensis intron possesses self-splicing ability in an in vitro assay. Taken together, these data support the idea that C. albicans and C. stellatoidea should be joined together as variants of the same species while C. dubliniensis is a distinct but closely related microorganism. To our knowledge, the C. albicans and C. dubliniensis introns are the first example of a pair of homologous group I introns differing only by the presence of apparently facultative sequences in some stem-loops suspected to be involved in stabilization of tertiary structure.     

Structural analysis of the three lipopolysaccharides produced by Salmonella madelia (1,6,14,25)

Salmonella madelia reported to express the O-antigenic factors 1, 6, 14, and 25, defined in the Kauffmann-White classification system, was found to produce three different homogeneous lipopolysaccharides, which differed in having three structurally distinct O-polysaccharide components. The O-polysaccharide fraction obtained by mild acetic acid hydrolysis of the S. madelia lipopolysaccharide was analyzed by chemical composition, nitrous acid deamination, periodate oxidation, methylation, and 1H and 13C nuclear magnetic resonance methods and was demonstrated to be composed of three polysaccharides, PS(I), PS(II), and PS(III), which had the structures of repeating oligosaccharide units: (formula; see text)     

Evaluation of polysaccharide antigens of Vibrio cholerae O139 of different origin by monoclonal antibodies

The epitope composition of O-polysaccharides in the lipopolysaccharide (LPS) of V. cholerae, serogroup O139, isolated from clinical material and water of surface reservoirs was analyzed with the use of monoclonal antibodies. The analysis demonstrated that these O-polysaccharides were similar in their structure and chemical composition. In LPS of V. cholerae O139 clinical strains O-polysaccharide determinants occurred more often. Among V. cholerae isolated from water strains on whose surface individual epitopes of O-polysaccharide occurred less frequently or were absent appeared to be more numerous. A decrease in the concentration of microbial cells in the process of their testing by immunological methods led to increased percent of negative reactions with specific antibodies. Some V. cholerae O139 strains isolated from water were similar in the epitope composition of their O-polysaccharide and binding activity to cultures isolated from humans. As indicated by the results of these studies, cholera vibrios Bengal and vibrios isolated from river water on the territory of Russia had quantitative differences due to a higher level of the production of O-polysaccharide determinants and their occurrence in V. cholerae of serogroup O139.     

Selection and preliminary characterization of β-glycosidases producer Patagonian wild yeasts

Four pre-selected indigenous yeast strains belonging to Candida guilliermondii (V₂ and V₅), Candida pulcherrima (V₆) and Kloeckera apiculata (V₉), were used as β-glucosidase (βGL) and β-xylosidase (βXL) sources. The optimization of yeast culture conditions was carried out and the effects of oenological parameters on β-glycosidase activities were evaluated. C. guilliermondii V₂ and C. pulcherrima V₆ strains were selected. These strains showed intracellular (C. pulcherrima V₆) and parietal (C. guilliermondii V₂) constitutive βGL and βXL. The enzymatic activities were active at pH, glucose, ethanol and SO₂ concentrations usually found in winemaking and they were able to release monoterpenols and alcohols from grape juice glycoside extracts. Additionally, these yeast strains were not able to produce volatile acidity and off flavour. Regional ecological relevance of these species was also discussed. Our results evidence that the selected C. guilliermondii V₂ and C. pulcherrima V₆ strains have interesting oenological characteristics and allow us to think in their potential application in winemaking.     

Identification of the A and M antigens of Brucella as the O-polysaccharides of smooth lipopolysaccharides

Rabbit antisera, cross-absorbed serotype-specific for the Brucella A and M antigens, precipitated respectively the smooth lipopolysaccharides from B. abortus 1119-3 and B. melitensis 16M. The antigenic A and M activity of these lipopolysaccharides was shown to reside within the O-chain region of the smooth lipopolysaccharides by inhibition experiments. Homologous O-polysaccharides showed the highest inhibitory activity in ELISA, although both the A and M antigens were active as heterologous inhibitors, showing that the antigenic determinants of the classical A and M antigens are therefore within the respective O-polysaccharide structures. Their cross-serological activity may be explained in terms of the distinct but related chemical structures of these polysaccharides.     

Structural and immunochemical homogeneity of Aeromonas salmonicida lipopolysaccharide.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the lipopolysaccharides of typical and atypical strains of the fish pathogen Aeromonas salmonicida. 32P intrinsically radiolabeled lipopolysaccharide in sarcosinate-extracted outer membrane preparations, lipopolysaccharide stained by silver in proteinase K-digested outer membrane preparations and whole cell lysates, as well as purified lipopolysaccharide, displayed O-polysaccharide chains which were unusually homogeneous with respect to chain length. Chemical analysis further revealed that the sugar composition of the smooth lipopolysaccharide purified from three typical strains was very similar. Immunoblotting and immunofluorescent staining with both polyclonal and monoclonal antibody showed that the O-polysaccharide chains were strongly immunogenic and were antigenically cross-reactive on typical and atypical strains from diverse origins. Immunofluorescence analysis and phage binding studies demonstrated that a number of these O-polysaccharide chains traversed the surface protein array of virulent strains of A. salmonicida and were exposed on the cell surface.     

A yeast clade near Candida kruisii uncovered: nine novel Candida species associated with basidioma-feeding beetles

Yeasts similar to Candida kruisii were isolated repeatedly from the digestive tracts of basidioma-feeding beetles, especially nitidulids inhabiting and feeding on a variety of agarics in the southeastern USA and Barro Colorado Island, Panama. Based on the identical sequences of the D1/D2 domains of the LSU rRNA gene (rDNA) and host beetle information, the isolates were grouped into 19 genotypes which varied from C. kruisii by up to 38 nucleotide differences in the D1/D2 region. Phylogenetic analysis of rDNA sequences and phenotypic traits placed the isolates in C. kruisii and in nine undescribed taxa. The new species and type strains are designated as Candida pallodes (NRRL Y-27653^T), C. tritomae (NRRL Y-27650^T), C. panamensis (NRRL Y-27657^T), C. lycoperdinae (NRRL Y-27658^T), C. atbi (NRRL Y-27651^T), C. barrocoloradensis (NRRL Y-27934^T), C. aglyptinia (NRRL Y-27935^T), C. stri (NRRL Y-48063^T), and C. gatunensis (NRRL Y-48064^T). A phylogeny based on analysis of a combined database of sequences of SSU and LSU rDNA and the ITS region showed that the nine new species formed a novel sister clade to C. kruisii that was strongly supported by bootstrap analysis. Candida pallodes, C. tritomae, C. panamensis, and C. lycoperdinae formed one subclade, while C. atbi, C. barrocoloradensis, C. aglyptinia, C. stri, and C. gatunensis formed a second distinct subclade within the larger clade. Candida pallodes and C. atbi showed a strong host specificity to beetle species in the genus Pallodes (Coleoptera: Nitidulidae) collected from a variety of agarics. On the other hand, C. panamensis, C. tritomae, and C. lycoperdinae were associated with several unrelated beetles in Erotylidae, Scarabaeidae, Tenebrionidae, and Curculionidae as well as Lycoperdina ferruginea (Nitidulidae). Candida pallodes, C. tritomae, C. lycoperdinae, and C. atbi have been isolated repeatedly in the USA, while the other five new species have been found only at Barro Colorado Island, Panama.     

新疆不同来源金黄壳囊孢的多样性

为明确新疆不同寄主及地理来源的金黄壳囊孢(Cytospora chrysosperma)的多样性, 探讨种内亲缘关系和多样性差异。作者通过记录菌株在PDA培养基上的菌落颜色、形状、子实体形态等特征, 并应用Biolog-FF技术及ISSR分子标记技术, 比较了来自新疆5个地区5种寄主上的47株金黄壳囊孢的培养特征、生理生化特征及遗传多样性。结果表明47株金黄壳囊孢依据培养特征可划分为15种类型。不同类型菌株在碳源利用及代谢能力上存在差异, 各菌株对碳源的利用数量随着培养时间的增长逐渐增多。菌株882利用的碳源数量最多, 培养120 h可利用28种不同碳源, 碳源代谢能力中等; 菌株812-1利用的碳源数量最少, 培养120 h仅利用7种碳源, 代谢能力较低; 菌株1074-2、847、934、891-1、896、740具有单独利用碳源的能力。基于遗传相似性系数进行聚类分析, 结果显示遗传相似性系数为0.58时, 47个菌株被划分为两大类群, 其中第二类群菌株的培养特征为: 菌落白色、子实体较小且分布密集。供试金黄壳囊孢的多样性主要受自身遗传结构的影响, 不同寄主种类和地理来源对多样性的影响不显著。     

Structure of the O-polysaccharide and serological studies of the lipopolysaccharide of Proteus mirabilis 2002

The structure of the O-polysaccharide of the lipopolysaccharide of Proteus mirabilis 2002 was elucidated by chemical methods and 1H and 13C NMR spectroscopy. It was found that the polysaccharide consists of branched pentasaccharide repeating units having the following structure: [structure in text]. The O-polysaccharide of P. mirabilis 2002 has a common tetrasaccharide fragment with that of P. mirabilis 52/57 from serogroup O29, and the lipopolysaccharides of the two strains are serologically related. Therefore, based on the structural and serological data, we propose to classify P. mirabilis 2002 into the Proteus O29 serogroup as a subgroup O29a,29b.     

Combining population genomics and ecological niche modeling to assess taxon limits between Carex jemtlandica and C. lepidocarpa

Carex section Ceratocystis (Cyperaceae) is a group of recently evolved plant species, in which hybridization is frequent, introgression is documented, taxonomy is complex, and morphological boundaries are vague. Within this section, a unified taxonomic treatment of the Carex jemtlandica–Carex lepidocarpa species complex does not exist, and Norway may currently be the sole country accepting species rank for both. Carex jemtlandica is mainly confined to Fennoscandia and is thus a Fennoscandian conservation responsibility. This motivated us to test the principal hypothesis that both C. jemtlandica and C. lepidocarpa represent evolutionary significant units, and that both deserve their current recognition at species level. We investigated their evolutionary distinctiveness in Norway,using restriction site-associated DNA sequencing and ecological niche modeling. Our genomic results reveal two genetic clusters, largely corresponding to C. jemtlandica and C. lepidocarpa that also remain distinct in sympatry, despite clear indications of ongoing hybridization and introgression. The ecological niche modeling suggests that they occupy different environmental niches. Jointly, our results clearly show that C. jemtlandica and C. lepidocarpa represent separately evolving entities that should qualify recognition as evolutionary significant units. Given the high level of introgression compared to other hybridizing species pairs in Carex we recommend treating C. jemtlandica as a subspecies of C. lepidocarpa.     

化肥减量配施有机肥对药用菊花产量、品质和药理活性的影响

采用田间小区试验,设置5个有机肥无机肥配施处理(100%化肥和14%、28%、56%、84%有机肥替代化肥处理),测定各处理对药用菊花农艺性状、产量、矿质元素吸收、有效成分含量的影响。并采用酶标仪和MTT试剂盒测定不同处理菊花水提物体外抗氧化活性及对H₂O₂致损的LO2肝细胞的保护作用。结果表明: 与100%化肥处理相比,化肥配施有机肥可以保证药用菊花产量,甚至低比例配施处理(14%有机肥替代化肥)还可以增产达8.3%。随着化肥减量配施有机肥比例的提高,菊花花中N、Mg含量呈上升趋势,而Ca和P含量分别在56%和28%有机肥替代处理有最大值。化肥减量配施有机肥可以显著增加药用菊花中绿原酸、木犀草苷和3,5-O-二咖啡酰基奎宁酸的含量,各成分含量随着有机肥比例的升高呈逐渐上升的趋势,上升幅度分别为3.3%～12.8%、15.7%～30.1%和9.5%～29.7%。各处理菊花水提液均有一定的体外抗氧化活性,且随着有机肥比例的升高呈先上升后下降的趋势;菊花水提液能显著提高H₂O₂致损的LO2肝细胞存活率,28%有机肥替代化肥处理细胞存活率最高,为91.2%,与模型组相比呈现极显著差异。综合产量、养分吸收、有效成分含量、体外抗氧化活性、对H₂O₂致损的LO2肝细胞的保护作用等指标,以及有机肥生态友好的特点,确定药用菊花栽培上以28%有机肥替代化肥的效果最佳。     

Structural analysis of lipopolysaccharides from Gram-negative bacteria

This review covers data on composition and structure of lipid A, core, and O-polysaccharide of the known lipopolysaccharides from Gram-negative bacteria. The relationship between the structure and biological activity of lipid A is discussed. The data on roles of core and O-polysaccharide in biological activities of lipopolysaccharides are presented. The structural homology of some oligosaccharide sequences of lipopolysaccharides to gangliosides of human cell membranes is considered.     

Immunological characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test and reactivity with patient sera of tsutsugamushi diseases.

Immunological analyses of lipopolysaccharides (LPS) isolated from Proteus strains OX2, OX19, and OXK used as antigens of Weil-Felix (WF) test, were performed by quantitative agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Antisera against LPS and whole cells (WC) of the three Proteus strains reacted with homologous LPS but not with heterologous LPS, and the reaction was inhibited by the O-polysaccharide fraction isolated from the homologous LPS except OX19-LPS, which lacked O-polysaccharide moiety. The immunological data support the findings that the O-polysaccharide moieties of LPS from OX2 and OXK strains possess different chemical composition (Mizushiri, Amano, Fujii, Fukushi, and Watanabe, Microbiol. Immunol. 34: 121-133, 1990). Antisera against Proteus strains reacted weakly with WC of Rickettsia prowazekii, Rickettsia typhi, and Rickettsia tsutsugamushi. Antisera from patients with tsutsugamushi disease reacted with OXK-WC by WF test when the sera were obtained 13 days after onset of fever. The immunoperoxidase (IP) test titers of these antisera began to rise 6 days after the onset of fever. By ELISA tests these antisera reacted with OXK-WC and OXK-LPS independently of the titers of WF or IP tests.     

Cryptococcus zeae, a new yeast species associated with Zea mays

A new yeast, Cryptococcus zeae (type strain HB 1207^T) is described. Six strains were isolated from corn and pests of corn in Austria. Microsatellite-primed polymerase chain reaction (MSP-PCR) fingerprints showed that the strains are members of the same species. Phylogenetical analyses of domains D1/D2 26S rDNA and ITS 1-5,8S–ITS 2 sequences showed C. zeae to have the closest relationship to C. luteolus. The D1/D2 sequences of C. zeae fit with three Korean Cryptococcus sp. strains (AF459690, AF459691, AF459692). The new species is separable from the closest relative C. luteolus using only two physiological tests.     

蛹虫草线粒体基因型快速检测体系的构建及线粒体遗传稳定性的初步研究

本研究的目的是建立一种快速确定蛹虫草菌株线粒体基因型的技术体系,并探讨蛹虫草连续传代培养后线粒体的遗传稳定性。从已知线粒体基因组的蛹虫草菌株中扩增线粒体内含子位点,将扩增产物混合并制作出两套DNA分子量标准,即在8个内含子位点分别具有内含子的8条扩增条带组成的M-I和在6个内含子位点分别缺失内含子的6条扩增条带组成的M-II。从待检测的蛹虫草菌株（包括3个已知和2个未知线粒体基因组的菌株）中扩增同样的（假定）内含子位点,然后通过琼脂糖凝胶电泳分别与制备好的两个DNA分子量标准进行比较,能够准确判断蛹虫草菌株的线粒体内含子分布模式,从而验证了所构建的线粒体基因型快速检测体系的有效性。选择10个蛹虫草组织分离菌株和8个单分生孢子菌株连续转接培养15代,没有发现线粒体内含子分布模式发生改变。本研究成功构建了快速检测蛹虫草线粒体基因型的技术体系,并发现蛹虫草线粒体具有很高的遗传稳定性,为开展蛹虫草线粒体遗传规律的研究奠定了基础。     

Allozyme differentiation in four species of the Crocus cartwrightianus group and in cultivated saffron (Crocus sativus)

The genetic variability and divergence of four species of the genus Crocus L., related to Crocus cartwrightianus group, namely Crocus thomasii Ten., Crocus hadriaticus Herbert, Crocus oreocreticus B.L. Burtt, C. cartwrightianus Herbert, has been studied by means of starch gel isozyme electrophoresis. For each population the following enzymatic loci were analyzed: PGI-1, PGI-2, G6PDH-1, G6PDH-2, IDH-1, IDH-2, 6PGDH-1, 6PGDH-2, SKDH-1, SKDH-2, AK-1 and AK-2. The genetic variability was estimated through the parameters A (mean number of alleles per locus), P (percent of polymorphic loci), H_o (mean observed heterozygosity), and H_e (mean expected heterozygosity). The genetic differentiation has been assayed by Wrigth's F-statistics, and the genetic divergence by Nei's index. Our data confirmed that the taxa are distinct species, in spite of their similar morphology and karyology. C. thomasii is more genetically similar to C. cartwrightianus and C. oreocreticus than to C. hadriaticus. We hypothesized an autopolyploid origin of saffron, probably from C. cartwrightianus, considering the genotypic classes of Crocus sativus and the other related species of the C. cartwrightianus group studied here.     

中国沿海洛氏角毛藻复合群的多样性组成及地理分布

洛氏角毛藻复合群(Chaetoceros lorenzianus complex)指具有与洛氏角毛藻相似形态学特征的物种集合, 它们广泛分布于全球近岸水域。近年国际上关于该复合群的分类学研究取得新进展, 而我国相关研究仍较为滞后。为了弄清我国沿海洛氏角毛藻复合群的物种多样性, 明确物种信息, 厘清种间界限, 为相关研究提供准确的物种鉴定依据, 本研究陆续在中国沿海建立了该复合群的332个单克隆培养株系, 利用光学显微镜、扫描电镜和透射电镜进行了较为详尽的形态学研究, 基于核糖体大亚基编码基因D1-D3区序列, 构建了分子系统学关系。结果表明其形态聚类与分子系统学结论相一致, 显示我国洛氏角毛藻复合群具有较高的物种多样性, 共鉴定到5个物种, 分别是并基角毛藻(C. decipiens)、优美角毛藻(C. elegans)、平孢角毛藻(C. laevisporus)、曼纳角毛藻(C. mannaii)和稀树角毛藻(C. pauciramosus)。研究表明传统认知的光镜下特征, 如群体特征、角毛走势等易变化, 其分类学价值需谨慎应用。角毛的超微结构, 如角毛孔纹的形状、大小、密度等是有效的种间区别特征, 休眠孢子亦是重要的物种识别依据。并基角毛藻和平孢角毛藻在我国沿岸的分布范围最为广泛, 而稀树角毛藻的分布较为有限。     

滇西太保山森林公园子囊菌门虫生真菌物种多样性及其消长动态

云南及青藏高原存在着丰富的虫生真菌资源。本文选择云南省保山市省级城市森林公园太保山森林公园为研究区域, 对子囊菌门虫生真菌物种多样性及其季节消长动态开展了系统研究。在2016年每月采集土样和罹病昆虫, 分离虫生真菌菌株, 采用多基因(nrSSU, nrLSU, EF-1α, RPB1和RPB2)系统发育分析进行物种鉴定, 通过α多样性分析来研究虫生真菌的数量特征和种群消长动态。太保山森林公园子囊菌门虫生真菌全年均有分布, 共获得395个菌株, 包括3科9属24种; 优势属为虫草属(Cordyceps) (6个种199个菌株), 白僵菌属(Beauveria) (5个种80个菌株), 优势种依次为Akanthomyces sp.、Cordyceps tenuipes、C. cicadae、C. fumosorosea和Beauveria bassiana。7月菌株检出率最高(85株, 占总数的21.4%), 8月物种丰富度(15种)和多样性指数(2.35)最高。在5个优势种中, 只有Cordyceps cicadae受季节变化影响较大, 集中分布于5-9月, 7月分布最丰富(35株, 占该月菌株总数的41.2%); 其他4个种一年大部分时间(11或12个月)都能检测到。结果表明, 太保山森林公园子囊菌门虫生真菌资源丰富, 多数优势种对季节变化适应能力较强。     

A phylogenetic study of the free-living amoeba

Immunological distances were determined for four strains of the free-living amoeba classified as Amoeba proteus, two strains classified as Polychaos dubia and a single strain classified as Chaos carolinensis. The data show that the _ShP strain does not belong to the proteus group; that A. proteus is more closely related to C. carolinensis and is derived from Chaos as is the _ShP strain; that P. dubia and C. carolinensis are the more distantly related species and appear to be the first of the above to have diverged from a common ancestor; and that the amoebae have a long evolutionary history. The accuracy of the phylogenetic tree and the distance Wagner network was discussed. Since the amoebae may be polyphyletic in origin, the latter was assumed to be a more accurate representation of the immunological distance data.     

Complex O-acetylation in Legionella pneumophila serogroup 1 lipopolysaccharide. Evidence for two genes involved in 8-O-acetylation of legionaminic acid.

A putative gene encoding an O-acetyl transferase, lag-1, is involved in biosynthesis of the O-polysaccharide (polylegionaminic acid) in some Legionella pneumophila serogroup 1 strains. To study the effect of the presence and absence of the gene on the O-polysaccharide O-acetylation, lag-1 from strain Philadelphia 1 was expressed in trans in the naturally lag-1-negative OLDA strain RC1, and immunoblot analysis revealed that the lag-1-encoded O-acetyl transferase is active. O-Polysaccharides of different size were prepared from the lipopolysaccharides of wild-type and transformant strains by mild acid degradation followed by gel-permeation chromatography. Using NMR spectroscopy and MALDI-TOF mass spectrometry, it was found that O-acetylation of the first three legionaminic acid residues next to the core occurs in the short-chain O-polysaccharide (<10 sugars) from both strains. Hence, there is another O-acetyl transferase encoded by a gene different from lag-1. In the longer-chain O-polysaccharide, a legionaminic acid residue proximal to the core is N-methylated and could be further 8-O-acetylated in the lag-1-dependent manner. Only strains expressing a functional lag-1 gene were recognized in Western blot analysis by monoclonal antibody 3/1 requiring 8-O-acetylated polylegionaminic acid for binding. The highly O-acetylated outer core region of the lipopolysaccharide is involved in the epitope of another serogroup 1-specific monoclonal antibody termed LPS-1. The O-acetylation pattern of the L. pneumophila serogroup 1 core oligosaccharide was revised using MALDI-TOF mass spectrometry. lag-1-independent O-acetylation of the core and short-chain O-polysaccharide was found to be a common feature of L. pneumophila serogroup 1 strains. The biological importance of conserved lag-1-independent and variable lag-1-dependent O-acetylation is discussed.