Molecular epidemiology of Legionella pneumophilia serogroup 1 by ribotyping with a non-radioactive probe and PCR fingerprinting

Abstract Hybridization with acetylaminofluorene-labelled 16 + 23 S rRNA from Escherichia coli was used to detect DNA polymorphism among Legionella pneumophila serogroup 1 isolates. Isolates from unrelated patients showed at least four different rRNA restriction patterns, whereas those from related patients showed a single pattern. Amplification of genomic regions with an arbitrary primer by polymerase chain reaction was used to further analyze the isolates. Related isolates showed closely related patterns while unrelated isolates displayed six distinct patterns. We could differentiate the majority of unrelated isolates with the combination of the patterns obtained with the ribotyping and the PCR fingerprinting, while strains from the same outbreak remained highly related. The ribotyping and the PCR fingerprinting are proposed as useful and easy to perform epidemiological markers of L. pneumophila serogroup 1 infection.     

Characterization of Trichoderma Isolates from Philippine Rice Fields by UP-PCR and rDNA-ITS1 Analysis: Identification of UP-PCR Markers

Forty-two isolates of Trichoderma from rice fields in four provinces in the Philippines were characterized using rDNA-ITS1 analysis and universally primed polymerase chain reaction (UP-PCR). Two groups were clearly distinguishable on the basis of length and restriction pattern of the internal transcribed spacer (ITS) region of the ribosomal DNA and UP-PCR banding profiles using UP primer, L45. The 40 isolates comprising the largest group were very similar with respect to their UP-PCR banding profiles and were assigned to Trichoderma harzianum Rifai following morphological identification of four of the isolates. The two isolates belonging to the second group were identified as Trichodermaviride Pers. ex. Gray on the basis of their morphology, rDNA-ITS1 analysis and distinct UP-PCR banding profiles. One of the T. harzianum isolates with good cellulolytic and competitive saprophytic abilities was analysed using single and pair-wise combinations of UP primers in order to distinguish it from the remaining 41 isolates. A suitable diagnostic marker was identified and this marker will be valuable for monitoring the isolate in field tests.     

Rapid identification of Rhizobium strains by targeted PCR fingerprinting

Numerous polymerase chain reaction (PCR) based procedures are routinely used to produce genomic fingerprints of prokaryotes. Many of them have drawbacks however such as sensitivity to experimental variation, lack of reproducibility, poor resolution and the inability to distinguish between closely related strains. To overcome these difficulties, we developed an alternative procedure, Targeted PCR Fingerprinting (TPF) which is based upon the amplification of few but carefully selected markers, followed by high resolution RFLP analysis of the amplified DNA fragments. In contrast to most fingerprinting protocols that use low resolution agarose gels, TPF patterns are produced on denaturing polyacrylamide gels which allow the precise recording of the genomic fingerprints. TPF analysis, which can simultaneously process 96 samples in less than 12 h and remains unaffected by slight experimental variations, is particularly adapted for the rapid identification of target strains amongst many field isolates. Using PCR primers specific for the nifH and recA genes, this procedure was also sufficiently sensitive to discriminate between Rhizobium species NGR234 and R. fredii USDA257, two closely related bacteria in which the symbiotic loci are 98% homologous. Interestingly, comparison of several of their symbiotic genes as well as the partial DNA sequences of their 16S rDNA and recA genes suggest that chromosomes and symbiotic plasmids did not co-evolve, but that symbiotic functions were acquired by lateral gene transfer long after NGR234 and USDA257 diverged from their common ancestors. In this respect, TPF fingerprints produced with distinct chromosomal and plasmid born markers, such as the recA and the nifH genes in NGR234 and USDA257, are probably more likely to detect lateral transfer of genes in bacterial field-populations than procedures relying on the amplification of numerous fragments of unknown genomic position and biological function.     

Genetic heterogeneity among Vibrio alginolyticus isolated from shrimp farms by PCR fingerprinting

AIMS: To study the strain variability among Vibrio alginolyticus isolates from different sources by insertion sequence-targeted PCR fingerprinting and whole cell protein profile analysis. METHODS AND RESULTS: Eleven strains of V. alginolyticus were isolated from seven different sources including healthy, infected, farm-reared and wild shrimps. Following biochemical characterization, the isolates were analysed by PCR fingerprinting and whole cell protein analysis by SDS-PAGE. The strains were genetically different irrespective of the sources of isolation. CONCLUSIONS: Strain variation exists in V. alginolyticus isolates obtained even from the same source, and PCR fingerprinting is a simple and efficient method in identifying strain-specific variations among the different isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio alginolyticus is implicated in severe vibriosis of marine aquaculture systems although many strains are avirulent and could be used as probiotic strains. As a wide variation exists among this species, differentiating the harmful and beneficial strains would help in finding ways of controlling the infections by eliminating harmful shrimp pathogenic vibrios.     

Randomly amplified polymorphic DNA fingerprinting identifies subgroups of Trichoderma viride and other Trichoderma sp. capable of chestnut blight biocontrol

Abstract Eleven strains of Trichoderma viride , 2 strains of the putative teleomorph Hypocrea rufa and 9 of several other Trichoderma sp. were characterized by random polymorphic DNA amplification (RAPD) fingerprinting and screened for their ability to antagonize growth of European strains of the chestnut blight causing fungus Cryphonectria parasitica , using a dual-culture assay. The best strains were found in the species T. harzianum, T. parceramosum , a distinguishable subgroup of T. viride and a not named Trichoderma sp. The successful application of these strains against chestnut blight in vivo is demonstrated.     

Identification of Trichoderma strains by image analysis of HPLC chromatograms

Forty-four Trichoderma strains from water-damaged building materials or indoor dust were classified with chromatographic image analysis on full chromatographic matrices obtained by high performance liquid chromatography with UV detection of culture extracts. The classes were compared with morphological identification and rDNA sequence data, and for each class all strains were of the same identity. With all three techniques each strain--except one--was identified as the same species. These strains belonged to Trichoderma atroviride (nine strains), Trichoderma viride (three strains), Trichoderma harzianum (10 strains), Trichoderma citrinoviride (12 strains), and Trichoderma longibrachiatum (nine strains). The odd strain was identified as Trichoderma hamatum by morphology and rDNA sequencing, but not by image analysis as no reference strains of this species were included. It is concluded that the secondary metabolite profile contains sufficient information for classification and species identification.     

Comparison of ribotyping, pulsed-field gel electrophoresis and random amplified polymorphic DNA for typing Clostridium difficile strains

Abstract Clostridium difficile is a Gram-positive sporulating anaerobic bacillus which causes pseudomembranous colitis. Nosocomial acquisition of this bacteria has proved frequent, and epidemiological markers are needed to recognize and control common-source outbreaks. We therefore compared the results of pulsed-field gel electrophoresis (PFGE) after restriction with Sma I or Nru I, random-amplified polymorphic DNA (RAPD) using 3 10-mer oligonucleotides, and ribotyping to differentiate between 30 unrelated strains of C. difficile belonging to 8 serotypes. The strains were separated into 26 different types by PFGE, 25 by RAPD, but into only 18 types by ribotyping. Median percentages of similarity between strains ranged from 27 in the PFGE assay to 90 in the ribotyping assay, but there was good agreement between the 3 methods for the clustering of strains. PFGE was more time-consuming than RAPD but its patterns were easier to analyze.     

Differentiation of Vibrio alginolyticus strains isolated from Sardinian waters by ribotyping and a new rapid PCR fingerprinting method

We investigated the usefulness of a novel PCR fingerprinting technique, based on the specific amplification of genomic regions, to differentiate 30 Vibrio alginolyticus strains isolated in Sardinian waters. The different profiles obtained were scanned and analyzed by a computer program in order to determine genetic relationships. The results were then compared with the patterns obtained by ribotyping with HindIII, KpnI, and XbaI restriction enzymes. PCR fingerprinting could differentiate the strains analyzed into 12 different patterns, whereas ribotyping with XbaI, which produced the highest number of patterns, generated only 7 different profiles. This study revealed the superior discriminative power of the proposed technique for the differentiation of related V. alginolyticus strains and the potential use of PCR fingerprinting in epidemiological studies.     

Ribotyping of Pseudomonas aeruginosa isolates from patients and water springs and genome fingerprinting of variants concerning mucoidy

Abstract Thirty-one strains of Pseudomonas aeruginosa , isolated from water springs, clinical isolates (some of which were from cystic fibrosis (CF) patients), and two type cultures, were characterized by ribotyping. After restriction of chromosomal DNA of the different isolates with Eco RI and hybridization of Southern transfer blots with 2-acetylaminofluorene labelled Escherichia coli 16S + 23S rRNA probe, eleven different ribopatterns were obtained, representing variations of a dominant profile. This largely predominant pattern included both type cultures, all six isolates from water springs, 33% of the nine CF isolates and 43% of fourteen other clinical isolates most of them from nosocomial infections. When the genomic macrorestriction fingerprints of three mucoid CF isolates, with Ase I, Dra I or Bfr I were compared with those of their spontaneous variants, concerning mucoidy, no differences were detected.     

瑞氏木霉木糖醇脱氢酶基因的分离与鉴定

将在木聚糖上生长的瑞氏木霉(Trichoderma reesei)RutC-30的cDNA文库全部质粒转化已携带有毕赤氏酵(Pithia stipitis)木糖还原酶基因的重组酿酒酵母(Saccharomycescerevisiae)菌株H475,在H475中构建了瑞氏木霉的cDNA表达亚文库。在以木糖为唯一碳源的选择性酵母合成培养基上,从该亚文库中筛选到瑞氏木霉木糖醇脱氢酶cDNA基因．该基因片段长为1．3kb。Southern、Norhern印迹杂交分析和蛋白质凝胶电泳结果表明该基因确实来源于瑞氏木霉,所编码蛋白质分子量约为40kDa。携带有毕赤氏酵母木糖还原酶和瑞氏木霉木糖醇脱氢酶基因的重组酵母能够在以木糖为唯一碳源的培养基上生长,并能将90％以上的木糖转化为木糖醇、乙醇和其它副产品。     

Desorption of zinc by extracellularly produced metabolites of Trichoderma harzianum, Trichoderma reesei and Coriolus versicolor

AIMS: To determine the role of fungal metabolites in the desorption of metals. METHODS AND RESULTS: Desorption of Zn from charcoal by three different fungi was compared against metal desorption with reverse osmosis water, a 0.1% Tween 80 solution and a 0.1 mol l(-1) CaCl(2) solution. All three fungal filtrates desorbed three times more Zn than either 0.1% Tween 80 or 0.1 mol l(-1) CaCl(2). Metal chelator production in Trichoderma harzianum and Coriolus versicolor was constitutively expressed while chelator production in Trichoderma reesei was induced by Zn. The presence of Zn inhibited the production of metal chelators by C. versicolor. Only C. versicolor was found to produce oxalic acid (a strong metal chelator). All fungi caused a marked decrease in pH, although this was not enough to explain the increased desorption of the metals by the different fungal filtrates. CONCLUSIONS: Metal chelation via organic acids and proteins are the main mechanisms by which the fungal filtrates increase zinc desorption. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study explain why plants inoculated with T. harzianum T22 take up more metal from soil, than noninoculated plants while metabolites produced by fungi could be used for metal leaching from contaminated soils.     

Antisense inhibition of xylitol dehydrogenase gene, xdh1 from Trichoderma reesei

AIMS: To inhibit xylitol dehydrogenase (XDH) in Trichoderma reesei by antisense inhibition strategy and construct novel strains capable of accumulating xylitol. METHODS AND RESULTS: The xdh1 antisense expression plasmid pGTA-xdh was constructed by inserting xdh1 DNA fragment inversely between the gpdA promoter and the trpC terminator from Aspergillus nidulans into a pUC19 plasmid backbone. Trichoderma reesei protoplasts were co-transformated with pGTA-xdh and hygromycin B resistance plasmid pAN7-1. Of 20 transformants screened from the selective medium, one transformant with the highest xylitol accumulation, designated ZY15, showed a distinct reduction (c. 52%) in XDH activity compared with the original strain Rut-C30. The results of Southern hybridization and PCR assay showed that the antisense expression cassette of xdh1 was integrated into the genome of T. reesei. The RT-PCR analysis proved that antisense RNA effectively inhibited XDH expression (c. 65%). Xylitol accumulation (2.37 mg ml(-1)) of ZY15 was five times higher than that (0.46 mg ml(-1)) of the original strain Rut-C30. CONCLUSIONS: Strain ZY15 successfully downregulated XDH production and exhibited xylitol accumulation in xylose liquid medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributed to the budding field of fungal genetics in two points. First, it confirmed that antisense RNA strategy could be used as a means of reducing gene expression in the filamentous fungus T. reesei. Secondly, it verified that the strategy appears most promising for creating novel filamentous fungi strains capable of accumulating intermediary metabolites.     

Characterization of pathogenic and nonpathogenic strains of Xanthomonas axonopodis pv. manihotis by PCR-based DNA fingerprinting techniques

Strains of Xanthomonas axonopodis pv. manihotis (Xam) were characterized for pathogenicity and for DNA polymorphism using different PCR-based techniques. Using amplified restriction fragment length polymorphism (AFLP), strains were distinguished from each other and also from other Xanthomonas strains. Cluster analysis showed a high correlation between DNA polymorphism and pathogenicity. Four Xam strains were further analyzed using three PCR-based techniques, AFLP, AFLP-pthB and RAPD-pthB. Various primer combinations were used including primers specific to a Xam pathogenicity gene (pthB) along with RAPD or AFLP primers. The AFLP primer combinations EcoRI+T/MseI+A and EcoRI+T/MseI+T were the most efficient to discriminate among pathogenic and nonpathogenic Xam strains. Polymorphic bands were excised from the gel, amplified and cloned. Sequences analysis showed significant homology with bacterial pathogenicity island, genes involved in pathogenic fitness and regulators of virulence. Three cloned AFLP fragments were used as probes in DNA blot experiments and two of them showed significant polymorphism.     

绿色木霉产生的葡萄糖苷酶类

由绿色木霉(Trichoderma viride)A10菌株的粗酶制剂中分离纯化得到 6个β-葡萄糖苷酶组分,对它们的物理、化学性质和对20种糖苷类化合物的水解作用进行了测试。其中5个组分都是既能水解β-构型的糖苷化合物,又能水解α-构型的糖苷化合物,表明它们对α-、β-异头专一性的要求并不象文献中报导的那样十分严格,对糖基和糖苷配基专一性的要求也并不十分严格。由此,对葡萄糖苷酶类的命名分类方法提出了讨论,并对在纤维素酶系研究中纤维二糖酶活力的检测方法进行了讨论。     

Production of mycotoxins on artificially and naturally infested building materials

In this study, the ability to produce mycotoxins during growth on artificially infested building materials was investigated for Penicillium chrysogenum, Pen. polonicum, Pen. brevicompactum, Chaetomium spp., Aspergillus ustus, Asp. niger, Ulocladium spp., Alternaria spp., and Paecilomyces spp., all isolated from water-damaged building materials. Spores from the different isolates of the above mentioned species were inoculated on gypsum board with and without wallpaper and on chipboard with and without wallpaper. Fungal material was scraped off the materials, extracted, and analyzed using high performance liquid chromatography-diode array detection and thin layer chromatography. All six isolates of C. globosum produced the toxic chaetoglobosins A and C, at levels of up to 50 and 7 g/cm² respectively. The quantities of secondary metabolites produced by Penicillia were generally low, and no toxin production was detected from any of the five isolates of Pen. chrysogenum. Both isolates of Pen. polonicum produced 3-methoxy-viridicatin, verrucosidin, and verrucofortine. Two of five isolates of Pen. brevicompactum produced mycophenolic acid. From five out of six isolates of Alternaria spp., alternariol and alternariol monomethyl ether were detected. From Ulocladium spp., Paecilomyces spp., and Asp. ustus no known mycotoxins were detected, although the latter two are known mycotoxin producers. Asp. niger produced several naphtho--pyrones and tetra-cyclic compounds. All investigated species, especially Asp. ustus and Asp. niger produced many unknown secondary metabolites on the building materials. Analyses of wallpaper and glass-fibre wallpaper naturally infested with Asp. versicolor revealed sterigmatocystin and 5-methoxysterigmatocystin. Analyses of naturally infested wallpaper showed that C. globosum produced the chaetoglobosins A and C, and Pen. chrysogenum produced the antibiotic meleagrin.This revised version was published online in October 2005 with corrections to the Cover Date.     

Cold adaptation of a mesophilic cellulase,EG Ⅲ from Trichoderma reesei,by directed evolution

Cold-active enzymes have received little research attention although they are very useful in industries. Since the structure bases of cold adaptation of enzymes are still unclear, it is also very difficult to obtain cold-adapted enzymes for industrial applications using routine protein engineering methods. In this work, we employed directed evolution method to randomly mutate a mesophilic cellulase, endoglucanase Ⅲ (EG Ⅲ) from Trichoderma reesei, and obtained a cold-adapted mutant, designated as w-3. DNA sequence analysis indicates that w-3 is a truncated form of native EG Ⅲ with a deletion of 25 consecutive amino acids at C-terminus. Further examination of enzymatic kinetics and thermal stability shows that mutant w-3 has a higher Kcat value and becomes more thermolabile than its parent. In addition, activation energies of w-3 and wild type EG Ⅲ calculated from Arrhenius equation are 13.3 kJ· mol-1 and 26.2 kJ ·mol-1, respectively. Therefore, the increased specific activity of w-3 at lower tempera