Heterozygosity and instability of amplified chromosomal insertions in the radioresistant bacterium Deinococcus radiodurans.

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. The circles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strains that are defective in natural transformation and sensitive to DNA-damaging agents were further characterized. Both strains were defective in duplication insertion. While on strain was normal for plasmid transformation, the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may require recombinational functions.     

Multiple resistance and biochemical mechanisms of pyridaben resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) (Acari: Tetranychidae) resistant to pyridaben was selected with pyridaben successively for 20 generations to produce the PR-20 strain. Resistance and multiple resistance levels of the PR-20 strain to 15 acaricides were determined using a spray bioassay. The PR-20 strain was extremely resistant to pyridaben (resistance ratio [RR] = 240]. The strain exhibited extremely strong resistance to fenpyroximate (RR=373) and acrinathrin (RR=329) and strong resistance to benzoximate (RR=84). An RR = 10-40 was observed with abamectin, fenazaquin, fenbutatin oxide, fenpropathrin, and tebufenpyrad. The PR-20 strain showed low levels of resistance (RR <10) to azocyclotin, bromopropylate, chlorfenapyr, dicofol, milbemectin, and propargite. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor, had the greatest effect on pyridaben resistance. PBO significantly caused pyridaben resistance in the PR-20 strain to drop to the full susceptibility level of the susceptible (S) strain. However, there was no significant difference in MFO activities measured using a model substrate between the S and PR-20 strains. These results suggest that use of certain acaricides with little multiple resistance or PBO will be useful for the management of pyridaben resistance in the field.     

Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of Clonostachys rosea and Development of a Strain-Specific PCR Detection Assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

The use of fecundity, lobe biometry and the RAPD-PCR technique in order to compare strains of Tetranychus sp.

In Belgium, recently two new red carmine forms of Tetranychus sp. have appeared on tomato crops in glasshouses and in maize crops. These forms maintain their red colour even during summer and on other host plants such as French bean. Our aim was to establish their status and to compare them with strains of Tetranychus urticae and Tetranychus cinnabarinus. A strain of Tetranychus kanzawai, a distant relative of the same genus, was used as a control. The five strains tested were characterized by their own demographic parameters, probably because of a longseparated evolution on different host plants. The white eye strain of T. urticae was differentiated from the four other strains by scanning electron microscopy studies of the dorsal integument folds. An investigation using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) showed T. cinnabarinus as a distinct group. However, the mixing of the individuals of the four other strains raises the question of the role of hybridization and host plant selection in the evolution of the Tetranychus complex. Exp Appl Acarol 22: 649–666 © 1998 Kluwer Academic Publishers     

Schistosoma mansoni: mice infected with different worm strains.

Infections with five geographical strains and substrains of Schistosoma mansoni were compared in mice. Two substrains (Lc-1 and Lt-1) were derived from the parent (L-1) St. Lucian strain on the basis of differing infectivity for various snail strains. The Puerto Rican strains (PR-1 and PR-2) were obtained with an interval of 25 years. Consistent differences among the lines were found in egg distribution and numbers of eggs in tissues and feces. One Puerto Rican strain (PR-2) and one St. Lucian substrain (Lc-1) had longer prepatent periods than the other strains. Mice infected with the PR-1 strain consistently had the highest egg accumulation in the tissues per worm pair. Relatively few eggs were passed in the feces of the Lt-1 strain. By Week 9 of infection, eggs were noted in the spleens of mice carrying each of the strains and substrains.     

Characterization of Erwinia amylovora strains from different host plants using repetitive-sequences PCR analysis, and restriction fragment length polymorphism and short-sequence DNA repeats of plasmid pEA29

AIMS: The three main aims of the study were the assessment of the genetic relationship between a deviating Erwinia amylovora strain isolated from Amelanchier sp. (Maloideae) grown in Canada and other strains from Maloideae and Rosoideae, the investigation of the variability of the PstI fragment of the pEA29 plasmid using restriction fragment length polymorphism (RFLP) analysis and the determination of the number of short-sequence DNA repeats (SSR) by DNA sequence analysis in representative strains. METHODS AND RESULTS: Ninety-three strains obtained from 12 plant genera and different geographical locations were examined by repetitive-sequences PCR using Enterobacterial Repetitive Intergenic Consensus, BOX and Repetitive Extragenic Palindromic primer sets. Upon the unweighted pair group method with arithmetic mean analysis, a deviating strain from Amelanchier sp. was analysed using amplified ribosomal DNA restriction analysis (ARDRA) analysis and the sequencing of the 16S rDNA gene. This strain showed 99% similarity to other E. amylovora strains in the 16S gene and the same banding pattern with ARDRA. The RFLP analysis of pEA29 plasmid using MspI and Sau3A restriction enzymes showed a higher variability than that previously observed and no clear-cut grouping of the strains was possible. The number of SSR units reiterated two to 12 times. The strains obtained from pear orchards showing for the first time symptoms of fire blight had a low number of SSR units. CONCLUSIONS: The strains from Maloideae exhibit a wider genetic variability than previously thought. The RFLP analysis of a fragment of the pEA29 plasmid would not seem a reliable method for typing E. amylovora strains. A low number of SSR units was observed with first epidemics of fire blight. SIGNIFICANCE AND IMPACT OF THE STUDY: The current detection techniques are mainly based on the genetic similarities observed within the strains from the cultivated tree-fruit crops. For a more reliable detection of the fire blight pathogen also in wild and ornamentals Rosaceous plants the genetic features of deviating E. amylovora strains have to be studied in detail.     

Biological properties of some nitrogen-fixing associative enterobacteria

Enterobacteria isolates from potato tubers were able to fix nitrogen, to protect plants against phytopathogens and to produce phytohormones thus increasing the plant yield. These isolates were previously phenotypically identified as Erwinia carotovora; however, they differed from typical E. carotovora in a number of biological characteristics and were found to be nonphytopathogenic (avirulent) due to the lack of pectate lyase activity. A data matrix, containing 31 strains and 105 biological characteristics was used for computer cluster analysis. The avirulent strains formed a separate cluster more closely related to Klebsiella spp. strains (with a 0.67 level of similarity) than to typical phytopathogenic bacteria of the E. carotovora group (with a 0.48 level of similarity). A phylogenetic analysis based on restriction polymorphisms of an amplified ribosomal DNA spacer region revealed that the avirulent strains studied here were different from all Erwinia, Klebsiella and other enterobacteria species strains. The AP PCR/hybridization technique showed cross homology of amplified DNA of these avirulent strains and a lack of such homology with the DNA from strains of other species. Numerical taxonomy data, rDNA analysis and AP PCR/hybridization assays confirmed that these avirulent bacteria may be regarded as an independent group of enterobacteria.     

Genomic organization of the yeast Yarrowia lipolytica

We produced electrophoretic karyotypes of the reference strain E150 and of seven other isolates from different geographical origins to study the genomic organization of the dimorphic yeast Yarrowia lipolytica. These karyotypes differed in the number and size of the chromosomal bands. The karyotype of the reference stain E150 consisted of five bands of between 2.6 and 4.9 Mb in size. This strain contained at least five rDNA clusters, from 190 to 620 kb in size, which were scattered over most of the chromosomes. The assignment of 43 markers, including rRNA genes and three centromeres, to the E150 bands defined five linkage groups. Hybridization to the karyotypes of other isolates with pools of markers of each linkage group showed that linkage groups I, II, IV and V were conserved in the strains tested whereas group III was not and was split between at least two chromosomes in most strains. Use of a meganuclease I-SceI site targeted to one locus of E150 linkage group III showed that two chromosomes actually comigrated in band III of this strain. Our results are compatible with six chromosomes defining the haploid complement of strains of Y. lipolytica and that, despite an unprecedented chromosome length polymorphism, the overall structure of the genome is conserved in different isolates. Received: 27 March 1997; in revised form: 8 July 1997 / Accepted: 9 July 1997     

鸭疫里默氏杆菌外膜蛋白生物学特性研究

血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。     

鸭疫里默氏杆菌外膜蛋白生物学特性研究

血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。     

温度对抗性朱砂叶螨发育和繁殖的影响

比较了朱砂叶螨Tetranychus cinnabarinus (Boiduval)敏感品系（S）、阿维菌素抗性品系（AR）和甲氰菊酯抗性品系（FR）在不同温度下的发育和繁殖情况,用王-兰-丁模型拟合了各品系发育速率与温度的关系。结果表明,在36℃下,S品系和FR品系卵均不能孵化,而AR品系却能完成世代发育。15℃下,AR和FR品系的世代历期分别比S品系延长8.0和6.2天,产卵前期均比S品系缩短约2.0天。在20～28℃下,S品系繁殖力大于抗性品系; 在34℃下, AR品系的产卵量显著大于S和FR品系。S、FR和AR品系发育临界高温分别为36.0℃、36.1℃和42.4℃。结果提示AR品系对高温的适应能力较S和FR品系明显增强。     

Molecular characterization of Yarrowia lipolytica and Candida zeylanoides isolated from poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.     

NDV HeB02分离株的生物学特性及其F基因的克隆与序列测定

对河北省新城疫分离株HeB0 2的生物学特性进行了鉴定 ,根据国外已发表的新城疫病毒F基因序列 ,设计了一对引物并以RT PCR特异性扩增出HeB0 2分离株的F基因 ,基因产物大小为 1 63kb ,与设计相符 ,对其进行序列测定后 ,与其它标准毒株F48E9、LaSota和Clone30的F基因进行同源性比较 ,结果表明 ,HeB0 2株与国内标准强毒株F48E9及目前广泛应用的弱毒疫苗LaSota和Clone30的F基因核苷酸序列的同源性分别为 88 1 %、84 9%和 83 8%,由此可以看出HeB0 2分离株与标准毒株和疫苗株在F基因上发生了变异。     

Identification and characterization of four strains of Acidithiobacillus ferrooxidans isolated from different sites in China

Four strains of Acidithiobacillus ferrooxidans (A. ferrooxidans), AF1, AF2, AF3 and AFc, were isolated from samples with different geological sources using a 9K medium. These four isolates were identified as A. ferrooxidans by phenotypic and 16S rDNA sequence analyses. All four isolates were able to use ferrous ion (Fe(2+)), elemental sulfur (S0) or pyrite as a sole energy source, but they showed differences in pH optima and range of activity, optimum temperature of activity, resistance to chloride (KCl) and heavy metal ions, and oxidation rates of Fe(2+), S0 and pyrite. AF3 was the most active strain when using Fe(2+) as the energy source, while AFc grew best using pyrite as the energy source. AF2 appeared to differ from the other three strains in substrate utilization, as it oxidizes S0 and pyrite more effectively than Fe(2+). RAPD analysis of genomic DNA from these isolates showed that banding profiles of their genomic DNA exhibited some differences, and the genomic banding profile of AF2 was significantly different from that of others. To obtain an insight into the molecular biology of the process of the energy production of these strains, several genes involved in the iron respiratory chain were cloned and sequenced, including Fe(2+) oxidase (iro), rusticyanin (rus) and subunit III of aa3-type cytochrome oxidase (cox C) genes. The results revealed that the iro gene can be cloned from all of the four strains and the nucleotide sequences were shown to be completely identical in each. However, rus and coxC genes could be amplified only from AF1, AF3 and AFc, not from AF2. These results suggested that the phenotypic differences of the four strains of A. ferrooxidans from different sites correlated with their genetic polymorphism, which may result from the different environments in which they lived, and that the strain AF2 was phenotypically and genetically significantly different from the other three strains.     

Chemical communication of emigration behavior of Drosophila melanogaster. II. Identification of chemical substances

Two chemical substances isolated from adult flies of Drosophila melanogaster differently affected the emigration activity of genetically different strains. These substances were identified as palmitic acid and oleic acid, respectively. Chemical and biological comparisons of the natural and authentic compounds showed them to be identical. The behavior response was dependent on the concentration of these fatty acids. The two chemical substances are excreted by adult flies, both male and female, but the emigration activity of one strain was affected by only palmitic acid and that of the other strain, by only oleic acid.     

The interaction of myotropic and macrophagotropic strains of Trypanosoma cruzi with myoblasts and fibers of skeletal muscle

The process of interaction of bloodstream trypomastigotes from the myotropic CL and Colombiana strains and the macrophagotropic Y strain of Trypanosoma cruzi with mouse myoblasts and myotubes was analysed. After 24 h of parasite-host cell interaction, parasites from the CL and Colombiana strains appeared to be more infective to myoblasts than those from the Y strain. Parasites from the Colombiana strain were more infective for myotubes than those from the Y strain, while those from the CL strain showed very a low ability to infect the cells. For all strains the infectivity was low for short periods of interaction, increasing with time. Myoblasts infected with parasites from the Y strain fused with other infected and uninfected cells to form myotubes. However, the process of fusion was blocked when the myoblasts were infected with parasites from the CL and Colombiana strains. These data indicate a different behavior of muscle cells when in contact with myotropic or non-myotropic strains of T. cruzi.     

Epitope analyses of pneumococcal surface protein A: a combination of two monoclonal antibodies detects 94% of clinical isolates

Immunisation of BALB/c mice with seven heat-treated Norwegian clinical isolates of Streptococcus pneumoniae of different serotypes elicited mainly monoclonal antibodies (mAbs) to pneumococcal surface protein A (PspA). It was remarkable that the fusions resulted only in a few mAbs directed against other protein antigens. Dot blot analysis with 16 mAbs using clinical isolates representing 23 different capsular types and the uncapsulated reference strain R36A showed that some of the mAbs bound to PspA epitopes expressed by a low number of strains whereas others bound to broadly distributed epitopes. On the basis of their reactivities, seven of these mAbs could be divided into two groups recognising different subsets of pneumococci. The three mAbs in the narrow reacting group bound to epitopes found in 21-25% of the strains whereas the four mAbs in the broad reacting group detected more than 57% of the analysed strains. The epitopes for these seven antibodies were surface exposed on live exponential phase grown pneumococci as shown by flow cytometry. The finding that a combination of mAb 180,C-1 (IgG2a) from the first group and mAb 170,E-11 (IgG2a) from the second group detected 94% of the examined strains is interesting because PspA has been reported by others to be a serological highly variable protein.     

Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.     

Development of new strains and related SCAR markers for an edible mushroom, Hypsizygus marmoreus

New fast-growing and less bitter varieties of Hypsizygus marmoreus were developed by crossing monokaryotic mycelia from a commercial strain (Hm1-1) and a wild strain (Hm3-10). Six of the better tasting new strains with a shorter cultivation period were selected from 400 crosses in a large-scale cultivation experiment. We attempted to develop sequence characterized amplified region (SCAR) markers to identify the new strain from other commercial strains. For the SCAR markers, we conducted molecular genetic analysis on a wild strain and the eight most cultivated H. marmoreus strains collected from various areas in East Asia by randomly amplified polymorphic DNA. Ten unique DNA bands for a commercial Hm1-1 strain and the Hm3-10 strain were extracted and their sequences were determined. Primer sets were designed based on the determined sequences. PCR reactions with the primer sets revealed that four primer sets successfully discriminated the new strains from other commercial strains and are thus suitable for commercial purposes.