DNA restriction patterns and DNA-DNA solution hybridization studies of Frankia isolates from Myrica pennsylvanica (bayberry).

Sixteen Frankia strains were isolated from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. Restriction pattern analysis of total genomic DNA was used to group the isolates into gel groups, and the genetic relatedness among the isolates was evaluated by DNA-DNA solution hybridization studies. Restriction pattern analysis provided a distinctive reproducible fingerprint for each isolate. Isolates fell into nine separate groups (strain types). More than one strain type was isolated from most sites. Isolates from two different gel groups were found in 3 of 10 nodules examined. Of the 16 isolates, 10 contained extrachromosomal DNA. Six different extrachromosomal DNA banding patterns were found. Genomically similar isolates carried related, but different, banding patterns. DNA hybridization studies indicated that isolates from a single plant species can be minimally related as determined by total genome homology. Homology ranged from 12 to 99%. Highly divergent strains were isolated from the same plant and found to cohabit the same nodule. Thus, this study demonstrated that Frankia strains which infect the same host plant are not only phenotypically different but also genetically diverse.     

Physiological, chemical, morphological, and plant infectivity characteristics of Frankia isolates from Myrica pennsylvanica: correlation to DNA restriction patterns.

The filter exclusion method was used to isolate Frankia strains from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. A total of 16 isolates from five locations were cultured. The isolates were characterized by morphological, chemical, physiological, and plant infectivity criteria and compared with genomic DNA restriction pattern data, which were used to assign the isolates into gel groups (see accompanying paper). The isolates from M. pennsylvanica evaluated in this study were characteristic of Frankia physiological group B strains and were indistinguishable on the basis of whole-cell wall chemistry and diaminopimelic acid isomer analysis. Distinct differences in the spectrum of utilized organic acids and carbohydrates were observed among the isolates and were the only phenotypic criteria by which the isolates could be separated and assigned into separate groups. In general, isolates within a restriction pattern gel group had identical utilization patterns, whereas intragroup isolates had different utilization patterns. Correlation of these phenotypic characteristics with the results of molecular analysis revealed an exclusive carbohydrate and organic acid utilization pattern for each gel group as established by restriction pattern analysis.     

Physiological, chemical, morphological, and plant infectivity characteristics of Frankia isolates from Myrica pennsylvanica: correlation to DNA restriction patterns

The filter exclusion method was used to isolate Frankia strains from Myrica pennsylvanica (bayberry) root nodules collected at diverse sites in New Jersey. A total of 16 isolates from five locations were cultured. The isolates were characterized by morphological, chemical, physiological, and plant infectivity criteria and compared with genomic DNA restriction pattern data, which were used to assign the isolates into gel groups (see accompanying paper). The isolates from M. pennsylvanica evaluated in this study were characteristic of Frankia physiological group B strains and were indistinguishable on the basis of whole-cell wall chemistry and diaminopimelic acid isomer analysis. Distinct differences in the spectrum of utilized organic acids and carbohydrates were observed among the isolates and were the only phenotypic criteria by which the isolates could be separated and assigned into separate groups. In general, isolates within a restriction pattern gel group had identical utilization patterns, whereas intragroup isolates had different utilization patterns. Correlation of these phenotypic characteristics with the results of molecular analysis revealed an exclusive carbohydrate and organic acid utilization pattern for each gel group as established by restriction pattern analysis.     

Restriction analysis of the Frankia spp. genome

Abstract Total cellular DNAs of 10 Frankia isolates from Alnus, Elaeagnus and Colletia spp. root nodules were cleaved with ten site-specific restriction endonucleases and analysed by agarose gel electrophoresis. Among the endonucleases tested, Bam HI, Bgl II, Sal I and Sma I proved to be the most suitable for the genome analysis in Frankia spp. DNA restriction banding patterns were reproducible and characteristic of each Frankia strain. The patterns of different strains differed marked indicating considerable genotypic heterogeneity among the isolates. The approach can be used for strain identification in Frankia spp. as well as for differentiation between phenotypically similar strains.     

Genetic heterogeneity among Frankia isolates from root nodules of individual actinorhizal plants

Abstract Genetic variations among selected Frankia isolates from nitrogen-fixing root nodules harvested from an individual actinorhizal plant ( Elaeagnus angustifolia L. or Shepherdia argentea Nutt.) were estimated by restriction fragment analysis of their total genomic DNA. The presence of plasmids and their restriction enzyme patterns were used as additional criteria. Certain isolates from separate nodules on the same plant were found indistinguishable, being probably clones of the same strain. An endophytic passage of a strain isolated from S. argentea on another host plant, Hippophaë rhamnoides L., did not modify the structural characteristics of the genome in the reisolates obtained. However, in some cases, especially when restriction endonucleases cleaving Frankia DNA into relatively small fragments were used, multiple infection of the actinorhizal plants with different Frankia strains and the presence of more than one strain in a nodule were demonstrated. Some aspects of variability in natural populations of Frankia are discussed.     

Identification and subgrouping of Frankia strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Polypeptide patterns of soluble proteins from 35 Frankia strains from different plants of various geographical origins, belonging to Alnus and Elaeagnus host-specificity groups were determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide pattern was qualitatively the same for each strain whatever the number of subcultures or the age. Two gel electrophoresis groups A and E were observed which matched with the Alnus and Elaeagnus host-specificity groups, but with some exceptions. The polypeptide patterns of the 35 Frankia strains tested were separated into 13 gel electrophoresis subgroups. Five Frankia strains were inoculated separately or in 3 mixed combinations of 2 strains on Alnus glutinosa (L.) Gaertn. plants. The polypeptide patterns of the re-isolates obtained from 5-month-old nodules were identical to the corresponding strains used initially in the inoculum. Dual infection was observed on single plantlets.     

Comparison of DNA-based typing methods to assess genetic diversity and relatedness among Candida albicans clinical isolates

Three serial isolates of Candida albicans were obtained from each of five HIV infected patients with recurrent oropharyngeal candidiasis from the same geographical area. Isolates from one patient remained susceptible to fluconazole whereas serial isolates from the other four patients showed decreasing susceptibilities to the drug. Strain identity was investigated by pulse-field gel electrophoretic (PFGE) separation of chromosomes, restriction fragment length polymorphism (RFLP) of chromosomal DNA, Southern blot analysis with the moderately repetitive probe Ca3 of the materials present in the RFLP gels after transfer to nylon membranes, and random amplification of polymorphic DNA (RAPD). All techniques were able to group isolates obtained from the same patient. Techniques resulting in more complex banding profiles exhibited increased discriminatory power allowing detection of strain variants. Methods resulting in less complex banding patterns, especially Southern hybridization of SfiI digested chromosomal DNA with the moderately repetitive probe Ca3, were more helpful to determine isogenicity among isolates obtained from the same patient. The combination of results from methods with high discriminatory power (to maximize detection of strain variants) and methods resulting in less complex banding patterns (to allow determination of isogenic isolates) should facilitate the delineation of the epidemiology of C. albicans infection.     

Diversity and specificity of Frankia strains in nodules of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis determined by rrs gene polymorphism

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.     

Protective effect of Brucella outer membrane complex-bearing liposomes against experimental murine brucellosis

Abstract Nodulation ability was tested for Frankia strains HFPCcI3 and ELI, and Frankia sources A.t. and G.a. from Allocasuarina torulosa and Gymnostoma australianum , respectively, on A. torulosa Miq., Casuarina cunninghamiana Miq., G. australianum L. Johnson and Elaeagnus triflora Roxb. It was shown that A. torulosa and C. cunninghamiana formed nodules only with the Frankia sources obtained from their own host plant, while E. triflora formed nodules with three of the four Frankia sources tested. All nodules formed were effectively fixing nitrogen. Specific nitrogenase activity was highest in E. triflora inoculated with the Frankia strain isolated from nodules of the same species. Identification of Frankia sources in the nodules was performed by use of PCR amplification of DNA with a random primer. PCR amplification of DNA isolated from nodules of G. australianum and E. triflora inoculated with Frankia strain EL1 revealed, when compared with DNA amplified from free living Frankia strain EL1, that there was only one Frankia strain causing the observed nodules.     

Pulsed-field gel electrophoresis for genotypic comparison of Rhizobium bacteria that nodulate leguminous trees

Abstract Pulsed-field gel electrophoresis (PFGE) was applied to characterize Rhizobium bacteria isolated from the root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Keya. For the electrophoresis, the total DNA of 42 isolates, embedded in agarose, was digested by a rare-cutting restriction endonuclease, Xba I. The PFGE run resulted in good resolution of the DNA fragments and gave the strains distinctive fingerprint patterns. The patterns were analysed visually and using automated clustering analysis, which divided the strains into groups resembling the results generated by numerical taxonomy. However, several strains had unique banding patterns, which indicates that these strains are genetically very diverse.     

福建省放线菌结瘤植物共生菌Frankia遗传多样性研究

[目的]了解福建省放线菌结瘤植物共生固氮菌Frankia的遗传多样性.[方法]利用16S-23SrDNA间隔区(rrn)和nifD-K基因间隔区的PCR扩增和RFLP技术,分析了福建省木麻黄、杨梅、桤木、胡颓子等共生Frankia纯培养菌株的遗传差异.[结果]17个菌株获得rrn扩增片段,2个杨梅菌株和1个胡颓子菌株扩增未成功,酶切图谱经聚类分析表明6个地点的细枝木麻黄、短枝木麻黄、粗枝木麻黄12个共生Frankia菌株同源性高,属于一个类群,2个地点的4个杨梅菌株和1个四川桤木菌株亲缘关系近,为另一类群.25个Frankia菌株的,nifD-K基因间隔区PCR-RFLP分析结果显示,7个地点的3种木麻黄14个菌株聚类为一个类群,4个地点的7个杨梅菌株、2个地点的2个四川桤木菌株以及1个台湾桤木菌株聚类为另一个类群,胡颓子菌株则为独立的类群.[结论]研究结果表明福建省共生Frankia遗传多样性丰富.     

Identification of atypical Frankia strains by fatty acid analysis

Abstract: Ineffective, non-infective actinomycetous isolates obtained from actinorhizal nodules of Coriaria nepalensis and Datisca cannabina were identified as Frankia using whole cell fatty acid analysis. The isolates exhibited fatty-acid patterns very similar to those of confirmed Frankia strains from other host plants ( Alnus, Casuarina, Colletia, Comptonia, Elaeagnus and Hippophae ). All Frankia strains, including Coriaria and Datisca isolates, showed fatty-acid profiles very distinct from those of other actinomycetes used as controls ( Actinomyces, Geodermatophilus, Nocardia, Mycobacterium and Streptomyces ). For the genus Frankia , a characteristic pattern of five fatty acids (15:0; 15:1; 16:0 iso; 17:0 and 17:1) was found. These fatty acids comprised 75% or more of the total content. All Frankia strains could be placed into three subgroups. Coriaria isolates were found in the largest subgroup which contained most Frankia strains from other hosts while ineffective strains from Alnus, Elaeagnus and Datisca were distributed in all three subgroups of Frankia .     

Study on Infectivity of the Nodule Endophyte of Some Non-Leguminous N2-Fixing Plants

Cross inoculations were made with Frankia spp. from the nodules of non-leguminous plants belonging to different families, genera and species. The results showed that there are no apparent host specificity in these strains. Under general cases, many strains can nodulate plants in different families, genera and species, but there also are some special results. Both infective ability of the same strains on diffierent host and the different strains on the same host are different. Different isolates from the same host plant were found in certain cases to have various degrees of infectivity. If the original host plant was replaced by others, both these Frankia infective ability and nitrogenase activity in new symbiotic system were lower. The strains that are higher N2-fixing activity in the nodules of the original host also possess stronger N2-fixing activity in the nodules of other hosts. Under test condition, there are positive correlation between the number of the nodules of host plants and N2-fixing activity of the root nodules. The morphology of the spores of the strains in the nodules of new host plant also change more or less.     

Danish Rhizobium leguminosarum strains nodulating 'Afghanistan' pea (Pisum sativum)

A wild pea ( Pisum sativum L.) native to Afghanistan normally known to be resistant to nodulation with European strains of Rhizobium leguminosarum was nodulated early and effectively in field soil in Denmark. Isolates from nodules formed effective nodules abundantly on 'Afghanistan' on reinfection under aseptic conditions. Five types differing in isoenzyme composition pattern were found among 15 isolates from 'Afghanistan' nodules. None were identical with the 'Tom' strain from Turkey, which also forms effective nodules with 'Afghanistan'. The five types were also different with respect to isoenzyme pattern from Rhizobium leguminosarum strains isolated from a modern pea variety cultivated in the same field.     

Molecular characterization of sylvatic isolates of Trichinella spiralis

Genetic relationships of 20 Trichinella isolates from Indiana wildlife were assessed and compared to Trichinella isolated from an infected swine herd. Trichinella larvae were isolated from coyotes, mink, raccoons, and red foxes. The larvae were maintained and amplified in white mice (ICR) and wild mice (Peromyscus leucopus). Differences in phenotypic characters of sylvatic isolates in the 2 laboratory hosts included an approximately 10-30-fold increase in parasite fecundity in wild mice. DNA for each isolate was extracted from Trichinella larvae and analyzed by dot-blot hybridization using a repetitive DNA probe pBP2 that recognizes DNA sequences specific for swine Trichinella. The probe hybridized only to Trichinella from swine and a single coyote isolate. Restriction endonucleases were used to digest DNA and the resulting fragments were separated by gel electrophoresis. Based on the presence of repetitive DNA sequences in the Trichinella genome, distinctive banding patterns were seen among the isolates. Trichinella isolated from swine had a pattern distinct from all sylvatic isolates except 1 from a coyote. Because this coyote was from the same general locality as the swine Trichinella outbreak, it was concluded that the isolate represents transmission of swine trichinellosis to the wildlife population. Further analysis using the enzyme Cla I identified unique banding patterns for wild isolates, suggesting that the sylvatic group is a genetically heterogeneous complex.     

Genetic polymorphism in Xanthomonas albilineans strains originating from 11 geographical locations, revealed by two DNA probes

Two DNA fragments from Xanthomonas albilineans were used as probes to study the molecular diversity among strains of this pathogen. Two serologically distinct groups, serovars I and II, could be differentiated by hybridization to the probes. These probes, designated 830 and 838, were cloned after subtractive DNA hybridization of common sequences of Xanthomonas campestris pv. vasculorum from a serovar I strain of X. albilineans. They did not hybridize to the DNA of several other xanthomonads or to sugarcane DNA under the conditions of hybridization used. Faint bands were observed upon hybridization of probe 830 with one strain of X. campestris pv. phaseoli. The same banding patterns were obtained with a strain of X. albilineans from Burkina Faso and the serovar II strains of Mauritius. The serovar I strains from Mauritius and two other strains each from Reunion and South Africa had similar pattern.     

Strain-specific differentiation of environmental Escherichia coli isolates via denaturing gradient gel electrophoresis (DGGE) analysis of the 16S-23S intergenic spacer region

Denaturing gradient gel electrophoresis (DGGE) was applied to the 16S-23S rRNA intergenic spacer region (ISR) as a means to evaluate strain level differences in Escherichia coli. The ISRs of 81 environmental E. coli isolates obtained from bovine, poultry, and human sources yielded a total of 41 unique DGGE banding patterns, with identical patterns and common bands within each source and no overlapping patterns among sources. An additional 51 isolates from two nearby streams yielded 45 unique banding patterns with no overlap between sites. However, two of the isolates from the streams had identical banding patterns to those from two of the source isolates, resulting in a total of 84 unique DGGE banding patterns out of 132 isolates identified in this study. These results revealed high diversity among environmental E. coli isolates, which made it difficult to unambiguously ascribe strains found in water samples to specific host organisms.     

Estimation of ribosomal RNA operon (rrn) copy number in Acinetobacter isolates and potential of patterns of rrn operon-containing fragments for typing strains of members of this genus

The copy number of the rrn operon in 70 strains of Acinetobacter including the type strains of almost all the genomic species with validated names was estimated after digestion of their genomic DNA by the restriction enzymes BglII and PstI, and Southern blotting. Copy number estimates varied between and among species, with between 3 and 7 rrn operon copies detected. Copy number estimates obtained from the same strain with the two enzymes sometimes varied. BglII generated RFLP patterns of the rrn containing fragments obtained from Southern blots after agarose gel electrophoresis were examined for their value in identifying Acinetobacter isolates. This method was very reproducible with the same fragment pattern always generated from the same isolate on repeated analysis. Often multiple strains of the same genomic species gave identical or very similar patterns (e.g. Acinetobacter baylyi), clustering closest together on the dendrogram generated after numerical analysis of these patterns. However, with some, like BG5 and BG8, the patterns derived from the different strains, some of which had been placed in this genomic species from DNA:DNA hybridization data, varied considerably to each other and to the type strain. Little similarity was seen when relationships between these strains based on these patterns were compared to those using DNA:DNA hybridization data. Often these patterns could be used to question earlier identification of strains using phenotypic characters. Thus, strain AB82 thought to belong to genomic species 5 gave an identical pattern to A. bouvetii(T) (DSM 14964). In some cases this pattern analysis suggested that novel species of Acinetobacter might exist among the strains examined.     

Natural diversity of Frankia strains in actinorhizal root nodules from promiscuous hosts in the family Myricaceae.

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N(2)-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

内蒙古沙棘共生菌Frankia的遗传多样性

利用RFLP分子标记方法,在自然条件下对内蒙古东、西部8个地点采集的24个沙棘根瘤样品进行沙棘共生菌Frankia的遗传多样性分析。结果表明,供试样品nifD-nifK基因间隔区(IGS)扩增片段的大小约1 100bp;不同样品的酶切图谱有明显差异;有些样品产生复合RFLP型,揭示在自然状态下不同基因型的Frankia菌株可共同侵染同一沙棘寄主。聚类分析显示,来源于相同地点和不同地点的根瘤样品内的Frankia菌株间均有遗传多样性;没有发现Frankia菌株遗传多样性的分布与采样地点有相关性。