scholarly journals Genotypic Characterization ofBradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing

1998 ◽  
Vol 64 (6) ◽  
pp. 2096-2104 ◽  
Author(s):  
Pablo Vinuesa ◽  
Jan L. W. Rademaker ◽  
Frans J. de Bruijn ◽  
Dietrich Werner
Keyword(s):  
Cluster Analysis ◽  
16S Rrna ◽  
16S Rdna ◽  
Canary Islands ◽  
Computer Assisted ◽  
Woody Legumes ◽  
Repetitive Extragenic Palindromic Pcr ◽  
Reference Strains ◽  
Restriction Fragment Length ◽  
Repetitive Extragenic Palindromic

ABSTRACT We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulatedGlycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus,Macroptilium atropurpureum, and Vigna unguiculata.

and inexpensively produced. During oligonucleotide synthesis a variety of marker or linker sequences can be introduced to the 5' end of the oligonucleotide. The principal conjugate fluorochromes are derivatives of fluorescein or rhodamine, although labels such as digoxigenin and biotin have also been used (Fuhrman et al. 1994, Amann et al. 1995). The design of rRNA-targeted group- or species-specific probes should be based on a good rRNA sequence database and be performed in a computer-assisted way using appropriate software. The organisms encompassed by a probe varies according to the region of the 16S rRNA selected as the hybridization target. Species-specific probes complement the most variable regions, while more general probes target more conserved regions of the molecule. The principal steps involved in the design of probes are: the alignment of rRNA gene sequences; the identification of sequence idiosyncrasies; the synthesis and labelling of complementary nucleic acid probes; and the experimental evaluation and optimization of the probe specificities and assay sensitivities using cultured reference strains (Manz et al. 1992, Devereux et al. 1992).

2003 ◽  
pp. 225-225
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Computer Assisted ◽  
Rrna Gene ◽  
Oligonucleotide Synthesis ◽  
Gene Sequences ◽  
Variable Regions ◽  
Species Specific ◽  
Derivatives Of ◽  
Reference Strains

scholarly journals Characterization of Nocardia asteroides Isolates from Different Ecological Habitats on the Basis of Repetitive Extragenic Palindromic-PCR Fingerprinting

2004 ◽  
Vol 70 (5) ◽  
pp. 3149-3151 ◽  
Author(s):  
Hideki Yamamura ◽  
Masayuki Hayakawa ◽  
Youji Nakagawa ◽  
Yuzuru Iimura
Keyword(s):  
Cluster Analysis ◽  
Type Strain ◽  
Dna Fingerprint ◽  
Nocardia Asteroides ◽  
Pcr Fingerprinting ◽  
And Cluster Analysis ◽  
Repetitive Extragenic Palindromic Pcr ◽  
Ecological Habitats ◽  
Repetitive Extragenic Palindromic

ABSTRACT Thirteen isolates of Nocardia asteroides from both soils and aquatic samples (lake and moat sediments, as well as scum from activated sludge), together with a type strain and two known clinical isolates of this species, were characterized by repetitive extragenic palindromic-PCR fingerprinting with the BOX-A1R primer. The resulting DNA fingerprint patterns proved to be strain specific, and cluster analysis distinguished the soil isolates, the aquatic isolates, and the known strains as being in separate groups.

scholarly journals Genetic Heterogeneity ofAlicyclobacillusStrains Revealed by RFLP Analysis ofvdcRegion andrpoBGene

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Agnieszka Dekowska ◽  
Jolanta Niezgoda ◽  
Barbara Sokołowska
Keyword(s):  
Cluster Analysis ◽  
16S Rdna ◽  
Genetic Heterogeneity ◽  
Reference Strain ◽  
Rflp Analysis ◽  
The Other ◽  
16S Rdna Gene ◽  
Restriction Patterns ◽  
Pcr Rflp ◽  
Reference Strains

PCR-RFLP targeting of the 16S rDNA andrpoBgenes, as well as thevdcregion, was applied to identify and differentiate between the spoilage and non-spoilageAlicyclobacillusspecies. Eight reference strains and 75 strains isolated from spoiled juices, juice concentrates, drinks, its intermediates, and fresh apples were subject to study. Hin6I restriction patterns of the 16S rDNA gene enabled distinguishing between all the species analyzed, while therpoBgene andvdcgene cluster analysis also revealed that there were two major types among theA. acidoterrestrisisolates, one similar to the reference strainA. acidoterrestrisDSM 2498, and the other similar to the reference strainA. acidoterrestrisATCC 49025. Heterogeneity was also observed among theA. acidocaldariusisolates. RFLP analysis of the 16S rDNA andrpoBgenes, as well asvdcregion, can be used successfully in the identification and research of intraspecies heterogeneity of theAlicyclobacillusspecies.

scholarly journals Restriction Fragment Length Polymorphisms and Sequence Analysis: an Approach for Genotyping Infectious Pancreatic Necrosis Virus Reference Strains and Other Aquabirnaviruses Isolated from Northwestern Spain

2004 ◽  
Vol 70 (2) ◽  
pp. 1059-1067 ◽  
Author(s):  
J. M. Cutrín ◽  
J. L. Barja ◽  
B. L. Nicholson ◽  
I. Bandín ◽  
S. Blake ◽  
...  
Keyword(s):  
Pancreatic Necrosis ◽  
Restriction Analysis ◽  
Infectious Pancreatic Necrosis Virus ◽  
Necrosis Virus ◽  
Genotype I ◽  
Northwestern Spain ◽  
Infectious Pancreatic Necrosis ◽  
Pancreatic Necrosis Virus ◽  
Reference Strains ◽  
Restriction Fragment Length

ABSTRACT Reference strains of infectious pancreatic necrosis virus resembling the 10 recognized serotypes and local isolates of aquabirnaviruses isolated in northwestern Spain from reservoirs (mollusks) and from asymptomatic and carrier cultured fish were genotyped by restriction fragment length polymorphism (RFLP) and nucleic acid sequence analyses. The RFLP analysis yielded seven genogroups, each of which was clearly correlated with a serotype. Sequence analysis of the three open reading frames provided quite similar results in terms of genogrouping. Based on the results of this study and in order to unify the two types of assays, we propose placing aquabirnaviruses into six genogroups, four of which can be subdivided into two genotypes based on a two-step restriction analysis. The genotyping corresponds with serotyping as follows: genogroup I includes two genotypes corresponding to serotypes A9 (genotype I.1) and A1 (genotype I.2); genogroup II corresponds to serotype A3; genogroup III includes genotypes III.1 (serotype A2) and III.2 (serotype B1); genogroups IV and V include two genotypes, each corresponding to serotypes A5, A6, A7, and A8 (genotypes IV.1, IV.2, V.1, and V.2, respectively);and genogroup VI corresponds to serotype A4. As expected, most local isolates belonged to genotype III.1 and genogroup II. However, a few local isolates corresponded to the American types of genogroup I. Finally, based on the results of this study and due to its simplicity, the two-step restriction analysis assay is proposed as a method for typing new isolates of aquabirnaviruses, and the results correspond to the results of conventional serotyping.

scholarly journals Terminal Restriction Fragment Length Polymorphism Data Analysis for Quantitative Comparison of Microbial Communities

2003 ◽  
Vol 69 (2) ◽  
pp. 926-932 ◽  
Author(s):  
Christopher B. Blackwood ◽  
Terry Marsh ◽  
Sang-Hoon Kim ◽  
Eldor A. Paul
Keyword(s):  
Cluster Analysis ◽  
Data Analysis ◽  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Redundancy Analysis ◽  
Fragment Length Polymorphism ◽  
Peak Height ◽  
Relative Peak Height ◽  
Restriction Fragment Length

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.

scholarly journals Marinobacter gudaonensis sp. nov., isolated from an oil-polluted saline soil in a Chinese oilfield

2007 ◽  
Vol 57 (2) ◽  
pp. 250-254 ◽  
Author(s):  
Jun Gu ◽  
Hua Cai ◽  
Su-Lin Yu ◽  
Ri Qu ◽  
Bin Yin ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Saline Soil ◽  
Sequence Similarity ◽  
Eastern China ◽  
Rrna Gene ◽  
Respiratory Quinone ◽  
Shengli Oilfield ◽  
Major Respiratory Quinone ◽  
Reference Strains

Two novel strains, SL014B61AT and SL014B11A, were isolated from an oil-polluted saline soil from Gudao in the coastal Shengli Oilfield, eastern China. Cells of strains SL014B61AT and SL014B11A were motile, Gram-negative and rod-shaped. Growth occurred at NaCl concentrations of between 0 and 15 % and at temperatures of between 10 and 45 °C. Strain SL014B61AT had Q9 as the major respiratory quinone and C16 : 0 (21.2 %), C18 : 1ω9c (20.3 %), C16 : 1ω7c (7.3 %) and C16 : 1ω9c (6.4 %) as predominant fatty acids. The G+C content of the DNA was 57.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SL014B61AT belonged to the genus Marinobacter in the class Gammaproteobacteria. Strain SL014B61AT showed the highest 16S rRNA gene sequence similarity with Marinobacter bryozoorum (97.9 %) and showed 97.8 % sequence similarity to Marinobacter lipolyticus. DNA–DNA relatedness to the reference strains Marinobacter bryozoorum and Marinobacter lipolyticus was 35.5 % and 33.8 %, respectively. On the basis of these data, it is proposed that strains SL014B61AT and SL014B11A represent a novel species, Marinobacter gudaonensis sp. nov. The type strain is strain SL014B61AT (=DSM 18066T=LMG 23509T=CGMCC 1.6294T).

scholarly journals Molecular Diversity Assessment of Plant Growth Promoting Rhizobacteria Using Denaturing Gradient Gel Electrophoresis (DGGE) of 16s rRNA Gene

2017 ◽  
Vol 5 (1) ◽  
pp. 72-80
Author(s):  
Umesh Prasad Shrivastava
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rdna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Uttar Pradesh ◽  
Plant Growth Promoting Rhizobacteria ◽  
Melting Behavior ◽  
Rrna Gene ◽  
Dgge Profile ◽  
Diversity Assessment

The rhizobacteria were isolated from rhizosphere of rice plant of different fields of 4 districts of Nepal and 5 districts of Bihar and Uttar Pradesh, adjoining states of India with Nepal. The DGGE analysis was performed for diversity analysis. For the construction of dendrogram, 16S rRNA gene was amplified by two different sets of primers. The DGGE ladder consisting of PCR amplified products of nine pure bacterial cultures were obtained. The first DGGE ladder was prepared by 400 bp fragment of 16S rDNA with GC clamp and the second DGGE ladder was prepared with 200 bp fragment of 16S rDNA with GC clamp. The perpendicular DGGE of these amplicons based on their melting behavior clearly demonstrated separation of different isolates. The 16S rDNA fragment amplified with primer set of V2-V3 regions with GC clamp showed separation between 40-60% of denaturant. The DGGE profile based on primer set F352T and 519r for various bacteria present in soil samples of 5 districts of India and 4 districts of Nepal revealed that the number of bands which might be specific for diazotrophic isolates varied from 2 to 11. The dendrogram constructed based on DGGE profile of various samples of 5 districts of India and 4 districts of Nepal showed that all the samples could be clustered in nine groups with 58-96% similarity to each other. Among all these 37 samples, only Var-4 and Var-5 showed 100% similarity, no other samples from any site showed 100% similarity. Int. J. Appl. Sci. Biotechnol. Vol 5(1): 72-80

scholarly journals Amplification of 16S rRNA gene sequences to differentiate two highly related bradyrhizobia species

2007 ◽  
Vol 42 (9) ◽  
pp. 1361-1364 ◽  
Author(s):  
Adriana Giongo ◽  
Adriana Ambrosini ◽  
João Ruy Jardim Freire ◽  
Maria Helena Bodanese Zanettini ◽  
Luciane Maria Pereira Passaglia
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bradyrhizobium Japonicum ◽  
Diagnostic Method ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Closely Related Species ◽  
Reference Strains ◽  
Commercial Inoculants

A 16S rRNA gene PCR-based assay was developed aiming at a fast molecular diagnostic method to differentiate the two phylogenetically closely related species Bradyrhizobium japonicum and B. elkanii, isolated from soybean nodules, in order to identify those more competitive and comprising greater nitrogen fixation ability for use in the formulation of commercial inoculants. The assay used was able to discriminate ten reference strains belonging to these two Bradyrhizobium species, as well as to efficiently identify 37 strains isolated from fields cultivated with soybean.

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