scholarly journals Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups

2007 ◽  
Vol 57 (8) ◽  
pp. 1855-1867 ◽  
Author(s):  
Wei Wei ◽  
Robert E. Davis ◽  
Ing-Ming Lee ◽  
Yan Zhao
Keyword(s):  
16S Rrna ◽  
Classification Scheme ◽  
Rflp Analysis ◽  
Plant Diseases ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Similarity Coefficients ◽  
Wide Range

Phytoplasmas are cell wall-less bacteria that cause numerous plant diseases. As no phytoplasma has been cultured in cell-free medium, phytoplasmas cannot be differentiated and classified by the traditional methods which are applied to culturable prokaryotes. Over the past decade, the establishment of a phytoplasma classification scheme based on 16S rRNA restriction fragment length polymorphism (RFLP) patterns has enabled the accurate and reliable identification and classification of a wide range of phytoplasmas. In the present study, we expanded this classification scheme through the use of computer-simulated RFLP analysis, achieving rapid differentiation and classification of phytoplasmas. Over 800 publicly available phytoplasma 16S rRNA gene sequences were aligned using the clustal_x program and the aligned 1.25 kb fragments were exported to pDRAW32 software for in silico restriction digestion and virtual gel plotting. Based on distinctive virtual RFLP patterns and calculated similarity coefficients, phytoplasma strains were classified into 28 groups. The results included the classification of hundreds of previously unclassified phytoplasmas and the delineation of 10 new phytoplasma groups representing three recently described and seven novel putative ‘Candidatus Phytoplasma’ taxa.

scholarly journals A Comparative Study: Taxonomic Grouping of Alkaline Protease Producing Bacilli

2017 ◽  
Vol 66 (1) ◽  
pp. 39-56
Author(s):  
Nilgun Tekin ◽  
Arzu Coleri Cihan ◽  
Basar Karaca ◽  
Cumhur Cokmus
Keyword(s):  
16S Rrna ◽  
Alkaline Protease ◽  
Phylogenetic Analyses ◽  
Molecular Techniques ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Protease Production ◽  
Rrna Gene ◽  
Wide Range ◽  
Its Pcr

Alkaline proteases have biotechnological importance due to their activity and stability at alkaline pH. 56 bacteria, capable of growing under alkaline conditions were isolated and their alkaline protease activities were carried out at different parameters to determine their optimum alkaline protease production conditions. Seven isolates were showed higher alkaline protease production capacity than the reference strains. The highest alkaline protease producing isolates (103125 U/g), E114 and C265, were identified as Bacillus licheniformis with 99.4% and Bacillus mojavensis 99.8% based on 16S rRNA gene sequence similarities, respectively. Interestingly, the isolates identified as Bacillus safensis were also found to be high alkaline protease producing strains. Genotypic characterizations of the isolates were also determined by using a wide range of molecular techniques (ARDRA, ITS-PCR, (GTG)5-PCR, BOX-PCR). These different techniques allowed us to differentiate the alkaliphilic isolates and the results were in concurrence with phylogenetic analyses of the 16S rRNA genes. While ITS-PCR provided the highest correlation with 16S rRNA groups, (GTG)5-PCR showed the highest differentiation at species and intra-species level. In this study, each of the biotechnologically valuable alkaline protease producing isolates was grouped into their taxonomic positions with multi-genotypic analyses.

scholarly journals Methanogenic Community Dynamics during Anaerobic Utilization of Agricultural Wastes

Acta Naturae ◽  
2012 ◽  
Vol 4 (4) ◽  
pp. 91-97 ◽  
Author(s):  
A. M. Ziganshin ◽  
E. E. Ziganshina ◽  
S. Kleinsteuber ◽  
J. Pröter ◽  
O. N. Ilinskaya
Keyword(s):  
16S Rrna ◽  
Anaerobic Degradation ◽  
Community Dynamics ◽  
Biological Diversity ◽  
Biogas Production ◽  
Rflp Analysis ◽  
Methanogenic Archaea ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene

This work is devoted to the investigation of the methanogenic archaea involved in anaerobic digestion of cattle manure and maize straw on the basis of terminal restriction fragment length polymorphism (TRFLP) analysis of archaeal 16S rRNA genes. The biological diversity and dynamics of methanogenic communities leading to anaerobic degradation of agricultural organic wastes with biogas production were evaluated in laboratory-scale digesters. T-RFLP analysis, along with the establishment of archaeal 16S rRNA gene clone libraries, showed that the methanogenic consortium consisted mainly of members of the genera Methanosarcina and Methanoculleus, with a predominance of Methanosarcina spp. throughout the experiment.

scholarly journals Structure and Topology of Microbial Communities in the Major Gut Compartments of Melolontha melolontha Larvae (Coleoptera: Scarabaeidae)

2005 ◽  
Vol 71 (8) ◽  
pp. 4556-4566 ◽  
Author(s):  
Markus Egert ◽  
Ulrich Stingl ◽  
Lars Dyhrberg Bruun ◽  
Bianca Pommerenke ◽  
Andreas Brune ◽  
...  
Keyword(s):  
16S Rrna ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Whole Cells ◽  
Melolontha Melolontha ◽  
And Topology ◽  
Radial Profiles ◽  
Reductase Gene

ABSTRACT Physicochemical gut conditions and the composition and topology of the intestinal microbiota in the major gut compartments of the root-feeding larva of the European cockchafer (Melolontha melolontha) were studied. Axial and radial profiles of pH, O2, H2, and redox potential were measured with microsensors. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes in midgut samples of individual larvae revealed a simple but variable and probably nonspecific community structure. In contrast, the T-RFLP profiles of the hindgut samples were more diverse but highly similar, especially in the wall fraction, indicating the presence of a gut-specific community involved in digestion. While high acetate concentrations in the midgut and hindgut (34 and 15 mM) corroborated the presence of microbial fermentation in both compartments, methanogenesis was confined to the hindgut. Methanobrevibacter spp. were the only methanogens detected and were restricted to this compartment. Bacterial 16S rRNA gene clone libraries of the hindgut were dominated by clones related to the Clostridiales. Clones related to the Actinobacteria, Bacillales, Lactobacillales, and γ-Proteobacteria were restricted to the lumen, whereas clones related to the β- and δ-Proteobacteria were found only on the hindgut wall. Results of PCR-based analyses and fluorescence in situ hybridization of whole cells with group-specific oligonucleotide probes documented that Desulfovibrio-related bacteria comprise 10 to 15% of the bacterial community at the hindgut wall. The restriction of the sulfate-reducer-specific adenosine-5′-phosphosulfate reductase gene apsA to DNA extracts of the hindgut wall in larvae from four other populations in Europe suggested that sulfate reducers generally colonize this habitat.

scholarly journals Identification and classification of the genus Bacteroides by multilocus sequence analysis

Microbiology ◽  
2011 ◽  
Vol 157 (12) ◽  
pp. 3388-3397 ◽  
Author(s):  
Mitsuo Sakamoto ◽  
Moriya Ohkuma
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Sequence Similarity ◽  
Single Gene ◽  
Housekeeping Genes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Multilocus Sequence Analysis ◽  
Rrna Gene

Multilocus sequence analysis (MLSA) was performed on representative species of the genus Bacteroides. Internal fragments of the genes selected, dnaJ, gyrB, hsp60, recA, rpoB and 16S rRNA, were amplified by direct PCR and then sequenced from 38 Bacteroides strains representing 35 species. Neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) phylogenies of the individual genes were compared. The data confirm that the potential for discrimination of Bacteroides species is greater using MLSA of housekeeping genes than 16S rRNA genes. Among the housekeeping genes analysed, gyrB was the most informative, followed by dnaJ. Analyses of concatenated sequences (4816 bp) of all six genes revealed robust phylogenetic relationships among different Bacteroides species when compared with the single-gene trees. The NJ, ML and MP trees were very similar, and almost fully resolved relationships of Bacteroides species were obtained, to our knowledge for the first time. In addition, analysis of a concatenation (2457 bp) of the dnaJ, gyrB and hsp60 genes produced essentially the same result. Ten distinct clades were recognized using the SplitsTree4 program. For the genus Bacteroides, we can define species as a group of strains that share at least 97.5 % gene sequence similarity based on the fragments of five protein-coding housekeeping genes and the 16S rRNA gene. This study demonstrates that MLSA of housekeeping genes is a valuable alternative technique for the identification and classification of species of the genus Bacteroides.

scholarly journals Genotypic Diversity of Haemophilus parasuis Field Strains

2006 ◽  
Vol 72 (6) ◽  
pp. 3984-3992 ◽  
Author(s):  
A. Olvera ◽  
M. Calsamiglia ◽  
V. Aragon
Keyword(s):  
16S Rrna ◽  
Genotypic Diversity ◽  
Epidemiological Studies ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Haemophilus Parasuis ◽  
Virulent Strains ◽  
Reliable Marker

ABSTRACT Haemophilus parasuis is the cause of Gl�sser's disease and other clinical disorders in pigs. It can also be isolated from the upper respiratory tracts of healthy pigs, and isolates can have significant differences in virulence. In this work, a partial sequence from the 60-kDa heat shock protein (Hsp60) gene was assessed as an epidemiological marker. We analyzed partial sequences of hsp60 and 16S rRNA genes from 103 strains of H. parasuis and other related species to obtain a better classification of the strains and examine the correlation with virulence. The results were compared with those obtained by enterobacterial repetitive intergenic consensus PCR. Our results showed that hsp60 is a reliable marker for epidemiological studies of H. parasuis and that the analysis of its sequence is a better approach than fingerprinting methods. Furthermore, the analysis of the hsp60 and 16S rRNA gene sequences revealed the presence of a separate lineage of virulent strains and indicated the occurrence of lateral gene transfer among H. parasuis and Actinobacillus strains.

scholarly journals Detection and Identification of Banana-associated Phytoplasma Using Nested-PCR Method

2019 ◽  
Vol 23 (1) ◽  
pp. 148
Author(s):  
Saurma Mona Astrid Sibarani ◽  
Tri Joko ◽  
Siti Subandiyah
Keyword(s):  
16S Rrna ◽  
Nested Pcr ◽  
Plant Diseases ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Aster Yellows ◽  
Detection And Identification ◽  
Pcr Method ◽  
The 16S Rrna Gene

Phytoplasma is known to be associated with plant diseases in about 300 plant species from various families. Information on the presence of phytoplasma in bananas as one of the pathogens that can cause disease in bananas in Indonesia has never been reported. This research was conducted with the aim to detect the presence of banana phytoplasma by the nested-PCR method and to identify phytoplasma obtained based on the sequence analysis of the 16S rRNA gene. Standard PCR was carried out using P1/P7 primary pairs, followed by nested-PCR using a pair of R16F2n/R16R2m23SR primers separately that could amplify the target 16S rRNA genes in a row at 1600 bp. BLAST analysis shows that the results of phylogenetic analysis of banana phytoplasmic nucleotide cv. manggala from Tasikmalaya and cv. Raja nangka from Banjar has a genetic relationship that is closer to lethal wilt oil palm Phytoplasma (Candidatus Phytoplasma asteris). This phytoplasma belongs to the 16SrI-B group (aster yellows).

Quantitatively evaluating mistaken clone assignments by RFLP analysis of 16S rRNA genes: a case study

2008 ◽  
Vol 54 (6) ◽  
pp. 479-482 ◽  
Author(s):  
Han-Bo Zhang ◽  
Chan-Wen Xu ◽  
Miao-Miao Wang ◽  
Tao Li ◽  
Zhi-Wei Zhao
Keyword(s):  
16S Rrna ◽  
Sequence Similarity ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Taxonomic Rank ◽  
Operational Taxonomic Units

We quantitatively evaluated the errors of clone assignment based on the restriction fragment length polymorphism (RFLP) pattern of 16S rRNA genes. Eighty clones were randomly selected from a 16S rRNA gene library and were categorized into 35 operational taxonomic units (OTU) based on their indistinguishable enzyme restriction patterns of 3 tetrameric restriction enzymes RsaI, BsuRI, and HinfI. All of these clones were then sequenced and were reassigned into 36–53 OTUs using the DOTUR program when sequence similarities of 95%–100% were used. The number of the identically assigned clones ranged from 53 to 61 and the percentage varied from 66.3% to 76.3%. The Shannon–Weaver index for the bacterial community observed by RFLP analysis was 2.75, equal to that estimated by DOTUR at a 97% sequence similarity. Compared with clones assigned with the DOTUR program at a 97% sequence similarity, only 61 clones (76.3%) were correctly assigned by RFLP analysis. Six clones (7.5%) were assigned mistakenly at the phylum level, and the positions of 13 clones (16.2%) were phylogenetically different at a lower taxonomic rank.

scholarly journals Gold Nanoparticle Clusters in Quasinematic Layers of Liquid-Crystalline Dispersion Particles of Double-Stranded Nucleic Acids

Acta Naturae ◽  
2012 ◽  
Vol 4 (4) ◽  
pp. 78-90 ◽  
Author(s):  
Yu. M. Yevdokimov ◽  
V. I. Salyanov ◽  
E. I. Katz ◽  
S. G. Skuridin
Keyword(s):  
16S Rrna ◽  
Liquid Crystalline ◽  
Biological Diversity ◽  
Biogas Production ◽  
Rflp Analysis ◽  
Methanogenic Archaea ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Liquid Crystalline Dispersion

This work is devoted to the investigation of the methanogenic archaea involved in anaerobic digestion of cattle manure and maize straw on the basis of terminal restriction fragment length polymorphism (TRFLP) analysis of archaeal 16S rRNA genes. The biological diversity and dynamics of methanogenic communities leading to anaerobic degradation of agricultural organic wastes with biogas production were evaluated in laboratory-scale digesters. T-RFLP analysis, along with the establishment of archaeal 16S rRNA gene clone libraries, showed that the methanogenic consortium consisted mainly of members of the genera Methanosarcina and Methanoculleus, with a predominance of Methanosarcina spp. throughout the experiment.

Acidianus Brierleyi is the Dominant Thermoacidophile in a Bioleaching Community Processing Chalcopyrite Containing Concentrates at 70°C

2009 ◽  
Vol 71-73 ◽  
pp. 67-70 ◽  
Author(s):  
Inez J.T. Dinkla ◽  
Mariekie Gericke ◽  
B.K. Geurkink ◽  
Kevin B. Hallberg
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Clone Library ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Qualitative Assessment ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Multi Stage ◽  
Acidianus Brierleyi

Bioleaching test work was performed in continuously operated multi-stage reactor systems at 70°C using a thermophilic culture treating an Aguablanca Ni-Cu concentrate from Spain and a blend of Cu concentrates from Bor, Serbia. The copper in both these concentrates occurs as chalcopyrite and therefore the use of thermophiles was applied, which resulted in copper recoveries of over 95%. Qualitative assessment of the microbial community in the bioreactors was performed by terminal restriction enzyme fragment length polymorphism (T-RFLP) and clone library analysis of the 16S rRNA genes amplified by PCR. T-RFLP analysis revealed that only archaea were present, and that the communities in both the Aguablanca and Bor systems consisted of two different microorganisms. A 16S rRNA gene clone library using DNA from the Aguablanca system was constructed and screened. Again, two archaea were detected in similar relative abundance in the population as found by T-RFLP analyses. The sequences of these two cloned genes revealed that the dominant archaeon (up to 98% of the total archaea detected) was Acidianus brierleyi, and the other was Metallosphaera sedula. Quantitative assessment of the microbial community was performed by Q-PCR and confirmed the dominance of archaea in the system with Acidianus being the dominant strain (98-99% of the total population) and a minor part of the population (1-2%) consisted of Metallosphaera. Additionally, very small amounts of Sulfolobus spp. were detected. This study, along with other recent studies on the diversity of thermoacidophiles involved in the solubilization of copper from chalcopyrite concentrates, revealed that a wider variety of thermoacidophiles are involved in bioprocessing of metal sulfides, and showed that A. brierleyi should be considered an important biomining acidophile.

Molecular Assessment of Microbial Diversity and Community Structure at Uranium Mines of Jaduguda, India

2007 ◽  
Vol 20-21 ◽  
pp. 413-416 ◽  
Author(s):  
Pinaki Sar ◽  
Paltu K. Dhal ◽  
Ekramul Islam ◽  
Sufia K. Kazy
Keyword(s):  
16S Rrna ◽  
Microbial Diversity ◽  
Molecular Level ◽  
Restriction Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Uranium Mine ◽  
Rrna Gene ◽  
Wide Range ◽  
Mine Sites

Microbial diversity associated with uranium mine areas of Jaduguda, India has been investigated using a culture independent molecular approach. Soil samples collected from existing and proposed mine sites were analyzed for physicochemical parameters. Community DNA was extracted from five samples. Small subunit rRNA gene (16S rRNA) was PCR amplified using bacterial primers. The diversity of the total bacterial community was described at molecular level by amplified ribosomal DNA restriction analysis (ARDRA). Dominant bacterial groups (represents by OTUs) selected by ARDRA were identified by sequencing the 16S rRNA genes. From the bacterial rDNA clone library around 230 clones were used for further analysis. The unique OTUs and number of clones representing such OTUs were determined. Dominant OTUs were sequenced and identified. These phylotypes spanned a wide range within the bacterial domain occupying Proteobacteria, Acidobacteria, Bacteroidetes, Firmicutes, Cyanobacteria as major phyla. About 46 % of clones sequenced from various sites were identified as Proteobacteria. The present findings on microbial diversity at the molecular level are the first of its kind for uranium mine sites of India. Around 20 % of the clone sequences showed little affiliation with known taxa and probably represent new organisms adapted to this habitat.

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