Species boundaries and molecular markers for the classification of 16SrI phytoplasmas inferred by genome analysis

AbstractPhytoplasmas are plant-pathogenic bacteria that impact agriculture worldwide. The commonly adopted classification system for phytoplasmas is based on the restriction fragment length polymorphism (RFLP) analysis of their 16S rRNA genes. With the increased availability of phytoplasma genome sequences, the classification system can now be refined. This work examined 11 strains in the 16SrI group within the genus ‘Candidatus Phytoplasma’ and investigated the possible species boundaries. We confirmed that the RFLP classification method is problematic due to intragenomic variation of the 16S rRNA genes and uneven weighing of different nucleotide positions. Importantly, our results based on the molecular phylogeny, differentiations in chromosomal segments and gene content, and divergence in homologous sequences, all supported that these strains may be classified into multiple operational taxonomic units (OTUs) equivalent to species. Strains assigned to the same OTU share >97% genome-wide average nucleotide identity (ANI) and >78% of their protein-coding genes. In comparison, strains assigned to different OTUs share <94% ANI and <75% of their genes. Reduction in homologous recombination between OTUs is one possible explanation for the discontinuity in genome similarities, and these findings supported the proposal that 95% ANI could serve as a cutoff for distinguishing species in bacteria. Additionally, critical examination of these results and the raw sequencing reads led to the identification of one genome that was presumably mis-assembled by combining two sequencing libraries built from phytoplasmas belonging to different OTUs. This finding provided a cautionary tale for working on uncultivated bacteria. Based on the new understanding of phytoplasma divergence and the current genome availability, we developed five molecular markers that could be used for multilocus sequence analysis (MLSA). By selecting markers that are short yet highly informative, and are distributed evenly across the chromosome, these markers provided a cost-effective system that is robust against recombination. Finally, examination of the effector gene distribution further confirmed the rapid gains and losses of these genes, as well as the involvement of potential mobile units (PMUs) in their molecular evolution. Future improvements on the taxon sampling of phytoplasma genomes will allow further expansions of similar analysis, and thus contribute to phytoplasma taxonomy and diagnostics.

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672 IDENTIFICATION OF POLYBACTERIAL 16S RRNA GENES FROM ASCITIC FLUIDS WITH T-RFLP ANALYSIS AND DIRECT SEQUENCING

Journal of Hepatology ◽

10.1016/s0168-8278(12)60685-0 ◽

2012 ◽

Vol 56 ◽

pp. S266

Author(s):

S. Krohn ◽

J. Hartmann ◽

A. Brodzinski ◽

A. Chatzinotas ◽

S. Bohm ◽

...

Keyword(s):

16S Rrna ◽

Direct Sequencing ◽

Rflp Analysis ◽

16S Rrna Genes ◽

Rrna Genes

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Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1787-1794.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1787-1794 ◽

Cited By ~ 113

Author(s):

Vanessa M. Conn ◽

Christopher M. M. Franco

Keyword(s):

Restriction Fragment Length Polymorphism ◽

16S Rrna ◽

Restriction Fragment ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Rflp Analysis ◽

16S Rrna Genes ◽

Rrna Genes ◽

Restriction Fragment Length

ABSTRACT The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.

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Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2555-2562.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2555-2562 ◽

Cited By ~ 213

Author(s):

Markus Egert ◽

Michael W. Friedrich

Keyword(s):

Restriction Fragment Length Polymorphism ◽

16S Rrna ◽

Length Polymorphism ◽

Fragment Length Polymorphism ◽

Rflp Analysis ◽

Restriction Enzymes ◽

16S Rrna Genes ◽

Rrna Genes ◽

Restriction Fragments ◽

Restriction Fragment Length

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

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Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1893-1901.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1893-1901 ◽

Cited By ~ 195

Author(s):

Gesche Braker ◽

Héctor L. Ayala-del-Rı́o ◽

Allan H. Devol ◽

Andreas Fesefeldt ◽

James M. Tiedje

Keyword(s):

Community Structure ◽

Restriction Fragment Length Polymorphism ◽

16S Rrna ◽

Fragment Length Polymorphism ◽

Puget Sound ◽

Rflp Analysis ◽

16S Rrna Genes ◽

Rrna Genes ◽

Restriction Fragment Length ◽

Redox Gradients

ABSTRACT Steep vertical gradients of oxidants (O2 and NO3 −) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers,Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs ofnirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis ofnirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity ofArchaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.

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Coyotes (Canis latrans) as the Reservoir for a Human Pathogenic Bartonella sp.: Molecular Epidemiology ofBartonella vinsonii subsp. berkhoffii Infection in Coyotes from Central Coastal California

Journal of Clinical Microbiology ◽

10.1128/jcm.38.11.4193-4200.2000 ◽

2000 ◽

Vol 38 (11) ◽

pp. 4193-4200 ◽

Cited By ~ 62

Author(s):

Chao-Chin Chang ◽

Rickie W. Kasten ◽

Bruno B. Chomel ◽

Darren C. Simpson ◽

Carrie M. Hew ◽

...

Keyword(s):

16S Rrna ◽

Canis Latrans ◽

Bacterial Genome ◽

Clinical Signs ◽

Rflp Analysis ◽

Domestic Dog ◽

16S Rrna Genes ◽

Rrna Genes ◽

Intergenic Spacer Region ◽

Pcr Rflp

Bartonella vinsonii subsp. berkhoffii was originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible with Bartonella infection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes (Canis latrans) from central coastal California, 31 animals (28%) were found to be bacteremic with B. vinsonii subsp.berkhoffii and 83 animals (76%) had B. vinsonii subsp. berkhoffii antibodies. These findings suggest these animals could be the wildlife reservoir of B. vinsonii subsp. berkhoffii. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA and 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those of B. vinsonii subsp.berkhoffii. Partial sequencing of the gltA and 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate and B. vinsonii subsp.berkhoffii (ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31Bartonella bacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case (B. vinsonii subsp. berkhoffii ATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) with SmaI restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, threeBartonella bacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission of B. vinsonii subsp. berkhoffii, especially to identify potential vectors, and to determine how humans become infected.

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Methanogenic Community Dynamics during Anaerobic Utilization of Agricultural Wastes

Acta Naturae ◽

10.32607/20758251-2012-4-4-91-97 ◽

2012 ◽

Vol 4 (4) ◽

pp. 91-97 ◽

Cited By ~ 10

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4556-4566.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4556-4566 ◽

Cited By ~ 71

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.

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

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.65000-0 ◽

2007 ◽

Vol 57 (8) ◽

pp. 1855-1867 ◽

Cited By ~ 233

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.

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Increase in Terminal Restriction Fragments of Bacteroidetes-Derived 16S rRNA Genes after Administration of Short-Chain Fructooligosaccharides

Applied and Environmental Microbiology ◽

10.1128/aem.00477-06 ◽

2006 ◽

Vol 72 (9) ◽

pp. 6271-6276 ◽

Cited By ~ 19

Author(s):

Yusuke Nakanishi ◽

Koichiro Murashima ◽

Hiroki Ohara ◽

Takahisa Suzuki ◽

Hidenori Hayashi ◽

...

Keyword(s):

16S Rrna ◽

Intestinal Microbiota ◽

Fragment Length Polymorphism ◽

Rflp Analysis ◽

Intestinal Bacteria ◽

Short Chain ◽

16S Rrna Genes ◽

Rrna Genes ◽

Restriction Fragments ◽

Culture Independent

ABSTRACT It is well known that short chain fructooligosaccharides (scFOS) modify intestinal microbiota in animals as well as in humans. Since most murine intestinal bacteria are still uncultured, it is difficult for a culturing method to detect changes in intestinal microbiota after scFOS administration in a mouse model. In this study, we sought markers of positive change in murine intestinal microbiota after scFOS administration using terminal restriction fragment length polymorphism (T-RFLP) analysis, which is a culture-independent method. The T-RFLP profiles showed that six terminal restriction fragments (T-RFs) were significantly increased after scFOS administration. Phylogenetic analysis of the 16S rRNA partial gene sequences of murine fecal bacteria suggested that four of six T-RFs that increased after scFOS administration were derived from the 16S rRNA genes of the class Bacteroidetes. Preliminary quantification of Bacteroidetes by real-time PCR suggests that the 16S rRNA genes derived from Bacteroidetes were increased by scFOS administration. Therefore, the T-RFs derived from Bacteroidetes are good markers of change of murine intestinal microbiota after scFOS administration.

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