scholarly journals First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7452
Author(s):  
Cleiziane Bispo da Silva ◽  
Hellen Ribeiro Martins dos Santos ◽  
Phellippe Arthur Santos Marbach ◽  
Jorge Teodoro de Souza ◽  
Valter Cruz-Magalhães ◽  
...  
Keyword(s):  
16S Rdna ◽  
Endophytic Bacteria ◽  
Restriction Analysis ◽  
Natural Populations ◽  
Amplicon Sequencing ◽  
Limiting Factor ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Restriction Profiles ◽  
Chi Square Analysis

Background Intragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner. Methods Total genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5–V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the corresponding AluI-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the ‘number of AluI bands’ and ‘type of eletropherogram’. Results Two types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with the AluI-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity. Conclusion The approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.

scholarly journals Genetic and Biochemical Diversity among Isolates ofPaenibacillus alvei Cultured from Australian Honeybee (Apis mellifera) Colonies

2000 ◽  
Vol 66 (3) ◽  
pp. 1098-1106 ◽  
Author(s):  
Steven P. Djordjevic ◽  
Wendy A. Forbes ◽  
Lisa A. Smith ◽  
Michael A. Hornitzky
Keyword(s):  
Apis Mellifera ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Whole Cell ◽  
Restriction Profiles ◽  
Dna Profiles

ABSTRACT Twenty-five unique CfoI-generated whole-cell DNA profiles were identified in a study of 30 Paenibacillus alvei isolates cultured from honey and diseased larvae collected from honeybee (Apis mellifera) colonies in geographically diverse areas in Australia. The fingerprint patterns were highly variable and readily discernible from one another, which highlighted the potential of this method for tracing the movement of isolates in epidemiological studies. 16S rRNA gene fragments (length, 1,416 bp) for all 30 isolates were enzymatically amplified by PCR and subjected to restriction analysis with DraI, HinfI,CfoI, AluI, FokI, andRsaI. With each enzyme the restriction profiles of the 16S rRNA genes from all 30 isolates were identical (one restriction fragment length polymorphism [RFLP] was observed in theHinfI profile of the 16S rRNA gene from isolate 17), which confirmed that the isolates belonged to the same species. The restriction profiles generated by using DraI,FokI, and HinfI differentiated P. alvei from the phylogenetically closely related speciesPaenibacillus macerans and Paenibacillus macquariensis. Alveolysin gene fragments (length, 1,555 bp) were enzymatically amplified from some of the P. alvei isolates (19 of 30 isolates), and RFLP were detected by using the enzymesCfoI, Sau3AI, and RsaI. Extrachromosomal DNA ranging in size from 1 to 10 kb was detected in 17 of 30 (57%) P. alvei whole-cell DNA profiles. Extensive biochemical heterogeneity was observed among the 28 P. alvei isolates examined with the API 50CHB system. All of these isolates were catalase, oxidase, and Voges-Proskauer positive and nitrate negative, and all produced acid when glycerol, esculin, and maltose were added. The isolates produced variable results for 16 of the 49 biochemical tests; negative reactions were recorded in the remaining 30 assays. The genetic and biochemical heterogeneity inP. alvei isolates may be a reflection of adaptation to the special habitats in which they originated.

Endophytic bacterial communities and spatiotemporal variations in cotton roots in Xinjiang, China

2020 ◽  
Author(s):  
Yingwu Shi ◽  
Hongmei Yang ◽  
Min Chu ◽  
Xinxiang Niu ◽  
Xiangdong Huo ◽  
...  
Keyword(s):  
Community Structure ◽  
Endophytic Bacteria ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Operational Taxonomic Units ◽  
Cotton Sample ◽  
Endophytic Bacterial Community ◽  
V3 Region ◽  
Illumina Amplicon Sequencing

Endophytic bacteria may be important for plant health and other ecologically relevant functions of cotton. However, the endophytic bacterial community structure and diversity in cotton is still poorly characterized. We investigated the community structure of endophytic bacteria in cotton roots growing in Xinjiang, China, using the Illumina amplicon sequencing. A total of 60.84 M effective sequences of 16S rRNA gene V3 region were obtained from cotton samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in cotton, that is, 81-338 OTUs in a cotton sample, at 3% cutoff level and sequencing depth of 50000 sequences. We identified 23 classes from the resulting 2,723,384 sequences. Gammaproteobacteria were the dominant class in all cottons, followed by Alphaproteobacteria, Actinobacteria and Bacilli. A marked difference in the diversity of endophytic bacteria in cotton for different growth periods was evident. The greatest number of OTUs was detected during seedling (654 OTUs) and budding (381 OTUs). Endophytic bacteria diversity was reduced during flowering (350 OTUs) and boll-opening (351 OTUs). 217 OTUs were common to all four periods. There were more tags of Pantoea in Shihezi than other locations. While there were more tags of Erwinia in Hami than other locations. The dynamics of endophytic bacteria communities were influenced by plant growth stage. These results show the complexity of the bacterial populations present in inner tissues of cotton.

scholarly journals Proof that Burkholderia Strains Form Effective Symbioses with Legumes: a Study of Novel Mimosa-Nodulating Strains from South America

2005 ◽  
Vol 71 (11) ◽  
pp. 7461-7471 ◽  
Author(s):  
Wen-Ming Chen ◽  
Sergio M. de Faria ◽  
Rosângela Straliotto ◽  
Rosa M. Pitard ◽  
Jean L. Simões-Araùjo ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Restriction Analysis ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Consensus Sequences ◽  
Mimosa Pigra ◽  
Green Fluorescent ◽  
Gene Restriction ◽  
Single Cluster ◽  
The 16S Rrna Gene

ABSTRACT Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other β-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known β-rhizobia, Burkholderia caribensis, Burkholderia phymatum, and Burkholderia tuberum. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus Burkholderia; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosis-related genes nodA, the NodD-dependent regulatory consensus sequences (nod box), and nifH. The nodA and nifH sequences were very close to each other and to those of B. phymatum STM815, B. caribensis TJ182, and Cupriavidus taiwanensis LMG19424 but were relatively distant from those of B. tuberum STM678. In addition to nodulating their original hosts, all five strains could also nodulate other Mimosa spp., and all produced nodules on Mimosa pudica that had nitrogenase (acetylene reduction) activities and structures typical of effective N2-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N2-fixing nodules on their original hosts, Mimosa bimucronata (Br3461) and Mimosa pigra (MAP3-5), and hence this confirms strongly that Burkholderia strains can form effective symbioses with legumes.

scholarly journals Two Groups of Phytoplasmas from Japan Distinguished on the Basis of Amplification and Restriction Analysis of 16S rDNA

Plant Disease ◽  
1997 ◽  
Vol 81 (3) ◽  
pp. 301-305 ◽  
Author(s):  
Seiichi Okuda ◽  
James P. Prince ◽  
Robert E. Davis ◽  
Ellen L. Dally ◽  
Ing-Ming Lee ◽  
...  
Keyword(s):  
16S Rdna ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Rrna Gene ◽  
Gene Group ◽  
Beet Leafhopper ◽  
Pcr Products ◽  
Elm Yellows

Phytoplasmas (mycoplasmalike organisms, MLOs) associated with mitsuba (Japanese hone-wort) witches'-broom (JHW), garland chrysanthemum witches'-broom (GCW), eggplant dwarf (ED), tomato yellows (TY), marguerite yellows (MY), gentian witches'-broom (GW), and tsu-wabuki witches'-broom (TW) in Japan were investigated by polymerase chain reaction (PCR) amplification of DNA and restriction enzyme analysis of PCR products. The phytoplasmas could be separated into two groups, one containing strains JHW, GCW, ED, TY, and MY, and the other containing strains GW and TW, corresponding to two groups previously recognized on the basis of transmission by Macrosteles striifrons and Scleroracus flavopictus, respectively. The strains transmitted by M. striifrons were classified in 16S rRNA gene group 16SrI, which contains aster yellows and related phytoplasma strains. Strains GW and TW were classified in group 16SrIII, which contains phytoplasmas associated with peach X-disease, clover yellow edge, and related phytoplasmas. Digestion of amplified 16S rDNA with HpaII indicated that strains GW and TW were affiliated with subgroup 16SrIII-B, which contains clover yellow edge phytoplasma. All seven strains were distinguished from other phytoplasmas, including those associated with clover proliferation, ash yellows, elm yellows, and beet leafhopper-transmitted virescence in North America, and Malaysian periwinkle yellows and sweet potato witches'-broom in Asia.

16S rRNA gene amplicon sequencing reveals dominance of Actinobacteria in Rhodnius pallescens compared to Triatoma maculata midgut microbiota in natural populations of vector insects from Colombia

Acta Tropica ◽  
2018 ◽  
Vol 178 ◽  
pp. 327-332 ◽  
Author(s):  
Luisa M. Montoya-Porras ◽  
Triana-Chavez Omar ◽  
Juan F. Alzate ◽  
Claudia X. Moreno-Herrera ◽  
Gloria E. Cadavid-Restrepo
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Natural Populations ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Rhodnius Pallescens

Bacterial diversity associated with subalpine fir (Abies lasiocarpa) ectomycorrhizae following wildfire and salvage-logging in central British Columbia

2002 ◽  
Vol 48 (7) ◽  
pp. 611-625 ◽  
Author(s):  
Madhukar B Khetmalas ◽  
Keith N Egger ◽  
Hugues B Massicotte ◽  
Linda E Tackaberry ◽  
M Jill Clapperton
Keyword(s):  
Bacterial Diversity ◽  
16S Rdna ◽  
Bacterial Communities ◽  
Restriction Analysis ◽  
Rrna Gene ◽  
Abies Lasiocarpa ◽  
Gram Positive ◽  
Salvage Logging ◽  
Gram Positive Bacteria ◽  
Significant Difference

To assess the effect of fire and salvage logging on the diversity of mycorrhizal–bacterial communities, bacteria associated with Cenococcum, Thelephora, Tomentella, Russulaceae, and E-strain ectomycorrhizae (ECM) of Abies lasiocarpa seedlings were characterized using two approaches. First, bacteria were isolated and characterized by Biolog©, gas chromatography fatty acid methyl ester (GC-FAME), and amplified 16S rDNA restriction analysis (ARDRA). The bacterial communities retrieved from ECM from both sites were dominated by Proteobacteria (groups gamma and beta). Pseudomonas was the most common genus isolated, followed by Variovorax, Burkholderia, and Xanthomonas. Gram-positive isolates (mostly high-G+C Gram-positive bacteria) were more frequently retrieved on the burned-salvaged site, many commonly associated with the two ascomycete ECM, Cenococcum and E-strain. Pseudomonas species were retrieved more frequently from Thelephora. Although actinomycetes were isolated from all sites, almost no actinomycetes or other Gram-positive bacteria were isolated from either Thelephora or Tomentella. Second, amplified 16S rRNA gene sequences were amplified directly from root tips and then cloned into the plasmid vector pAMP1, followed by restriction analysis. This technique distinguished more genotypes than isolates retrieved by culturing methods, but generally, results were similar in that the largest proportion of the bacteria were putatively Gram-negative; putative Gram-positive bacteria were fewer and most were from the burned–salvaged site. Direct cloning resulted in many patterns that did not match any identified isolates, suggesting that a large proportion of clones were unique or not culturable by the methods used. Analysis for both protocols showed no significant difference in bacterial diversity between the burned–salvaged and unburned sites. Key words: rhizosphere bacteria, ARDRA, 16S rDNA, Biolog©, GC-FAME.

scholarly journals ddPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Isabel Abellan-Schneyder ◽  
Andrea Janina Schusser ◽  
Klaus Neuhaus
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
Bacterial Biomass ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Limiting Factor ◽  
Rrna Gene ◽  
Rrna Gene Sequencing ◽  
Dna Amounts

Abstract Background One limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols. Results Using a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts. Conclusion Our improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.

scholarly journals Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases

2000 ◽  
Vol 38 (3) ◽  
pp. 1094-1104 ◽  
Author(s):  
Andreas Roth ◽  
Udo Reischl ◽  
Anna Streubel ◽  
Ludmila Naumann ◽  
Reiner M. Kroppenstedt ◽  
...  
Keyword(s):  
16S Rdna ◽  
Restriction Analysis ◽  
Diagnostic Algorithm ◽  
Species Level ◽  
23S Rrna ◽  
Rrna Gene ◽  
Correct Identification ◽  
Mycobacterium Fortuitum ◽  
Mycobacterium Chelonae ◽  
Rdna Sequencing

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence,HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found inMycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium aviumcomplex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, orAvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus,M. chelonae, Mycobacterium farcinogenes,Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.

scholarly journals Unlocking a high bacterial diversity in the coralloid root microbiome from the cycad genusDioon

2018 ◽  
Author(s):  
Pablo de Jesús Suárez-Moo ◽  
Andrew P. Vovides ◽  
M. Patrick Griffith ◽  
Francisco Barona-Gómez ◽  
Angélica Cibrián-Jaramillo
Keyword(s):  
Bacterial Diversity ◽  
Natural Populations ◽  
Amplicon Sequencing ◽  
Botanical Garden ◽  
Taxonomic Composition ◽  
Rrna Gene ◽  
Biological Interactions ◽  
Plant Growth Promoting ◽  
Coralloid Roots ◽  
Root Microbiome

AbstractCycads are among the few plants that have developed specialized roots to host nitrogen-fixing bacteria. We describe the bacterial diversity of the coralloid roots from sevenDioonspecies and their surrounding rhizosphere and soil. Using 16S rRNA gene amplicon sequencing, we found that all coralloid roots are inhabited by a broad diversity of bacterial groups, including cyanobacteria and Rhizobiales among the most abundant groups. The diversity and composition of the endophytes are similar in the six Mexican species ofDioonthat we evaluated, suggesting a recent divergence ofDioonpopulations and/or similar plant-driven restrictions in maintaining the coralloid root microbiome. Botanical garden samples and natural populations have a similar taxonomic composition, although the beta diversity differed between these populations. The rhizosphere surrounding the coralloid root serves as a reservoir and source of mostly diazotroph and plant growth-promoting groups that colonize the coralloid endosphere. In the case of cyanobacteria, the endosphere is enriched withNostocspp andCalothrixspp that are closely related to previously reported symbiont genera in cycads and other early divergent plants. The data reported here provide an in-depth taxonomic characterization of the bacterial community associated with coralloid root microbiome. The functional aspects of the endophytes, their biological interactions, and their evolutionary history are the next research step in this recently discovered diversity within the cycad coralloid root microbiome.

scholarly journals Iron Bacterial Phylogeny and their Execution Towards Iron Availability in Equatorial Indian Ocean and Coastal Arabian Sea

2013 ◽  
Vol 62 (4) ◽  
pp. 391-400 ◽  
Author(s):  
RAJU RAJASABAPATHY ◽  
CHELLANDI MOHANDASS ◽  
AJAKKALAMOOLE SRINIVAS VIJAYARAJ ◽  
VARSHA VINAYAK MADIVAL ◽  
RAM MURTI MEENA
Keyword(s):  
Indian Ocean ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Arabian Sea ◽  
Sequence Data ◽  
Restriction Analysis ◽  
Equatorial Indian Ocean ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Bacterial Strains

Based on distinct colony morphology, color, size, shape and certain other traits, 92 bacterial isolates were investigated to understand their managerial ability on iron from the Arabian Sea and Equatorial Indian Ocean samples. The ARDRA (amplified rDNA restriction analysis) applied to eliminate the duplication of the bacterial strains, resulted 39 different banding patterns. The 16S rRNA gene sequencing data indicate the dominancy of three phylogenetic groups, alpha-Proteobacteria (10.25%), gamma-Proteobacteria (35.89%) and Bacilli (53.84%) in these waters. Marinobacter and Bacillus were the only common genera from both of the regions. Pseudoalteromonas, Halomonas, Rheinheimera, Staphylococcus and Idiomarina were some of the other genera obtained from the Arabian Sea. Erythrobacter, Roseovarius, Sagittula and Nitratireductor were found mostly in Equatorial Indian Ocean. In addition, 16S rRNA gene sequence data of some of our iron bacterial strains belong to novel species and one isolate ASS2A could form a new genus. Close to 23% of the isolates were able to produce high affinity sets of ligands like siderophores to mediate iron transport into the cell. The current study indicated that the Equatorial Indian Ocean species were well adapted to oxidize iron as an electron acceptor and the Arabian Sea species preferably go through siderophore production.

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