To see the other types of publications on this topic, follow the link: Sequencing of 16S RNAr fragments.
Journal articles on the topic 'Sequencing of 16S RNAr fragments'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Sequencing of 16S RNAr fragments.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Armani, A., L. Tinacci, X. Xiong, L. Castigliego, D. Gianfaldoni, and A. Guidi. "Fish species identification in canned pet food by BLAST and Forensically Informative Nucleotide Sequencing (FINS) analysis of short fragments of the mitochondrial 16s ribosomal RNA gene (16S rRNA)." Food Control 50 (April 2015): 821–30. http://dx.doi.org/10.1016/j.foodcont.2014.10.018.
Full text
2
Zduńczyk, Zenon. "Functioning of the Intestinal Ecosystem: From New Technologies in Microbial Research to Practical Poultry Feeding – A Review." Annals of Animal Science 19, no. 2 (2019): 239–56. http://dx.doi.org/10.2478/aoas-2019-0007.
Full textAbstract:
AbstractUnlike classical microbiology which focuses on bacteria capable of growing in vitro, metagenomics is a study of genetic information originating from microflora which aims to characterise the microbiome, namely the common genome of bacteria, archaea, fungi, protozoa and viruses living in the host. Metagenomics relies on next-generation sequencing (NGS), a large-scale sequencing technique which allows millions of sequential reactions to be carried out in parallel to decode entire communities of microorganisms. Metagenomic analyses support taxonomic analyses (involving gene fragments encoding ribosomal RNAs 5S and 16S in bacteria) or functional analyses for identifying genes encoding proteins that participate in the regulation of metabolic pathways in the body. New metagenomics technologies expand our knowledge of the phylogenetic structure of microflora in the gastrointestinal tract of poultry, and they support the identification of previously unknown groups of microbiota, mainly those occurring in small numbers. Next-generation sequencing also provides indirect information about the quantitative structure of the genes of gut microorganisms, but microbial activity and changes in the proportions of microbial metabolites that affect the host’s intestinal integrity and metabolism remain insufficiently investigated. Therefore, research studies are undertaken to investigate the proportions of the key microbial metabolites in the intestinal contents of poultry relative to changes in the population size of the most important bacterial groups, including those determined by cheaper techniques.
3
Koizumi, Yoshikazu, John J. Kelly, Tatsunori Nakagawa, et al. "Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology." Applied and Environmental Microbiology 68, no. 7 (2002): 3215–25. http://dx.doi.org/10.1128/aem.68.7.3215-3225.2002.
Full textAbstract:
ABSTRACT A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.
4
Hori, Tomoyuki, Matthias Noll, Yasuo Igarashi, Michael W. Friedrich, and Ralf Conrad. "Identification of Acetate-Assimilating Microorganisms under Methanogenic Conditions in Anoxic Rice Field Soil by Comparative Stable Isotope Probing of RNA." Applied and Environmental Microbiology 73, no. 1 (2006): 101–9. http://dx.doi.org/10.1128/aem.01676-06.
Full textAbstract:
ABSTRACT Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH4 and/or CO2. Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope probing with 13C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25°C for 12 days, during which 13C-labeled acetate was added at a concentration of 500 μM every 3 days. 13C-labeled CH4 and CO2 were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate 13C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the 13C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO2 was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions.
5
Janda, Ivan, and Karel Mikulík. "Preparation and sequencing of the cloacin fragment of Streptomycesaureofaciens 16S RNA." Biochemical and Biophysical Research Communications 137, no. 1 (1986): 80–86. http://dx.doi.org/10.1016/0006-291x(86)91178-2.
Full text
6
Arakawa, Shizuka, Kohsuke Kamizaki, Yusuke Kuwana, et al. "Application of solid-phase DNA probe method with cleavage by deoxyribozyme for analysis of long non-coding RNAs." Journal of Biochemistry 168, no. 3 (2020): 273–83. http://dx.doi.org/10.1093/jb/mvaa048.
Full textAbstract:
Abstract The solid-phase DNA probe method is a well-established technique for tRNA purification. We have applied this method for purification and analysis of other non-coding RNAs. Three columns for purification of tRNAPhe, transfer-messenger RNA (tmRNA) and 16S rRNA from Thermus thermophilus were connected in tandem and purifications were performed. From each column, tRNAPhe, tmRNA and 16S rRNA could be purified in a single step. This is the first report of purification of native tmRNA from T. thermophilus and the purification demonstrates that the solid-phase DNA probe method is applicable to non-coding RNA, which is present in lower amounts than tRNA. Furthermore, if a long non-coding RNA is cleaved site-specifically and the fragment can be purified by the solid-phase DNA probe method, modified nucleosides in the long non-coding RNA can be analysed. Therefore, we designed a deoxyribozyme (DNAzyme) to perform site-specific cleavage of 16S rRNA, examined optimum conditions and purified the resulting RNA fragment. Sequencing of complimentary DNA and mass spectrometric analysis revealed that the purified RNA corresponded to the targeted fragment of 16S rRNA. Thus, the combination of DNAzyme cleavage and purification using solid-phase DNA probe methodology can be a useful technique for analysis of modified nucleosides in long non-coding RNAs.
7
Schloss, Patrick D., Matthew L. Jenior, Charles C. Koumpouras, Sarah L. Westcott, and Sarah K. Highlander. "Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system." PeerJ 4 (March 28, 2016): e1869. http://dx.doi.org/10.7717/peerj.1869.
Full textAbstract:
Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.
8
Kozich, James J., Sarah L. Westcott, Nielson T. Baxter, Sarah K. Highlander, and Patrick D. Schloss. "Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform." Applied and Environmental Microbiology 79, no. 17 (2013): 5112–20. http://dx.doi.org/10.1128/aem.01043-13.
Full textAbstract:
ABSTRACTRapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
9
Callahan, Benjamin J., Joan Wong, Cheryl Heiner, et al. "High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution." Nucleic Acids Research 47, no. 18 (2019): e103-e103. http://dx.doi.org/10.1093/nar/gkz569.
Full textAbstract:
AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.
10
Darwish, Ahmed M., and Adnan A. Ismaiel. "Genetic diversity of Flavobacterium columnare examined by restriction fragment length polymorphism and sequencing of the 16S ribosomal RNA gene and the 16S–23S rDNA spacer." Molecular and Cellular Probes 19, no. 4 (2005): 267–74. http://dx.doi.org/10.1016/j.mcp.2005.04.003.
Full text
11
Tung, Sheng Kai, Lee Jene Teng, Mario Vaneechoutte, Hung Mo Chen, and Tsung Chain Chang. "Identification of species of Abiotrophia, Enterococcus, Granulicatella and Streptococcus by sequence analysis of the ribosomal 16S–23S intergenic spacer region." Journal of Medical Microbiology 56, no. 4 (2007): 504–13. http://dx.doi.org/10.1099/jmm.0.47027-0.
Full textAbstract:
The feasibility of sequence analysis of the ribosomal 16S–23S intergenic spacer region (ITS) was evaluated for identification of 24 species of Streptococcus, one species of Abiotrophia, 18 species of Enterococcus and three species of Granulicatella. As GenBank currently lacks ITS sequence entries for many species of these four genera, the ITS sequences of 38 type strains were first sequenced and submitted to GenBank to facilitate species identification of these genera. Subsequently, the ITS sequences of 217 strains (84 reference strains and 133 clinical isolates) were determined and species identification was made by blast search for homologous sequences in public databases. Species other than Streptococcus contained multiple ITS fragments and only the shortest fragment was analysed. A total of 25 isolates (11.5 %) produced discrepant identification by ITS sequencing. The 25 discordant strains were analysed further by sequencing of the 16S rRNA gene for species clarification, and 21 were found to be identified correctly by ITS sequence analysis. The correct identification rate by ITS sequencing was 98.2 % (213/217). Several closely related enterococcal and streptococcal species/subspecies contained specific ITS signature sequences that were useful for differentiating these bacteria. In conclusion, ITS sequencing provides a useful approach towards identifying this group of pathogens on a molecular platform alongside 16S rRNA gene sequencing.
12
Thanh Viet, Nguyen, and Vo Thi Bich Thuy. "Detection of 16S rRNA and 23S rRNA gene mutations in multidrug resistant Salmonella serovars isolated from different sources using RNA sequencing method." Vietnam Journal of Biotechnology 16, no. 4 (2020): 737–44. http://dx.doi.org/10.15625/1811-4989/16/4/13513.
Full textAbstract:
The rapid emergence of resistant bacteria is occurring worldwide. Antibiotic resistance is a serious problem for human beings because pathogenic microorganisms that acquire such resistance void antibiotic treatments. Bacterial antibiotic resistance mechanisms include efflux, reduced influx, modification and degradation of the drug, as well as mutation, modification or overexpression of the target. However, our knowledge as to how bacteria acquire antibiotic resistance is still fragmented, especially for ribosome-targeting drugs. Salmonella is a leading cause of foodborne salmonellosis in the world. The number of antibiotic resistant isolates identified in humans is steadily increasing, suggesting that the spread of antibiotic resistant strains is a major threat to public health. Salmonella is commonly identified in a wide range of animal hosts, food sources, and environments, but our knowledge as to how Salmonella resistance to antibiotics is still fragmented in this ecologically complex serovar. Therefore, the aim of this study was to support for finding novel mechanisms that render bacteria resistant to the ribosome targeting antibiotics, we screen for antibiotic resistant 16S and 23S ribosomal RNAs (rRNAs) in multidrug resistant Salmonella serovars isolated from raw retail meats isolated from Hanoi, Vietnam. Bioinformatic analysis identified 193 unknown novel mutations (64 mutations in 16S rRNA and 129 mutations in 23S rRNA genes). These mutations might play a role in streptomycin resistant in Salmonella serovars. These results suggest that uncharacterized antibiotic resistance mutations still exist, even for traditional antibiotics. This study is only a preliminary kind, further validation before they are applied in Salmonella or other closely related species are required.
13
Jahan, Hawa, Maria Akter, Rowshan Ara Begum, and Reza Md Shahjahan. "Identification and comparison of three carp fishes based on mitochondrial 16S rRNA gene." Dhaka University Journal of Biological Sciences 26, no. 2 (2017): 167–74. http://dx.doi.org/10.3329/dujbs.v26i2.46396.
Full textAbstract:
Identification of Labeo rohita, L. bata and L. gonius is sometimes problematic when usual morphological features are lost and it is difficult to differentiate them with traditional morphological features at their diverse developmental stages. PCR-sequencing provides an authentic alternative means of identification of individuals at species level. Three local carp fishes were collected and 16S rRNA genes were sequenced by sanger sequencing method after PCR amplification using universal primers. Obtained sequences were found accurate with blast search result which showed maximum range of similarity with the existing respective gene fragments present in GenBank database. Sequences were compared and multiple sequence alignment has revealed some polymorphic sites which can be used to differentiate these three species from one another. This study may provide valuable understanding to study their population in future.
 Dhaka Univ. J. Biol. Sci. 26(2): 167-174, 2017 (July)
14
Poyart, Claire, Gilles Quesne, Stephane Coulon, Patrick Berche, and Patrick Trieu-Cuot. "Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase." Journal of Clinical Microbiology 36, no. 1 (1998): 41–47. http://dx.doi.org/10.1128/jcm.36.1.41-47.1998.
Full textAbstract:
We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodAint ) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus,S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus,S. canis, S. cricetus, S. downei,S. dysgalactiae, S. equi subsp.equi, S. equi subsp. zooepidemicus,S. equinus, S. gordonii, S. iniae,S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis,S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis,S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodAint fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of theirsodAint fragments on the phylogenetic tree of the sodAint fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.
15
Tochilina, A. G., I. V. Belova, I. V. Solovieva, I. S. Gorlova, T. P. Ivanova, and V. A. Zhirnov. "CHARACTERISTICS OF BIOLOGICAL AND MOLECULAR-GENETIC PROPERTIES OF LACTOBACILLUS FERMENTUM 90 TC-4 PROBIOTIC STRAIN." Journal of microbiology, epidemiology and immunobiology, no. 2 (April 28, 2016): 16–23. http://dx.doi.org/10.36233/0372-9311-2016-2-16-23.
Full textAbstract:
Aim. Confirmation of taxonomic position of Lactobacillus fermentum 90 TC-4 strain using phenotypic (classic microbiological, MALDI TOF mass-spectrometry) and genetic (16S rRNA gene segment sequencing and full genome sequencing) methods. Materials and methods. Object of the study - Lactobacillus fermentum 90 TC-4 strains from various collections. Mass-spectrometric analysis was carried out using Autoflex MALDI TOF mass-spectrometer (Bruker Daltonics, Germany), study of biochemical properties of the strain was carried out using API 50 CHL strips (Biomerueux, France), “DNA-sorb B” kit was used for isolation ofgenome DNA (CRIE, Moscow). Sequencing of the accumulated fragments of 16S rRNA gene was carried out using GenomeLab GeXP sequencing (Beckman Coulter, USA), full genome sequencing was carried out in MiSeq platform (Illumina). Assembly ofgenome and bioinformation analysis was carried out using BLAST program (www.blast.ncbi.nlm.nih.gov/blast.cgi), «CLC Bio Assembly» and genome server RAST (rast.nmpdr.org). Results. L. fermentum 90 TC-4 strain was established to be contaminated by L. plantarum culture in a series of cases. As a result of identification of a pure culture of L. fermentum 90 TC-4 strain using a specter of high-technology methods, membership of the strain in L. fermentum species has been proven. Conclusion. Taxonomic status of L. fermentum 90 TC-4 strain was confirmed.
16
Awad, Mohamed, Osama Ouda, Ali El-Refy, Fawzy A. El-Feky, Kareem A. Mosa, and Mohamed Helmy. "FN-Identify: Novel Restriction Enzymes-Based Method for Bacterial Identification in Absence of Genome Sequencing." Advances in Bioinformatics 2015 (December 31, 2015): 1–14. http://dx.doi.org/10.1155/2015/303605.
Full textAbstract:
Sequencing and restriction analysis of genes like 16S rRNA and HSP60 are intensively used for molecular identification in the microbial communities. With aid of the rapid progress in bioinformatics, genome sequencing became the method of choice for bacterial identification. However, the genome sequencing technology is still out of reach in the developing countries. In this paper, we propose FN-Identify, a sequencing-free method for bacterial identification. FN-Identify exploits the gene sequences data available in GenBank and other databases and the two algorithms that we developed, CreateScheme and GeneIdentify, to create a restriction enzyme-based identification scheme. FN-Identify was tested using three different and diverse bacterial populations (members of Lactobacillus, Pseudomonas, and Mycobacterium groups) in an in silico analysis using restriction enzymes and sequences of 16S rRNA gene. The analysis of the restriction maps of the members of three groups using the fragment numbers information only or along with fragments sizes successfully identified all of the members of the three groups using a minimum of four and maximum of eight restriction enzymes. Our results demonstrate the utility and accuracy of FN-Identify method and its two algorithms as an alternative method that uses the standard microbiology laboratories techniques when the genome sequencing is not available.
17
Ninane, Véronique, Radegonde Mukandayambaje, and Gilbert Berben. "Identification of Lactic Acid Bacteria within the Consortium of a Kefir Grain by Sequencing 16S rDNA Variable Regions." Journal of AOAC INTERNATIONAL 90, no. 4 (2007): 1111–17. http://dx.doi.org/10.1093/jaoac/90.4.1111.
Full textAbstract:
Abstract The microflora of a kefir grain was identified using a polymerase chain reaction-based strategy combined with 16S rRNA gene sequencing. DNA was extracted from the kefir grain and amplified in its 16S rDNA V1 and V2 regions. To guarantee a good representation of the overall lactic acid bacteria populations, DNA amplification was performed separately with primers specific either to the dominant or to the less abundant bacterial groups. The amplified fragments were cloned in Escherichia coli and then sequenced. Sequences of the V1 region were gathered into 5 groups of similarity and identified by aligning with the sequences of a public library. The V1 region allowed the identification of Lactobacillus kefiranofaciens, L. kefir, L. parakefir, and Lactococcus lactis but was inappropriate for the identification of leuconostocs at species level. Among 16S rDNA variable regions, the V2 region showed the highest variability between Leuconostoc species. Nevertheless, even in the V2 region, differences were too tenuous for effective identification of L. mesenteroides. The methodology described here allowed detection of the dominant species within each targeted microbial group.
18
Bhakta, Sonali, Md Thoufic Anam Azad, and Toshifumi Tsukahara. "Genetic code restoration by artificial RNA editing of Ochre stop codon with ADAR1 deaminase." Protein Engineering, Design and Selection 31, no. 12 (2018): 471–78. http://dx.doi.org/10.1093/protein/gzz005.
Full textAbstract:
Abstract Site directed mutagenesis is a very effective approach to recode genetic information. Proper linking of the catalytic domain of the RNA editing enzyme adenosine deaminase acting on RNA (ADAR) to an antisense guide RNA can convert specific adenosines (As) to inosines (Is), with the latter recognized as guanosines (Gs) during the translation process. Efforts have been made to engineer the deaminase domain of ADAR1 and the MS2 system to target specific A residues to restore G→A mutations. The target consisted of an ochre (TAA) stop codon, generated from the TGG codon encoding amino acid 58 (Trp) of enhanced green fluorescent protein (EGFP). This system had the ability to convert the stop codon (TAA) to a readable codon (TGG), thereby restoring fluorescence in a cellular system, as shown by JuLi fluorescence and LSM confocal microscopy. The specificity of the editing was confirmed by polymerase chain reaction-restriction fragment length polymorphism, as the restored EGFP mRNA could be cleaved into fragments of 160 and 100 base pairs. Direct sequencing analysis with both sense and antisense primers showed that the restoration rate was higher for the 5′ than for the 3′A. This system may be very useful for treating genetic diseases that result from G→A point mutations. Successful artificial editing of RNA in vivo can accelerate research in this field, and pioneer genetic code restoration therapy, including stop codon read-through therapy, for various genetic diseases.
19
Alam, Mohammad Shamimul, Hawa Jahan, Rowshan Ara Begum, and Reza M. Shahjahan. "Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene." Journal of the Asiatic Society of Bangladesh, Science 41, no. 1 (2015): 51–58. http://dx.doi.org/10.3329/jasbs.v41i1.46190.
Full textAbstract:
Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future.
 Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015
20
Poyart, Claire, Gilles Quesnes, and Patrick Trieu-Cuot. "Sequencing the Gene Encoding Manganese-Dependent Superoxide Dismutase for Rapid Species Identification of Enterococci." Journal of Clinical Microbiology 38, no. 1 (2000): 415–18. http://dx.doi.org/10.1128/jcm.38.1.415-418.2000.
Full textAbstract:
ABSTRACT Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 438-bp-long DNA fragment internal ( sodA int ) to the sodA gene encoding the manganese-dependent superoxide dismutase in 19 enterococcal type strains ( Enterococcus avium , Enterococcus casseliflavus , Enterococcus cecorum , Enterococcus columbae , Enterococcus dispar , Enterococcus durans , Enterococcus faecalis , Enterococcus faecium , Enterococcus flavescens , Enterococcus gallinarum , Enterococcus hirae , Enterococcus malodoratus , Enterococcus mundtii , Enterococcus pseudoavium , Enterococcus raffinosus , Enterococcus saccharolyticus , Enterococcus seriolicida , Enterococcus solitarius , and Enterococcus sulfureus ). Sequence analysis of the sodA int fragments enabled reliable identification of 18 enterococcal species, including E. casseliflavus-E. flavescens and E. gallinarum . The sodA int fragments of E. casseliflavus and E. flavescens were almost identical (99.5% sequence identity), which suggests that they should be associated in a single species. Our results confirm that the sodA gene constitutes a more discriminative target sequence than 16S rRNA gene in differentiating closely related bacterial species.
21
Links, Matthew G., Tim J. Dumonceaux, E. Luke McCarthy, Sean M. Hemmingsen, Edward Topp, and Jennifer R. Town. "CaptureSeq: Hybridization-Based Enrichment of cpn60 Gene Fragments Reveals the Community Structures of Synthetic and Natural Microbial Ecosystems." Microorganisms 9, no. 4 (2021): 816. http://dx.doi.org/10.3390/microorganisms9040816.
Full textAbstract:
Background. The molecular profiling of complex microbial communities has become the basis for examining the relationship between the microbiome composition, structure and metabolic functions of those communities. Microbial community structure can be partially assessed with “universal” PCR targeting taxonomic or functional gene markers. Increasingly, shotgun metagenomic DNA sequencing is providing more quantitative insight into microbiomes. However, both amplicon-based and shotgun sequencing approaches have shortcomings that limit the ability to study microbiome dynamics. Methods. We present a novel, amplicon-free, hybridization-based method (CaptureSeq) for profiling complex microbial communities using probes based on the chaperonin-60 gene. Molecular profiles of a commercially available synthetic microbial community standard were compared using CaptureSeq, whole metagenome sequencing, and 16S universal target amplification. Profiles were also generated for natural ecosystems including antibiotic-amended soils, manure storage tanks, and an agricultural reservoir. Results. The CaptureSeq method generated a microbial profile that encompassed all of the bacteria and eukaryotes in the panel with greater reproducibility and more accurate representation of high G/C content microorganisms compared to 16S amplification. In the natural ecosystems, CaptureSeq provided a much greater depth of coverage and sensitivity of detection compared to shotgun sequencing without prior selection. The resulting community profiles provided quantitatively reliable information about all three domains of life (Bacteria, Archaea, and Eukarya) in the different ecosystems. The applications of CaptureSeq will facilitate accurate studies of host-microbiome interactions for environmental, crop, animal and human health. Conclusions: cpn60-based hybridization enriched for taxonomically informative DNA sequences from complex mixtures. In synthetic and natural microbial ecosystems, CaptureSeq provided sequences from prokaryotes and eukaryotes simultaneously, with quantitatively reliable read abundances. CaptureSeq provides an alternative to PCR amplification of taxonomic markers with deep community coverage while minimizing amplification biases.
22
Saha, Mihir Lal, Mohammad Nurul Islam, Chaman Binta Aziz, and Mohammad Zabed Hossain. "Molecular identification of bacteria present in the soils of the Madhupur Sal and the Sunderban mangrove forests of Bangladesh." Dhaka University Journal of Biological Sciences 21, no. 2 (2012): 117–23. http://dx.doi.org/10.3329/dujbs.v21i2.11509.
Full textAbstract:
The present study aimed at identifying the soil bacterial communities in the Madhupur Sal and the Sunderban mangrove forests, the two important forests of Bangladesh. Soil bacteria were identified through sequencing of PCR?amplified fragments of bacterial 16S rDNA. The results showed that the bacterial communities of the Madhupur Sal forest soils and that of the Sunderban mangrove forest soils differed in the morphology of the cells. Data presented here also showed that the bacterial communities of the Sunderban mangrove forest soils were more diverse than that of the Madhupur Sal forest soils as revealed by phylogenetic tree analysis of the 16S rDNA sequences.DOI: http://dx.doi.org/10.3329/dujbs.v21i2.11509Dhaka Univ. J. Biol. Sci. 21(2): 117?123, 2012 (July)
23
Palinska, Katarzyna A., Christian F. Thomasius, Jürgen Marquardt, and Stjepko Golubic. "Phylogenetic evaluation of cyanobacteria preserved as historic herbarium exsiccata." International Journal of Systematic and Evolutionary Microbiology 56, no. 10 (2006): 2253–63. http://dx.doi.org/10.1099/ijs.0.64417-0.
Full textAbstract:
Dried herbarium specimens of cyanobacteria (exsiccata) deposited over 100 years ago were analysed and characterized using combined morphological and molecular approaches. Six representative coccoid and filamentous cyanobacteria from two historic collections and a 15-year-old air-dried environmental sample were studied. Morphological features observed by light and electron microscopy were correlated with the results of 16S rRNA gene sequencing. Historic identifications achieved by means of classical morphology could thus be confirmed by extracted, amplified and sequenced 16S rRNA gene fragments. The results of this study open the possibility of providing genotypic characterizations to botanical type specimens, thus reconciling the botanical and bacteriological approaches to the taxonomic treatment of these micro-organisms.
24
Mikhailov, I. S., Y. R. Zakharova, N. A. Volokitina, D. P. Petrova, and Y. V. Likhoshway. "Bacteria associated with planktonic diatoms from Lake Baikal." Acta Biologica Sibirica 4, no. 4 (2018): 89–94. http://dx.doi.org/10.14258/abs.v4.i4.4880.
Full textAbstract:
Algal-bacterial associations were studied in unialgal xenic cultures of Synedra acus subsp. radians, Asterionella formosa and Fragillaria crotonensis planktonic diatoms from Lake Baikal, using epifluorescent and scanning electron microscopy. It was found that rod- and ovoid-shaped bacteria colonized cell walls of diatoms. Cloning and sequencing of fragments of 16S rRNA gene in diatom cultures revealed members of Gammaproteobacteria (Pseudomonas sp.), Betaproteobacteria (Janthinobacterium sp., Hydrogenophaga sp., Methylophilus sp.), Bacteroidetes (Flavobacterium sp., Pedobacter sp.), and Acinobacteria (Nocardioides sp.).
25
Liu, Xinmei, Zhiyang Liu, Yiyu Cheng, et al. "Application of Next-Generation Sequencing Technology Based on Single Gene Locus in Species Identification of Mixed Meat Products." Journal of Food Quality 2021 (August 31, 2021): 1–5. http://dx.doi.org/10.1155/2021/4512536.
Full textAbstract:
Polymerase chain reaction (PCR) detection is a commonly used method for species identification of meat products. However, this method is not suitable for the analysis of meat products containing multiple mixtures. This study aimed to test whether next-generation sequencing (NGS) technology could be used as a method for the certification of mixed meat products. In this study, five kinds of common meat (pigs, cattle, sheep, chickens, and ducks) were mixed as samples with different proportions. The primers designed from mitochondrial 16S rRNA and nuclear genome gene (growth hormone receptor, GHR), respectively, were used to detect these meats. The sequencing results of NGS were analyzed using a self-designed bioinformatics program. The fragments with similar sequences were classified and compared with the database to determine their species. The results showed that all five kinds of meat components could be correctly identified using these two primers. The meat composition could be detected as low as 0.5% in the mixed samples using the NGS technology targeting GHR gene fragments, which was superior to those targeting mitochondrial 16S rRNA. However, the quantitative detection of species in the mixture was not likely to be quite accurate due to the amplification bias of PCR amplification. These results showed that the NGS technology could be applied to identify meat species in mixtures.
26
Mau, Margit, and Kenneth N. Timmis. "Use of Subtractive Hybridization To Design Habitat-Based Oligonucleotide Probes for Investigation of Natural Bacterial Communities." Applied and Environmental Microbiology 64, no. 1 (1998): 185–91. http://dx.doi.org/10.1128/aem.64.1.185-191.1998.
Full textAbstract:
ABSTRACT We describe a rapid oligonucleotide probe design strategy based on subtractive hybridization which yields probes for 16S rRNA or rRNA genes of individual members of microbial communities that are specific within the context of those communities. This strategy circumvents the need to sequence many similar or identical clones of dominant members of a community. Radioactively labeled subfragments of a cloned 16S rRNA gene sequence for which a probe is required (target) were hybridized with biotinylated total 16S ribosomal DNA (rDNA) amplified from the microbial community, and the hybrids formed were subsequently discarded. The remaining enriched fragments were used to screen a library consisting of cloned subfragments of the target sequence by colony hybridization in order to identify the variable regions of the 16S rRNA gene with the required specificity. The sequencing of random clones in one 16S rDNA library demonstrated that only those clones with 100% sequence identity with the probe fragment were detected by it. Moreover, sequencing of other, randomly selected, probe-positive clones revealed 100% sequence identity with the probe. Probes developed in this way tended to correspond to more variable regions of the 16S rRNA if the target sequences were similar to the sequences of other clones in the library and to less variable regions if the target sequences were phylogenetically isolated within the clone library. Although the absolute specificity of the latter probes, as assessed by comparison with available database sequences, was lower than the absolute specificity of the probes from the more variable regions, they were specific within the context of the environmental samples from which they were derived.
27
Satokari, Reetta M., Elaine E. Vaughan, Antoon D. L. Akkermans, Maria Saarela, and Willem M. de Vos. "Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis." Applied and Environmental Microbiology 67, no. 2 (2001): 504–13. http://dx.doi.org/10.1128/aem.67.2.504-513.2001.
Full textAbstract:
ABSTRACT We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE).Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.
28
Said, Layla Abdulhamed, Sawsan Saeed Hasan, and Saad L. Hamed. "Identification of Clinical Pseudomonas spp. by VITEK 2 Compact System and Species-specific Polymerase Chain Reaction Assay for Identification of Pseudomonas aeruginosa." International Journal of Drug Delivery Technology 10, no. 03 (2020): 383–88. http://dx.doi.org/10.25258/ijddt.10.3.14.
Full textAbstract:
The objective of this study was to isolate, identify, and diagnose Pseudomonas spp. from different clinical sources in Baghdad, Iraq. VITEK 2 compact system identification gram-negative bacteria (ID gNB) cards were used to confirm the identification. Polymerase chain reaction (PCR) technique and sequencing were used for recognition of the 16S rDNA gene, by two pairs of primers, universal primers (930 bp fragments) for recognition of Pseudomonas spp., and Pseudomonas aeruginosa specific species (PASS) primers (956 bp fragments) for differentiation of P. aeruginosa from other species. Amplified PCR products of PASS primers were sent for DNA Sanger sequencing to Macrogen Company, Seoul, Korea; data were compared with the database using the Basic Local Alignment Search Tool (BLAST). Ninety-two Pseudomonas spp., including 86 isolates of P. aeruginosa and 1, 2, 3 isolates of Pseudomonas luteola, Pseudomonas putida, and Pseudomonas fluorescens, respectively, were obtained using VITEK 2 compact system ID gNB cards with a percentage of identification ranging from 91 to 99%. Gene amplification and sequencing results confirm identification ranging from 99 to 100%. After sequencing analysis, eleventh of the P. aeruginosa isolates had been submitted in GenBank National Centre for Biotechnology Information (NCBI) under accession numbers MN630696–MN630707. It was observed that phenotypic tests supported by PCR techniques have enabled to conduct a detailed characterization of Pseudomonas bacteria isolates.
29
da Silva, Cleiziane Bispo, Hellen Ribeiro Martins dos Santos, Phellippe Arthur Santos Marbach, et al. "First-tier detection of intragenomic 16S rRNA gene variation in culturable endophytic bacteria from cacao seeds." PeerJ 7 (November 20, 2019): e7452. http://dx.doi.org/10.7717/peerj.7452.
Full textAbstract:
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.
30
Foley, J. E., D. Borjesson, T. L Gross, C. Rand, M. Needham, and A. Poland. "Clinical, Microscopic, and Molecular Aspects of Canine Leproid Granuloma in the United States." Veterinary Pathology 39, no. 2 (2002): 234–39. http://dx.doi.org/10.1354/vp.39-2-234.
Full textAbstract:
Leproid granulomas from seven dogs in the United States were evaluated. Gross characteristics included nodular and ulcerated dermal and subcutaneous lesions primarily on the caudal aspects of the pinnae and to a lesser extent on the muzzle, face, and forelimbs. In all except one dog, there was complete regression of the lesions within 6 months, either with no therapy or after surgical resection. Cytology or histopathology revealed pyogranulomatous inflammation with few to many acid-fast mycobacterial bacilli within macrophages. The organisms could not be cultivated in vitro. DNA sequencing of part of the 16S ribosomal RNA gene region revealed 99–100% homology among fragments from five of these dogs and fragments from dogs in the South Pacific. This syndrome occurs in dogs in North America and the prognosis is excellent, in contrast to the prognosis for rapid-growing or tuberculous mycobacteriosis.
31
Schloss, Patrick D., Sarah L. Westcott, Thomas Ryabin, et al. "Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities." Applied and Environmental Microbiology 75, no. 23 (2009): 7537–41. http://dx.doi.org/10.1128/aem.01541-09.
Full textAbstract:
ABSTRACT mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the α and β diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.
32
Peng, Guixiang, Huarong Wang, Guoxia Zhang, et al. "Azospirillum melinis sp. nov., a group of diazotrophs isolated from tropical molasses grass." International Journal of Systematic and Evolutionary Microbiology 56, no. 6 (2006): 1263–71. http://dx.doi.org/10.1099/ijs.0.64025-0.
Full textAbstract:
Fifteen bacterial strains isolated from molasses grass (Melinis minutiflora Beauv.) were identified as nitrogen-fixers by using the acetylene-reduction assay and PCR amplification of nifH gene fragments. These strains were classified as a unique group by insertion sequence-PCR fingerprinting, SDS-PAGE protein patterns, DNA–DNA hybridization, 16S rRNA gene sequencing and morphological characterization. Phylogenetic analysis of the 16S rRNA gene indicated that these diazotrophic strains belonged to the genus Azospirillum and were closely related to Azospirillum lipoferum (with 97.5 % similarity). In all the analyses, including in addition phenotypic characterization using Biolog MicroPlates and comparison of cellular fatty acids, this novel group was found to be different from the most closely related species, Azospirillum lipoferum. Based on these data, a novel species, Azospirillum melinis sp. nov., is proposed for these endophytic diazotrophs of M. minutiflora, with TMCY 0552T (=CCBAU 5106001T=LMG 23364T=CGMCC 1.5340T) as the type strain.
33
Suzuki, Yasuhiko, Chihiro Katsukawa, Aki Tamaru, et al. "Detection of Kanamycin-ResistantMycobacterium tuberculosis by Identifying Mutations in the 16S rRNA Gene." Journal of Clinical Microbiology 36, no. 5 (1998): 1220–25. http://dx.doi.org/10.1128/jcm.36.5.1220-1225.1998.
Full textAbstract:
In Mycobacterium smegmatis and a limited number ofMycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, ≧200 μg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying therrs gene and the intervening sequence between therrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI andTsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI orDdeI.
34
Webster, Nicole S., Kate J. Wilson, Linda L. Blackall, and Russell T. Hill. "Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile." Applied and Environmental Microbiology 67, no. 1 (2001): 434–44. http://dx.doi.org/10.1128/aem.67.1.434-444.2001.
Full textAbstract:
ABSTRACT Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of theActinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria,Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.
35
Hahn, Martin W., Matthias Pöckl, and Qinglong L. Wu. "Low Intraspecific Diversity in a Polynucleobacter Subcluster Population Numerically Dominating Bacterioplankton of a Freshwater Pond." Applied and Environmental Microbiology 71, no. 8 (2005): 4539–47. http://dx.doi.org/10.1128/aem.71.8.4539-4547.2005.
Full textAbstract:
ABSTRACT Cultivation-dependent and -independent methods were combined to investigate the microdiversity of a Polynucleobacter subcluster population (Betaproteobacteria) numerically dominating the bacterioplankton of a small, humic freshwater pond. Complete coverage of the population by cultivation allowed the analysis of microdiversity beyond the phylogenetic resolution of ribosomal markers. Fluorescent in situ hybridization with two probes specific for the narrow subcluster C (PnecC bacteria) of the Polynucleobacter cluster revealed that this population contributed up to 60% to the total number of bacterioplankton cells. Microdiversity was investigated for a date at which the highest relative numbers of PnecC were observed. A clone library of fragments of the ribosomal operon (16S rRNA genes, complete 16S-23S internal transcribed spacer 1 [ITS1], partial 23S rRNA genes) amplified with universal bacterial primers was constructed. The library was stepwise screened for fragments from PnecC bacteria and for different ITS genotypes of PnecC bacteria. The isolated PnecC strains were characterized by sequencing of the 16S rRNA genes and the ITS1. Both the clone library and the established culture collection contained only the same three ITS genotypes, and one of them contributed 46% to the entire number of clones. Genomic fingerprinting of the isolates with several methods always resulted in the detection of only one fingerprint per ITS genotype. We conclude that a Polynucleobacter population with an extremely low intraspecific diversity and an uneven structure numerically dominated the bacterioplankton community in the investigated habitat. This low intraspecific diversity is in strong contrast to the high intraspecific diversities found in marine bacterial populations.
36
Fisher, Madeline M., and Eric W. Triplett. "Automated Approach for Ribosomal Intergenic Spacer Analysis of Microbial Diversity and Its Application to Freshwater Bacterial Communities." Applied and Environmental Microbiology 65, no. 10 (1999): 4630–36. http://dx.doi.org/10.1128/aem.65.10.4630-4636.1999.
Full textAbstract:
ABSTRACT An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.
37
Arcuri, Pedro Braga, Agnes Awino Odenyo, Edna Froeder Arcuri, Marlice Teixeira Ribeiro, Marta Fonseca Martins Guimarães, and Jailton da Costa Carneiro. "Tannin-tolerant bacteria from crossbred Holstein x Zebu cows." Pesquisa Agropecuária Brasileira 46, no. 3 (2011): 272–79. http://dx.doi.org/10.1590/s0100-204x2011000300007.
Full textAbstract:
The objective of this work was to isolate and characterize tannin-tolerant ruminal bacteria from crossbred Holstein x Zebu cows fed a chopped mixture of elephant grass (Pennisetum purpureum), young stems of "angico-vermelho" (Parapiptadenia rigida), and banana tree (Musa sp.) leaves. A total of 117 bacteria strains were isolated from enrichment cultures of rumen microflora in medium containing tannin extracts. Of these, 11 isolates were able to tolerate up to 3 g L-1 of tannins. Classical characterization procedures indicated that different morphological and physiological groups were represented. Restriction fragments profiles using Alu1 and Taq1 of 1,450 bp PCR products from the 16S rRNA gene grouped the 11 isolates into types I to VI. Sequencing of 16S rRNA PCR products was used for identification. From the 11 strains studied, seven were not identifiable by the methods used in this work, two were strains of Butyrivibrio fibrisolvens, and two of Streptococcus bovis.
38
MAI, TRUC, JEFFREY R. JOHANSEN, NICOLE PIETRASIAK, MARKÉTA BOHUNICKÁ, and MICHAEL P. MARTIN. "Revision of the Synechococcales (Cyanobacteria) through recognition of four families including Oculatellaceae fam. nov. and Trichocoleaceae fam. nov. and six new genera containing 14 species." Phytotaxa 365, no. 1 (2018): 1. http://dx.doi.org/10.11646/phytotaxa.365.1.1.
Full textAbstract:
A total of 48 strains of thin, filamentous cyanobacteria in Synechococcales were studied by sequencing 16S rRNA and rpoC1 sequence fragments. We also carefully characterized a subset of these by morphology. Phylogenetic analysis of the 16S rRNA gene data using Bayesian inference of a large Synechococcales alignment (345 OTU’s) was in agreement with the phylogeny based on the rpoC1 gene for 59 OTU’s. Both indicated that the large family-level grouping formerly classified as the Leptolyngbyaceae could be further divided into four family-level clades. Two of these family-level clades have been recognized previously as Leptolyngbyaceae and Prochlorotrichaceae. Oculatellaceae fam. nov. and Trichocoleaceae fam. nov. are proposed for the other two families. The Oculatellaceae was studied in greater detail, and six new genera containing 14 species were characterized and named. These new taxa are: Pegethrix botrychoides, P. olivacea, P. convoluta, P. indistincta, Drouetiella lurida, D. hepatica, D. fasciculata, Cartusia fontana, Tildeniella torsiva, T. nuda, Komarkovaea angustata, Kaiparowitsia implicata, Timaviella obliquedivisa, and T. radians.
39
Cambronero-Heinrichs, Juan Carlos, Laura Sofía Sánchez-Portilla, Ólger Calderón-Arguedas, and Adriana Troyo. "Cimex lectularius Linnaeus, 1758 (Hemiptera: Cimicidae) in Costa Rica: First Case Report Confirmed by Molecular Methods in Central America." Journal of Medical Entomology 57, no. 3 (2020): 969–73. http://dx.doi.org/10.1093/jme/tjz247.
Full textAbstract:
Abstract Cimex lectularius and Cimex hemipterus are the most common species of bedbugs that infest homes. Although case reports decreased substantially by the end of the 20th century, bed bugs, and especially C. lectularius, are currently suffering a resurgence mostly attributed to insecticide resistance, inadequate pest control, and increased travel. Here, we report, to the best of our knowledge, the first molecular confirmation of C. lectularius in Central America. Specimens were obtained from an apartment located in Heredia, Costa Rica. These specimens were identified morphologically as C. lectularius. The species identification was confirmed by amplifying and sequencing fragments of the cytochrome oxidase subunit I (COI) and the 16S rRNA (16S) genes. The phylogenetic analysis showed that the sequences obtained were more closely related to a C. lectularius mitochondrial complete genome sequence from China, with similarities of 98.84% (686/694) for COI and 98.97% (387/391) for 16S. The finding of C. lectularius in Costa Rica will require further investigation in order to determine the extent of current infestations and the susceptibility to insecticides, especially due to the impact that this species can have in human health, as well as the tourism industry in the region.
40
Brim, H., H. Heuer, E. Krögerrecklenfort, M. Mergeay, and K. Smalla. "Characterization of the bacterial community of a zinc-polluted soil." Canadian Journal of Microbiology 45, no. 4 (1999): 326–38. http://dx.doi.org/10.1139/w99-012.
Full textAbstract:
The bacterial community of a zinc-contaminated soil (Maatheide soil in Lommel, Belgium) was studied using cultivation as well as cultivation-independent techniques. Colony-forming units (CFU) were determined by plating on media with or without metals. Dominant isolates were characterized by fatty acid methyl ester analysis (FAME analysis) and PCR fingerprinting using repetitive extragenic palindromic sequences as primers. DNA was directly extracted from soil samples and used as a template for the PCR amplification of the 16S rDNA (8-1511) or a 16S rDNA fragment (968-1401). Clones resulting from cloning the 16S rDNA from soil DNA were sequenced. Temperature gradient gel electrophoresis (TGGE analysis) was performed for 16S rDNA fragments (968-1401) amplified from the dominant isolates, the clones, and the total soil DNA extracted according to two protocols differing in strength of lysis. Total CFU ranged from 104to 105/g soil. The majority of the isolates were identified by FAME analysis as Arthrobacter spp. (18 out of 23). None of the isolates were identified as a Ralstonia eutropha like strain (formerly Alcaligenes eutrophus). MetalloresistantRastomia eutropha like strains were previously shown to be dominant in the analyzed biotope. Most of the isolates were zinc tolerant but only seven could be considered zinc resistant. Sequences of the 16S rDNA clones obtained from total soil DNA were affiliated with genes of different bacteria such as alpha-proteobacteria, beta-proteobacteria, and the Cytophaga-Flexibacter-Bacteroides group. None of the sequenced clones aligned with the Ralstonia eutropha 16S rRNA gene. TGGE analysis of the 16S rDNA fragments (968-1401) amplified from the dominant strains, the clones, and the total soil DNA showed that isolates and clones represented only a part of the bands present in the TGGE pattern from total DNA. The 968-1401 fragment amplified from all Arthrobacter strains had a similar electrophoretic mobility. This band was seen as a major band in the pattern of DNA extracted from soil using a harsh cell lysis, whereas it did not appear, or appeared only as a weak band, in patterns obtained from soil DNA extracted using gentle lysis. The previously reported predominance of a Ralstonia eutropha like strain in this soil was no longer observed. This may suggest a population replacement by less resistant bacteria, concomitant with a progressive decrease of the zinc toxicity in the Maatheide soil.Key words: microbial community analysis, cultivation, 16S rDNA analysis, TGGE, sequencing, Zn-polluted soil.
41
Whiteson, Katrine L., Vladimir Lazarevic, Manuela Tangomo-Bento, et al. "Noma Affected Children from Niger Have Distinct Oral Microbial Communities Based on High-Throughput Sequencing of 16S rRNA Gene Fragments." PLoS Neglected Tropical Diseases 8, no. 12 (2014): e3240. http://dx.doi.org/10.1371/journal.pntd.0003240.
Full text
42
Di Finizio, Alessandra, Giulia Guerriero, Gian Luigi Russo, and Gaetano Ciarcia. "Identification of gadoid species (Pisces, Gadidae) by sequencing and PCR–RFLP analysis of mitochondrial 12S and 16S rRNA gene fragments." European Food Research and Technology 225, no. 3-4 (2006): 337–44. http://dx.doi.org/10.1007/s00217-006-0420-z.
Full text
43
Tomimura, Kenta, Shin-ichi Miyata, Noriko Furuya, et al. "Evaluation of Genetic Diversity Among ‘Candidatus Liberibacter asiaticus’ Isolates Collected in Southeast Asia." Phytopathology® 99, no. 9 (2009): 1062–69. http://dx.doi.org/10.1094/phyto-99-9-1062.
Full textAbstract:
The aim of this study was to investigate the genetic diversity and relationships among ‘Candidatus Liberibacter asiaticus’ isolates from different hosts and distinct geographical areas in Southeast Asia. Genetic diversity among ‘Ca. Liberibacter asiaticus’ was estimated by sequencing four well-characterized DNA fragments: the 16S ribosomal DNA (rDNA) and 16S/23S intergenic spacer regions; the outer membrane protein (omp) gene region; the trmU-tufB-secE-nusG-rplKAJL-rpoB region (gene cluster region); and the bacteriophage-type DNA polymerase region. The sequences of the 16S rDNA and 16S/23S intergenic spacer regions were identical among all ‘Ca. Liberibacter asiaticus’ isolates. In contrast, nucleotide substitutions were observed in both the omp gene and the gene cluster regions. However, extended bacteriophage-type DNA polymerase sequences acquired by thermal asymmetric interlaced polymerase chain reaction provided the most sequence diversity among isolates. Phylogenetic analysis of the bacteriophage-type DNA polymerase sequences revealed three clusters in the Southeast Asian ‘Ca. Liberibacter asiaticus’ population. All Indonesian ‘Ca. Liberibacter asiaticus’ isolates clustered in one group. The other clusters were not correlated with geographic distribution. The differences in genetic sequences did not reflect differences in the original citrus host (mandarin or pummelo). These results suggest that the bacteriophage-type DNA polymerase region would be useful for molecular differentiation between different Southeast Asian ‘Ca. Liberibacter asiaticus’ isolates.
44
Bouity-Voubou, Maurice Dieudonné, Jean Claude Frigon, Serge Guiot, and Roland Brousseau. "Comparison of treatment efficacy and stability of microbial populations between raw and anaerobically treated liquid pig manure, using PCR–DGGE and 16S sequencing." Canadian Journal of Microbiology 54, no. 2 (2008): 83–90. http://dx.doi.org/10.1139/w07-120.
Full textAbstract:
The effects of adding an adapted inoculum to liquid pig manure (LPM) prior to anaerobic digestion were evaluated by standard analytical methods. In parallel, the phylogenetic diversity of the microbial community of raw and anaerobically digested pig manure was studied by both denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA fragments amplified by polymerase chain reaction. Gas production, volative fatty acid production, removal of soluble chemical oxygen demand, and removal of volatile soluble solids were measured on raw and on inoculated liquid pig manure subjected to anaerobic digestion. DGGE profiles of 16S rRNA genes were used to compare the major elements of the bacterial community composition in raw LPM with those present under various incubation conditions. Major bands were excised and sequenced to gain insight into the identities of the bacterial populations from LPM treated under different conditions. The results show that the addition of an adapted inoculum did not have a major impact on the conversion of pig manure into soluble organic matter and did not significantly change the microbial populations present during anaerobic digestion of LPM. Bacterial composition also indicated that Clostridium species are important constituents of the LPM community.
45
Qiao, Y. J., Z. H. Li, X. Wang, B. Zhu, Y. G. Hu, and Z. H. Zeng. " Effect of legume-cereal mixtures on the diversity of bacterial communities in the rhizosphere." Plant, Soil and Environment 58, No. 4 (2012): 174–80. http://dx.doi.org/10.17221/351/2011-pse.
Full textAbstract:
Aboveground plant diversity is known to influence belowground diversity and ecosystem processes. However, there is little knowledge of soil microbial succession in legume-grass mixtures. Therefore, this study was designed to determine the effect of oat and common vetch binary mixtures at three seeding rates on soil bacterial communities. Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments was used to profile the structure of the bacterial community in the rhizosphere. Compared with a monoculture of common vetch and oat, the Shannon-Weaver index and species richness of the mixtures were increased. Thirteen cloned monocultures and mixtures of oat and common vetch soil 16S rDNA sequences were deposited to NCBI. Based on the sequencing results, the bands could be identified as related to Proteobacteria, Bacteroidetes and Cyanobacteria. Common vetch did not have some bacteria relatives to Sphingomonas spp. Some bacterial taxa could be detected in the ratio of 1:1 and 1:2, but not in the ratio of 1:3, e.g. Myxococcales. The results suggested that the belowground diversity could be promoted by mixed cropping systems.  
46
Burton, Jeremy P., Peter A. Cadieux, and Gregor Reid. "Improved Understanding of the Bacterial Vaginal Microbiota of Women before and after Probiotic Instillation." Applied and Environmental Microbiology 69, no. 1 (2003): 97–101. http://dx.doi.org/10.1128/aem.69.1.97-101.2003.
Full textAbstract:
ABSTRACT The vaginal bacterial microbiota of 19 premenopausal women was examined by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of the V2-V3 region of the 16S rRNA gene. Ten of the women were studied further to investigate the effect and persistence of vaginally inserted capsules containing viable lactobacilli. PCR-DGGE indicated that most subjects had a microbiota represented by one to three dominant DNA fragments. Analysis of these fragments revealed that 79% of the women possessed sequences with high levels of similarity to Lactobacillus species sequences. Sequences homologous to Lactobacillus iners sequences were the most common and were detected in 42% of the women tested. Alteration of the vaginal microbiota could be detected by PCR-DGGE in several women after the instillation of lactobacilli. Additionally, randomly amplified polymorphic DNA analysis of lactobacilli isolated from selective media demonstrated that the exogenous strains could be detected for up to 21 days in some subjects. This study demonstrates that non-culture-based techniques, such as PCR-DGGE, are useful adjuncts for studies of the vaginal microbiota.
47
Penny, Christian, Thierry Nadalig, Malek Alioua, Christelle Gruffaz, Stéphane Vuilleumier, and Françoise Bringel. "Coupling of Denaturing High-Performance Liquid Chromatography and Terminal Restriction Fragment Length Polymorphism with Precise Fragment Sizing for Microbial Community Profiling and Characterization." Applied and Environmental Microbiology 76, no. 3 (2009): 648–51. http://dx.doi.org/10.1128/aem.01556-09.
Full textAbstract:
ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.
48
Devulder, G., M. Pérouse de Montclos, and J. P. Flandrois. "A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model." International Journal of Systematic and Evolutionary Microbiology 55, no. 1 (2005): 293–302. http://dx.doi.org/10.1099/ijs.0.63222-0.
Full textAbstract:
Advances in DNA sequencing and the increasing number of sequences available in databases have greatly enhanced the bacterial identification process. Several species within the genus Mycobacterium cause serious human and animal diseases. In order to assess their relative positions in the evolutionary process, four gene fragments, from the 16S rRNA (564 bp), hsp65 (420 bp), rpoB (396 bp) and sod (408 bp) genes, were sequenced from 97 strains, including all available type strains of the genus Mycobacterium. The results demonstrate that, in this case, the concatenation of different genes allows significant increases in the power of discrimination and the robustness of the phylogenetic tree. The sequential and/or combined use of sequences of several genes makes it possible to refine the phylogenetic approach and provides a molecular basis for accurate species identification.
49
N� Chadhain, Sin�ad M., R. Sean Norman, Karen V. Pesce, Jerome J. Kukor, and Gerben J. Zylstra. "Microbial Dioxygenase Gene Population Shifts during Polycyclic Aromatic Hydrocarbon Biodegradation." Applied and Environmental Microbiology 72, no. 6 (2006): 4078–87. http://dx.doi.org/10.1128/aem.02969-05.
Full textAbstract:
ABSTRACT The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.
50
Ashelford, Kevin E., Nadia A. Chuzhanova, John C. Fry, Antonia J. Jones, and Andrew J. Weightman. "At Least 1 in 20 16S rRNA Sequence Records Currently Held in Public Repositories Is Estimated To Contain Substantial Anomalies." Applied and Environmental Microbiology 71, no. 12 (2005): 7724–36. http://dx.doi.org/10.1128/aem.71.12.7724-7736.2005.
Full textAbstract:
ABSTRACT A new method for detecting chimeras and other anomalies within 16S rRNA sequence records is presented. Using this method, we screened 1,399 sequences from 19 phyla, as defined by the Ribosomal Database Project, release 9, update 22, and found 5.0% to harbor substantial errors. Of these, 64.3% were obvious chimeras, 14.3% were unidentified sequencing errors, and 21.4% were highly degenerate. In all, 11 phyla contained obvious chimeras, accounting for 0.8 to 11% of the records for these phyla. Many chimeras (43.1%) were formed from parental sequences belonging to different phyla. While most comprised two fragments, 13.7% were composed of at least three fragments, often from three different sources. A separate analysis of the Bacteroidetes phylum (2,739 sequences) also revealed 5.8% records to be anomalous, of which 65.4% were apparently chimeric. Overall, we conclude that, as a conservative estimate, 1 in every 20 public database records is likely to be corrupt. Our results support concerns recently expressed over the quality of the public repositories. With 16S rRNA sequence data increasingly playing a dominant role in bacterial systematics and environmental biodiversity studies, it is vital that steps be taken to improve screening of sequences prior to submission. To this end, we have implemented our method as a program with a simple-to-use graphic user interface that is capable of running on a range of computer platforms. The program is called Pintail, is released under the terms of the GNU General Public License open source license, and is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/ .