scholarly journals Novel Bacterial Lineages at the (Sub)Division Level as Detected by Signature Nucleotide-Targeted Recovery of 16S rRNA Genes from Bulk Soil and Rice Roots of Flooded Rice Microcosms

2001 ◽  
Vol 67 (2) ◽  
pp. 623-631 ◽  
Author(s):  
Manigee Derakshani ◽  
Thomas Lukow ◽  
Werner Liesack
Keyword(s):  
16S Rrna ◽  
Bacterial Diversity ◽  
16S Rdna ◽  
The Other ◽  
Bulk Soil ◽  
Rrna Genes ◽  
Pcr Assay ◽  
Rice Roots ◽  
Flooded Rice ◽  
Bacterial Lineages

ABSTRACT Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus Pirellulawithin the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the orderPlanctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.

scholarly journals Vineyard soil bacterial diversity and composition revealed by 16S rRNA genes: Differentiation by vineyard management

2016 ◽  
Vol 103 ◽  
pp. 337-348 ◽  
Author(s):  
Kayla N. Burns ◽  
Nicholas A. Bokulich ◽  
Dario Cantu ◽  
Rachel F. Greenhut ◽  
Daniel A. Kluepfel ◽  
...  
Keyword(s):  
16S Rrna ◽  
Bacterial Diversity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Soil Bacterial Diversity ◽  
Vineyard Soil ◽  
Soil Bacterial ◽  
Vineyard Management

scholarly journals Bacterial Diversity and Function of Aerobic Granules Engineered in a Sequencing Batch Reactor for Phenol Degradation

2004 ◽  
Vol 70 (11) ◽  
pp. 6767-6775 ◽  
Author(s):  
He-Long Jiang ◽  
Joo-Hwa Tay ◽  
Abdul Majid Maszenan ◽  
Stephen Tiong-Lee Tay
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Bacterial Diversity ◽  
Sequencing Batch Reactor ◽  
Batch Reactor ◽  
Phenol Degradation ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Aerobic Granules ◽  
And Function

ABSTRACT Aerobic granules are self-immobilized aggregates of microorganisms and represent a relatively new form of cell immobilization developed for biological wastewater treatment. In this study, both culture-based and culture-independent techniques were used to investigate the bacterial diversity and function in aerobic phenol- degrading granules cultivated in a sequencing batch reactor. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes demonstrated a major shift in the microbial community as the seed sludge developed into granules. Culture isolation and DGGE assays confirmed the dominance of β-Proteobacteria and high-G+C gram-positive bacteria in the phenol-degrading aerobic granules. Of the 10 phenol-degrading bacterial strains isolated from the granules, strains PG-01, PG-02, and PG-08 possessed 16S rRNA gene sequences that matched the partial sequences of dominant bands in the DGGE fingerprint belonging to the aerobic granules. The numerical dominance of strain PG-01 was confirmed by isolation, DGGE, and in situ hybridization with a strain-specific probe, and key physiological traits possessed by PG-01 that allowed it to outcompete and dominate other microorganisms within the granules were then identified. This strain could be regarded as a functionally dominant strain and may have contributed significantly to phenol degradation in the granules. On the other hand, strain PG-08 had low specific growth rate and low phenol degradation ability but showed a high propensity to autoaggregate. By analyzing the roles played by these two isolates within the aerobic granules, a functional model of the microbial community within the aerobic granules was proposed. This model has important implications for rationalizing the engineering of ecological systems.

scholarly journals rDNA analyses of planktonic heterocystous cyanobacteria, including members of the genera Anabaenopsis and Cyanospira The GenBank accession numbers of the 16S rDNA gene sequences reported in this paper are AY038032–AY038037.

Microbiology ◽  
2002 ◽  
Vol 148 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Isabelle Iteman ◽  
Rosmarie Rippka ◽  
Nicole Tandeau de Marsac ◽  
Michael Herdman
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Genomic Sequence ◽  
Sequence Similarity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Heterocystous Cyanobacteria

The taxonomic coherence and phylogenetic relationships of 11 planktonic heterocystous cyanobacterial isolates were examined by investigating two areas of the rRNA operon, the 16S rRNA gene (rrnS) and the internal transcribed spacer (ITS) located between the 16S rRNA and 23S rRNA genes. The rrnS sequences were determined for five strains, including representatives of Anabaena flos-aquae, Aphanizomenon flos-aquae, Nodularia sp. and two alkaliphilic planktonic members of the genera Anabaenopsis and Cyanospira, whose phylogenetic position was previously unknown. Comparison of the data with those previously published for individual groups of planktonic heterocystous cyanobacteria showed that, with the exception of members assigned to the genus Cylindrospermopsis, all the planktonic strains form a distinct subclade within the monophyletic clade of heterocystous cyanobacteria. Within this subclade five different phylogenetic clusters were distinguished. The phylogenetic groupings of Anabaena and Aphanizomenon strains within three of these clusters were not always consistent with their generic or specific assignments based on classical morphological definitions, and the high degree of sequence similarity between strains of Anabaenopsis and Cyanospira suggests that they may be assignable to a single genus. Ribotyping and additional studies performed on PCR amplicons of the 16S rDNA or the ITS for the 11 planktonic heterocystous strains demonstrated that they all contain multiple rrn operons and ITS regions of variable size. Finally, evidence is provided for intra-genomic sequence heterogeneity of the 16S rRNA genes within most of the individual isolates.

scholarly journals Status of the Archaeal and Bacterial Census: an Update

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Patrick D. Schloss ◽  
Rene A. Girard ◽  
Thomas Martin ◽  
Joshua Edwards ◽  
J. Cameron Thrash
Keyword(s):  
16S Rrna ◽  
Bacterial Diversity ◽  
High Throughput Sequencing ◽  
New Technologies ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Short Read ◽  
Sequencing Technologies

ABSTRACT A census is typically carried out for people across a range of geographical levels; however, microbial ecologists have implemented a molecular census of bacteria and archaea by sequencing their 16S rRNA genes. We assessed how well the census of full-length 16S rRNA gene sequences is proceeding in the context of recent advances in high-throughput sequencing technologies because full-length sequences are typically used as references for classification of the short sequences generated by newer technologies. Among the 1,411,234 and 53,546 full-length bacterial and archaeal sequences, 94.5% and 95.1% of the bacterial and archaeal sequences, respectively, belonged to operational taxonomic units (OTUs) that have been observed more than once. Although these metrics suggest that the census is approaching completion, 29.2% of the bacterial and 38.5% of the archaeal OTUs have been observed more than once. Thus, there is still considerable diversity to be explored. Unfortunately, the rate of new full-length sequences has been declining, and new sequences are primarily being deposited by a small number of studies. Furthermore, sequences from soil and aquatic environments, which are known to be rich in bacterial diversity, represent only 7.8 and 16.5% of the census, while sequences associated with host-associated environments represent 55.0% of the census. Continued use of traditional approaches and new technologies such as single-cell genomics and short-read assembly are likely to improve our ability to sample rare OTUs if it is possible to overcome this sampling bias. The success of ongoing efforts to use short-read sequencing to characterize archaeal and bacterial communities requires that researchers strive to expand the depth and breadth of this census. IMPORTANCE The biodiversity contained within the bacterial and archaeal domains dwarfs that of the eukaryotes, and the services these organisms provide to the biosphere are critical. Surprisingly, we have done a relatively poor job of formally tracking the quality of the biodiversity as represented in full-length 16S rRNA genes. By understanding how this census is proceeding, it is possible to suggest the best allocation of resources for advancing the census. We found that the ongoing effort has done an excellent job of sampling the most abundant organisms but struggles to sample the rarer organisms. Through the use of new sequencing technologies, we should be able to obtain full-length sequences from these rare organisms. Furthermore, we suggest that by allocating more resources to sampling environments known to have the greatest biodiversity, we will be able to make significant advances in our characterization of archaeal and bacterial diversity.

Phylogenetic Differentiation of Two Closely RelatedNitrosomonas spp. That Inhabit Different Sediment Environments in an Oligotrophic Freshwater Lake

1999 ◽  
Vol 65 (11) ◽  
pp. 4855-4862 ◽  
Author(s):  
Corinne B. Whitby ◽  
Jon R. Saunders ◽  
Juana Rodriguez ◽  
Roger W. Pickup ◽  
Alan McCarthy
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Freshwater Lake ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ammonia Oxidizing Bacteria ◽  
Relative Distribution ◽  
Sediment Samples ◽  
Oligonucleotide Hybridization

ABSTRACT The population of ammonia-oxidizing bacteria in a temperate oligotrophic freshwater lake was analyzed by recovering 16S ribosomal DNA (rDNA) from lakewater and sediment samples taken throughout a seasonal cycle. Nitrosospira and Nitrosomonas16S rRNA genes were amplified in a nested PCR, and the identity of the products was confirmed by oligonucleotide hybridization.Nitrosospira DNA was readily identified in all samples, and nitrosomonad DNA of the Nitrosomonas europaea-Nitrosomonas eutropha lineage was also directly detected, but during the summer months only. Phylogenetic delineation with partial (345 bp) 16S rRNA gene sequences of clones obtained from sediments confirmed the fidelity of the amplified nitrosomonad DNA and identified two sequence clusters closely related to either N. europaea or N. eutropha that were equated with the littoral and profundal sediment sites, respectively. Determination of 701-bp sequences for 16S rDNA clones representing each cluster confirmed this delineation. A PCR-restriction fragment length polymorphism (RFLP) system was developed that enabled identification of clones containing N. europaea and N. eutropha 16S rDNA sequences, including subclasses therein. It proved possible to analyze 16S rDNA amplified directly from sediment samples to determine the relative abundance of each species compared with that of the other. N. europaea and N. eutropha are very closely related, and direct evidence for their presence in lake systems is limited. The correlation of each species with a distinct spatial location in sediment is an unusual example of niche adaptation by two genotypically similar bacteria. Their occurrence and relative distribution can now be routinely monitored in relation to environmental variation by the application of PCR-RFLP analysis.

Polynucleotide Probes That Target a Hypervariable Region of 16S rRNA Genes To Identify Bacterial Isolates Corresponding to Bands of Community Fingerprints

1999 ◽  
Vol 65 (3) ◽  
pp. 1045-1049 ◽  
Author(s):  
Holger Heuer ◽  
Kathrin Hartung ◽  
Gabriele Wieland ◽  
Ina Kramer ◽  
Kornelia Smalla
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Hypervariable Region ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Isolates ◽  
Closely Related Species ◽  
Gradient Gel Electrophoresis ◽  
Exonuclease Digestion ◽  
Temperature Gradient Gel Electrophoresis

ABSTRACT Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in hybridizations with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (Escherichia coli 16S rDNA sequence), which is a subset of the region used for TGGE (positions 968 to 1401), best met the criteria of high phylogenetic variability, required for sufficient probe specificity, and closely flanking conserved priming sites for amplification. Removal of flanking conserved bases was necessary to enable the differentiation of closely related species. This was achieved by 5′ exonuclease digestion, terminated by phosphorothioate bonds which were synthesized into the primers. The remaining complementary strand was removed by single-strand-specific digestion. Standard hybridization with truncated probes allowed differentiation of bacteria which differed by only two bases within the probe target site and 1.2% within the complete 16S rDNA. However, a truncated probe, derived from an excised TGGE band of a rhizosphere community, hybridized with three phylogenetically related isolates with identical V6 sequences. Only one of the isolates comigrated with the excised band in TGGE, which was shown to be due to identical sequences, demonstrating the utility of a combined TGGE and V6 probe approach.

scholarly journals Metagenomic Analysis of Slovak Bryndza Cheese Using Next-Generation 16S rDNA Amplicon Sequencing

2016 ◽  
Vol 15 (1) ◽  
pp. 23-34 ◽  
Author(s):  
Matej Planý ◽  
Tomáš Kuchta ◽  
Katarína Šoltýs ◽  
Tomáš Szemes ◽  
Domenico Pangallo ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Lactococcus Lactis ◽  
Microbial Population ◽  
Starter Culture ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Next Generation ◽  
16S Rdna Amplicon Sequencing

Abstract Knowledge about diversity and taxonomic structure of the microbial population present in traditional fermented foods plays a key role in starter culture selection, safety improvement and quality enhancement of the end product. Aim of this study was to investigate microbial consortia composition in Slovak bryndza cheese. For this purpose, we used culture-independent approach based on 16S rDNA amplicon sequencing using next generation sequencing platform. Results obtained by the analysis of three commercial (produced on industrial scale in winter season) and one traditional (artisanal, most valued, produced in May) Slovak bryndza cheese sample were compared. A diverse prokaryotic microflora composed mostly of the genera Lactococcus, Streptococcus, Lactobacillus, and Enterococcus was identified. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were the dominant taxons in all tested samples. Second most abundant species, detected in all bryndza cheeses, were Lactococcus fujiensis and Lactococcus taiwanensis, independently by two different approaches, using different reference 16S rRNA genes databases (Greengenes and NCBI respectively). They have been detected in bryndza cheese samples in substantial amount for the first time. The narrowest microbial diversity was observed in a sample made with a starter culture from pasteurised milk. Metagenomic analysis by high-throughput sequencing using 16S rRNA genes seems to be a powerful tool for studying the structure of the microbial population in cheeses.

scholarly journals Intragenomic homogeneity on Liberibacter 16S rDNA confirms phylogeny and explains ecological strategy

2015 ◽  
Author(s):  
Warrick R Nelson ◽  
Nelson A Wulff ◽  
Joseph M Bové
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Phylogenetic Signal ◽  
Metabolic Cost ◽  
Growth Conditions ◽  
The Other ◽  
Population Increase ◽  
Rrna Gene ◽  
Reverse Complement ◽  
Multiple Copies

Three of the five currently recognized “Candidatus Liberibacter” spp., “Ca. L. asiaticus” (Las), “Ca. L. americanus” (Lam) and “Ca. L. solanacearum”, and the newly erected genus Liberibacter species, L. crescens (Lcr), have had their genomes sequenced. In all four cases there are three homogeneous copies of the 16S rRNA gene, one present as the reverse complement of the other two. 16S intragenic homogeneity is common within the α-Proteobacteria. The presence of three 16S rRNA copies indicates an advantage for a rapid response of population increase to favourable growth conditions. The metabolic cost of carrying multiple copies is avoided during periods of low cellular activity as this situation occurs at low temperatures, for example overwintering in deciduous plants or in a dormant insect host. A large insertion in the 16S rDNA sequence of three species compared to the other three species indicates a dichotomy in the Liberibacter genus and provides a phylogenetic signal of closeness to the proximal node within the Rhizobiaceae. In spite of similar symptoms in Citrus crops associated with Lam and Las infections, these species belong on either side of this dichotomy, thus confirming Lam as phylogenetically closer to the proximal node than Las.

Analyses of microbial activity in biomass-recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products

2002 ◽  
Vol 48 (4) ◽  
pp. 333-341 ◽  
Author(s):  
Christine A Morgan ◽  
Andre Hudson ◽  
Allan Konopka ◽  
Cindy H Nakatsu
Keyword(s):  
Community Structure ◽  
16S Rrna ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Community Response ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Specific Content ◽  
Gradient Gel Electrophoresis

The relationship between mixed microbial community structure and physiology when grown under substrate-limited conditions was investigated using continuous-flow bioreactors with 100% biomass recycle. Community structure was analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR and RT-PCR amplified V3 region of 16S rDNA and 16S rRNA templates, respectively. Comparisons were made of communities exposed to different types of transient conditions (e.g., long- and short-term starvation, increasing nutrients). With progressively more stringent substrate limitation over time, the specific content of community RNA declined by more than 10-fold and closely followed the decline in specific growth rate. In contrast, the DNA content was variable (up to 3-fold differences) and did not follow the same trend. Cluster analysis of the presence or absence of individual bands indicated that the fingerprints generated by the two templates were different, and community response was first observed in the rRNA fraction. However, both the rDNA and rRNA fingerprints provided a picture of temporal population dynamics. Dice similarity coefficients gave a quantitative measure of the differences and changes between the communities. In comparison, standard cultivation techniques yielded only a quarter of the phylotypes detected by DGGE, but included the most dominant population based on rRNA. Nucleotide-sequence analyses of the almost complete 16S rRNA genes of these isolates place them in the same group of organisms that is typically cultivated from environmental samples: α, β, and γ Proteobacteria and the high GC and the low GC Gram-positive divisions.Key words: 16S rRNA, DGGE, community analysis, biomass-recycle reactor.

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