An assessment of the hypervariable domains of the 16S rRNA genes for their value in determining microbial community diversity: the paradox of traditional ecological indices

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

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

Applied and Environmental Microbiology ◽

10.1128/aem.70.11.6767-6775.2004 ◽

2004 ◽

Vol 70 (11) ◽

pp. 6767-6775 ◽

Cited By ~ 98

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.

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Bacterial Community Diversity of Oil-Contaminated Soils Assessed by High Throughput Sequencing of 16S rRNA Genes

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph121012002 ◽

2015 ◽

Vol 12 (10) ◽

pp. 12002-12015 ◽

Cited By ~ 48

Author(s):

Mu Peng ◽

Xiaoxue Zi ◽

Qiuyu Wang

Keyword(s):

Bacterial Community ◽

16S Rrna ◽

High Throughput ◽

Contaminated Soils ◽

High Throughput Sequencing ◽

Community Diversity ◽

16S Rrna Genes ◽

Rrna Genes ◽

Bacterial Community Diversity

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Comparison of Microbial Community Compositions of Two Subglacial Environments Reveals a Possible Role for Microbes in Chemical Weathering Processes

Applied and Environmental Microbiology ◽

10.1128/aem.71.11.6986-6997.2005 ◽

2005 ◽

Vol 71 (11) ◽

pp. 6986-6997 ◽

Cited By ~ 186

Author(s):

Mark Skidmore ◽

Suzanne P. Anderson ◽

Martin Sharp ◽

Julia Foght ◽

Brian D. Lanoil

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Microbial Communities ◽

Blot Hybridization ◽

16S Rrna Genes ◽

Rrna Genes ◽

Solute Flux ◽

Carbonate Dissolution ◽

Dot Blot ◽

Dot Blot Hybridization

ABSTRACT Viable microbes have been detected beneath several geographically distant glaciers underlain by different lithologies, but comparisons of their microbial communities have not previously been made. This study compared the microbial community compositions of samples from two glaciers overlying differing bedrock. Bulk meltwater chemistry indicates that sulfide oxidation and carbonate dissolution account for 90% of the solute flux from Bench Glacier, Alaska, whereas gypsum/anhydrite and carbonate dissolution accounts for the majority of the flux from John Evans Glacier, Ellesmere Island, Nunavut, Canada. The microbial communities were examined using two techniques: clone libraries and dot blot hybridization of 16S rRNA genes. Two hundred twenty-seven clones containing amplified 16S rRNA genes were prepared from subglacial samples, and the gene sequences were analyzed phylogenetically. Although some phylogenetic groups, including the Betaproteobacteria, were abundant in clone libraries from both glaciers, other well-represented groups were found at only one glacier. Group-specific oligonucleotide probes were developed for two phylogenetic clusters that were of particular interest because of their abundance or inferred biochemical capabilities. These probes were used in quantitative dot blot hybridization assays with a range of samples from the two glaciers. In addition to shared phyla at both glaciers, each glacier also harbored a subglacial microbial population that correlated with the observed aqueous geochemistry. These results are consistent with the hypothesis that microbial activity is an important contributor to the solute flux from glaciers.

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Different Active Microbial Communities in Two Contrasted Subantarctic Fjords

Frontiers in Microbiology ◽

10.3389/fmicb.2021.620220 ◽

2021 ◽

Vol 12 ◽

Author(s):

Claudia Maturana-Martínez ◽

Camila Fernández ◽

Humberto E. González ◽

Pierre E. Galand

Keyword(s):

Climate Change ◽

Microbial Community ◽

16S Rrna ◽

Microbial Communities ◽

Standing Stock ◽

16S Rrna Genes ◽

Rrna Genes ◽

Biogeochemical Processes ◽

Biogeographic Patterns ◽

Group I

Microorganisms play a crucial role in biogeochemical processes affecting the primary production and biogeochemical cycles of the ocean. In subpolar areas, the increment of the water temperature induced by climate change could lead to changes in the structure and activity of planktonic microbial communities. To understand how the structure of the microbial community in Chilean Patagonian fjords could be affected by climate change, we analyzed the composition of the prokaryotic community (bacteria-archaea) in two fjords (Pia and Yendegaia) with contrasting morphological and hydrological features. We targeted both the standing stock (16S rRNA genes) and the active fraction (16S rRNA transcripts) of the microbial communities during two consecutive austral winters. Our results showed that in both fjords, the active community had higher diversity and stronger biogeographic patterns when compared to the standing stock. Members of the Alpha-, Gamma-, and Deltaproteobacteria followed by archaea from the Marine Group I (Thaumarchaeota) dominated the active communities in both fjords. However, in Pia fjord, which has a marine-terminating glacier, the composition of the microbial community was directly influenced by the freshwater discharges from the adjacent glacier, and indirectly by a possible upwelling phenomenon that could bring deep sea bacteria such as SAR202 to the surface layer. In turn, in the Yendegaia, which has a land-terminating glacier, microbial communities were more similar to the ones described in oceanic waters. Furthermore, in Yendegaia fjord, inter-annual differences in the taxonomic composition and diversity of the microbial community were observed. In conclusion, Yendegaia fjord, without glacier calving, represents a fjord type that will likely be more common under future climate scenarios. Our results showing distinct Yendegaia communities, with for example more potential nitrogen-fixing microorganisms (Planctomycetes), indicate that as a result of climate change, changing planktonic communities could potentially impact biogeochemical processes and nutrient sources in subantarctic fjords.

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Microbial community diversity of Jinghong laying hens at peak production based on 16S rRNA sequencing

Journal of Applied Animal Research ◽

10.1080/09712119.2018.1520713 ◽

2018 ◽

Vol 46 (1) ◽

pp. 1430-1436 ◽

Cited By ~ 2

Author(s):

Shijun Fu ◽

Shijin Guo ◽

Jianjun Wang ◽

Yumao Wang ◽

Zhimei Zhang ◽

...

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Laying Hens ◽

Community Diversity ◽

16S Rrna Sequencing ◽

Microbial Community Diversity ◽

Rrna Sequencing

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Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State

Applied and Environmental Microbiology ◽

10.1128/aem.00455-08 ◽

2008 ◽

Vol 74 (15) ◽

pp. 4877-4888 ◽

Cited By ~ 57

Author(s):

Pedro A. Dimitriu ◽

Holly C. Pinkart ◽

Brent M. Peyton ◽

Melanie R. Mormile

Keyword(s):

16S Rrna ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Community Composition ◽

Soda Lake ◽

Community Diversity ◽

16S Rrna Genes ◽

Rrna Genes ◽

Most Probable Number ◽

Rrna Gene ◽

Probable Number

ABSTRACT The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.

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The relationship between benthic nutrient fluxes and bacterial community in Aquaculture tail-water treatment systems

10.1101/2021.08.18.456883 ◽

2021 ◽

Author(s):

Regan Nicholaus ◽

Betina Lukwambe ◽

Wen Yang ◽

Zhongming Zheng

Keyword(s):

Microbial Community ◽

Bacterial Community ◽

16S Rrna ◽

Constructed Wetland ◽

High Throughput Sequencing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Nutrient Fluxes ◽

Benthic Nutrient Fluxes ◽

Tail Water

Constructed-wetlands, Biofilms, and sedimentation are potential aquaculture tail-water treatments however their roles on the distribution of benthic microbial community and the way they affect the interaction between microbial community and inorganic nutrient fluxes have not been fully explored. This study applied 16S rRNA high-throughput sequencing technology to investigate the microbial community distribution and their link with nutrient fluxes in an aquaculture tail- water bioremediation system . Results showed that bacterial community compositions were significantly different in constructed-wetland and biofilm treatments (p<0.05) relative to sedimentation. The composition of the 16S rRNA genes among all the treatments was enriched with Proteobacteria, Bacteroidetes, Firmicutes, and Flavobacteria . NMDS analysis showed that the bacterial composition in constructed-wetland and biofilm samples clustered separately compared to those in sedimentation. The Functional-Annotation-of-Prokaryotic-Taxa analysis indicated that the proportions of sediment-microbial-functional groups (aerobic-chemoheterophy, chemoheterotrophy, and nitrate-ammonification combined) in the constructed-wetland treatment were 47%, 32% in biofilm and 13% in sedimentation system. Benthic-nutrient fluxes for phosphate, ammonium, nitrite, nitrate and sediment oxygen consumption differed markedly among the treatments ( p<0.05 ). Canonical correspondence analysis indicated constructed-wetland had the strongest association between biogeochemical contents and the bacterial community relative to other treatments. This study suggests that the microbial community distributions and their interactions nutrient fluxes were most improved in the constructed-wetland followed by the area under biofilm and sedimentation treatment.

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Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.66.7.2943-2950.2000 ◽

2000 ◽

Vol 66 (7) ◽

pp. 2943-2950 ◽

Cited By ~ 315

Author(s):

John Dunbar ◽

Lawrence O. Ticknor ◽

Cheryl R. Kuske

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rdna ◽

Restriction Fragment ◽

Community Diversity ◽

Detection Sensitivity ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Clone Libraries

ABSTRACT The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662–1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.

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