The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice using a 454 GS FLX Titanium pyrosequencer

Chun, Jongsik; Kim, Kap Y; Lee, Jae-Hak; Choi, Youngnim

doi:10.1186/1471-2180-10-101

Cited by 213 publications

(152 citation statements)

References 24 publications

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“…All three isolates or their close relatives have been identified in mice oral cavities from a separate study using 454-pyrosequecning analysis (Chun et al, 2010), however, their physiological or biological functions have not been investigated. Our synthetic community data demonstrated that the E. coli-induced colonization resistance required the participation of these three Microbial social structure involved in colonization resistance X He et al players, and missing any one of them resulted in a greatly reduced colonization resistance against the integration of E. coli.…”

Section: Discussionmentioning

confidence: 99%

The social structure of microbial community involved in colonization resistance

He¹,

McLean

Guo³

et al. 2013

The ISME Journal

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It is well established that host-associated microbial communities can interfere with the colonization and establishment of microbes of foreign origins, a phenomenon often referred to as bacterial interference or colonization resistance. However, due to the complexity of the indigenous microbiota, it has been extremely difficult to elucidate the community colonization resistance mechanisms and identify the bacterial species involved. In a recent study, we have established an in vitro mice oral microbial community (O-mix) and demonstrated its colonization resistance against an Escherichia coli strain of mice gut origin. In this study, we further analyzed the community structure of the O-mix by using a dilution/regrowth approach and identified the bacterial species involved in colonization resistance against E. coli. Our results revealed that, within the O-mix there were three different types of bacterial species forming unique social structure. They act as 'Sensor', 'Mediator' and 'Killer', respectively, and have coordinated roles in initiating the antagonistic action and preventing the integration of E. coli. The functional role of each identified bacterial species was further confirmed by E. coli-specific responsiveness of the synthetic communities composed of different combination of the identified players. The study reveals for the first time the sophisticated structural and functional organization of a colonization resistance pathway within a microbial community. Furthermore, our results emphasize the importance of 'Facilitation' or positive interactions in the development of community-level functions, such as colonization resistance.

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Section: Discussionmentioning

confidence: 99%

The social structure of microbial community involved in colonization resistance

He¹,

McLean

Guo³

et al. 2013

The ISME Journal

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“…To ensure the relative symbiont purity in the nucleic acid extractions, we examined the microbial composition of the 16S ribosomal RNA gene (V1-V3 region) via pyrosequencing, according to Chun et al (2010) (see Supplementary Methods for more details). Subsequent genomic analysis was performed on two samples that were determined to be relatively pure, one from each symbiont type, based on dominance of the target Oceanospirillales symbiont, which comprised 84% and 56% of the recovered 16S rRNA barcodes for symbionts Rs1 and Rs2, respectively (Supplementary Figure S1).…”

Section: Sample Collectionmentioning

confidence: 99%

Genomic versatility and functional variation between two dominant heterotrophic symbionts of deep-sea Osedax worms

Goffredi

Zhang

et al. 2013

The ISME Journal

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An unusual symbiosis, first observed at B3000 m depth in the Monterey Submarine Canyon, involves gutless marine polychaetes of the genus Osedax and intracellular endosymbionts belonging to the order Oceanospirillales. Ecologically, these worms and their microbial symbionts have a substantial role in the cycling of carbon from deep-sea whale fall carcasses. Microheterogeneity exists among the Osedax symbionts examined so far, and in the present study the genomes of the two dominant symbionts, Rs1 and Rs2, were sequenced. The genomes revealed heterotrophic versatility in carbon, phosphate and iron uptake, strategies for intracellular survival, evidence for an independent existence, and numerous potential virulence capabilities. The presence of specific permeases and peptidases (of glycine, proline and hydroxyproline), and numerous peptide transporters, suggests the use of degraded proteins, likely originating from collagenous bone matter, by the Osedax symbionts. 13 C tracer experiments confirmed the assimilation of glycine/ proline, as well as monosaccharides, by Osedax. The Rs1 and Rs2 symbionts are genomically distinct in carbon and sulfur metabolism, respiration, and cell wall composition, among others. Differences between Rs1 and Rs2 and phylogenetic analysis of chemotaxis-related genes within individuals of symbiont Rs1 revealed the influence of the relative age of the whale fall environment and support possible local niche adaptation of 'free-living' lifestages. Future genomic examinations of other horizontally-propogated intracellular symbionts will likely enhance our understanding of the contribution of intraspecific symbiont diversity to the ecological diversification of the intact association, as well as the maintenance of host diversity.

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“…At present, the 454 pyrosequencing technology has been widely used to reveal microbial diversity and ecology in different natural environments, such as the deep sea (Sogin et al, 2006;Huber et al, 2007), extreme hydrogeological conditions (Edwards et al, 2006) and soil (Leininger et al, 2006;Roesch et al, 2007). In addition, multiplexed high-throughput pyrosequencing of individual genes (for example, 16S rRNA) by tagging or bar coding with short nucleotides (also called pyrotag sequencing ) Parameswaran et al, 2007;Roesch et al, 2007;Hamady et al, 2008) has been developed to process many samples simultaneously and has been widely used in microbial community studies (Youssef et al, 2009;Cheung et al, 2010;Chun et al, 2010;He et al, 2010a, b;Koopman et al, 2010;Schutte et al, 2010;Teixeira et al, 2010;Uroz et al, 2010). A similar strategy has also been used for sequencing functional genes (Iwai et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Reproducibility and quantitation of amplicon sequencing-based detection

et al. 2011

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To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2% ± 2.3% between two technical replicates, and 8.2% ± 2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating b-diversity but less on a-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative analysis of the b-diversity of microbial communities.

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The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice using a 454 GS FLX Titanium pyrosequencer

Cited by 213 publications

References 24 publications

The social structure of microbial community involved in colonization resistance

The social structure of microbial community involved in colonization resistance

Genomic versatility and functional variation between two dominant heterotrophic symbionts of deep-sea Osedax worms

Reproducibility and quantitation of amplicon sequencing-based detection

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