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A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community.

Vital M, Penton CR, Wang Q, Young VB, Antonopoulos DA, Sogin ML, Morrison HG, Raffals L, Chang EB, Huffnagle GB, Schmidt TM, Cole JR, Tiedje JM - Microbiome (2013)

Bottom Line: As a result, reliable information on this important bacterial group is often lacking in microbiome research.Most butyrate producers identified in previous studies were detected and the general patterns of taxa found were supported by 16S rRNA gene pyrotag analysis, but the gene-targeted approach provided more detail about the potential butyrate-producing members of the community.Furthermore, our analysis refines but and buk reference annotations found in central databases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA. colej@msu.edu.

ABSTRACT

Background: Butyrate, which is produced by the human microbiome, is essential for a well-functioning colon. Bacteria that produce butyrate are phylogenetically diverse, which hinders their accurate detection based on conventional phylogenetic markers. As a result, reliable information on this important bacterial group is often lacking in microbiome research.

Results: In this study we describe a gene-targeted approach for 454 pyrotag sequencing and quantitative polymerase chain reaction for the final genes in the two primary bacterial butyrate synthesis pathways, butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk). We monitored the establishment and early succession of butyrate-producing communities in four patients with ulcerative colitis who underwent a colectomy with ileal pouch anal anastomosis and compared it with three control samples from healthy colons. All patients established an abundant butyrate-producing community (approximately 5% to 26% of the total community) in the pouch within the 2-month study, but patterns were distinctive among individuals. Only one patient harbored a community profile similar to the healthy controls, in which there was a predominance of but genes that are similar to reference genes from Acidaminococcus sp., Eubacterium sp., Faecalibacterium prausnitzii and Roseburia sp., and an almost complete absence of buk genes. Two patients were greatly enriched in buk genes similar to those of Clostridium butyricum and C. perfringens, whereas a fourth patient displayed abundant communities containing both genes. Most butyrate producers identified in previous studies were detected and the general patterns of taxa found were supported by 16S rRNA gene pyrotag analysis, but the gene-targeted approach provided more detail about the potential butyrate-producing members of the community.

Conclusions: The presented approach provides quantitative and genotypic insights into butyrate-producing communities and facilitates a more specific functional characterization of the intestinal microbiome. Furthermore, our analysis refines but and buk reference annotations found in central databases.

No MeSH data available.


Related in: MedlinePlus

Analysis of obtained butyrate kinase (buk) sequences.(A) Neighbor joining tree of all buk reference sequences(closest hit from FrameBot) matching our amplicon data. Amount of ampliconsequences per closest match and reference sequence GI number (GenBank) areprovided. Sequences marked with * or + highlight known butyrateproducers [5,29].Pie charts illustrate the origin of amplicon sequences (red: patient 200,green: patient 206, blue: patient 207, yellow: patient 210 and grey: healthycontrols). Note: relative abundance was investigated and the proportion of eachcolor in the pie charts does not correspond to actual abundance of genes insamples. Relative community patterns per individual sample are presented inAdditional file 1: Figure S6. Bootstrap values areindicated. (B) Conservation analysis of reference sequences from PanelA (dashed line) and of obtained amplicon sequences (thick grey line).The displayed sequence on top corresponds to the consensus sequence of allreference sequences. Bold amino acids demonstrate conserved sites (>95%) inboth reference and amplicon sequences. Blue amino acids on both sequence endsillustrate primer binding sites.
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Figure 2: Analysis of obtained butyrate kinase (buk) sequences.(A) Neighbor joining tree of all buk reference sequences(closest hit from FrameBot) matching our amplicon data. Amount of ampliconsequences per closest match and reference sequence GI number (GenBank) areprovided. Sequences marked with * or + highlight known butyrateproducers [5,29].Pie charts illustrate the origin of amplicon sequences (red: patient 200,green: patient 206, blue: patient 207, yellow: patient 210 and grey: healthycontrols). Note: relative abundance was investigated and the proportion of eachcolor in the pie charts does not correspond to actual abundance of genes insamples. Relative community patterns per individual sample are presented inAdditional file 1: Figure S6. Bootstrap values areindicated. (B) Conservation analysis of reference sequences from PanelA (dashed line) and of obtained amplicon sequences (thick grey line).The displayed sequence on top corresponds to the consensus sequence of allreference sequences. Bold amino acids demonstrate conserved sites (>95%) inboth reference and amplicon sequences. Blue amino acids on both sequence endsillustrate primer binding sites.

Mentions: For each gene, the frameshift-corrected protein sequences were aligned using HMMER3and clustered using RDP mcClust with the complete-linkage algorithm. Only ampliconswith an identity of ≥70% to the closest matches in the reference (97% ofbut and 93% of buk sequences) were used for additionalphylogenetic tree and ordination analysis, as we were not confident that more distantmatches were bona fide but or buk. Additional filtering wasperformed based on neighbor joining tree analysis of reference sequences (see Resultsand Additional file 1). The remaining sequences were binnedaccording to closest match assignments with reference sequences showing less than 2%dissimilarity merged (based on Figures 1 and 2). Results of both genes were combined and the entire butyratecommunity of each sample was used for ordination analysis. The nonmetricmultidimensional scaling based on Chao corrected Jaccard index distance was performedusing the vegan package in the R environment [21]. Both patients and time points were grouped for analysis.Diversity analysis (Shannon index) was calculated using the Biodiversity Rpackage.


A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community.

Vital M, Penton CR, Wang Q, Young VB, Antonopoulos DA, Sogin ML, Morrison HG, Raffals L, Chang EB, Huffnagle GB, Schmidt TM, Cole JR, Tiedje JM - Microbiome (2013)

Analysis of obtained butyrate kinase (buk) sequences.(A) Neighbor joining tree of all buk reference sequences(closest hit from FrameBot) matching our amplicon data. Amount of ampliconsequences per closest match and reference sequence GI number (GenBank) areprovided. Sequences marked with * or + highlight known butyrateproducers [5,29].Pie charts illustrate the origin of amplicon sequences (red: patient 200,green: patient 206, blue: patient 207, yellow: patient 210 and grey: healthycontrols). Note: relative abundance was investigated and the proportion of eachcolor in the pie charts does not correspond to actual abundance of genes insamples. Relative community patterns per individual sample are presented inAdditional file 1: Figure S6. Bootstrap values areindicated. (B) Conservation analysis of reference sequences from PanelA (dashed line) and of obtained amplicon sequences (thick grey line).The displayed sequence on top corresponds to the consensus sequence of allreference sequences. Bold amino acids demonstrate conserved sites (>95%) inboth reference and amplicon sequences. Blue amino acids on both sequence endsillustrate primer binding sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126176&req=5

Figure 2: Analysis of obtained butyrate kinase (buk) sequences.(A) Neighbor joining tree of all buk reference sequences(closest hit from FrameBot) matching our amplicon data. Amount of ampliconsequences per closest match and reference sequence GI number (GenBank) areprovided. Sequences marked with * or + highlight known butyrateproducers [5,29].Pie charts illustrate the origin of amplicon sequences (red: patient 200,green: patient 206, blue: patient 207, yellow: patient 210 and grey: healthycontrols). Note: relative abundance was investigated and the proportion of eachcolor in the pie charts does not correspond to actual abundance of genes insamples. Relative community patterns per individual sample are presented inAdditional file 1: Figure S6. Bootstrap values areindicated. (B) Conservation analysis of reference sequences from PanelA (dashed line) and of obtained amplicon sequences (thick grey line).The displayed sequence on top corresponds to the consensus sequence of allreference sequences. Bold amino acids demonstrate conserved sites (>95%) inboth reference and amplicon sequences. Blue amino acids on both sequence endsillustrate primer binding sites.
Mentions: For each gene, the frameshift-corrected protein sequences were aligned using HMMER3and clustered using RDP mcClust with the complete-linkage algorithm. Only ampliconswith an identity of ≥70% to the closest matches in the reference (97% ofbut and 93% of buk sequences) were used for additionalphylogenetic tree and ordination analysis, as we were not confident that more distantmatches were bona fide but or buk. Additional filtering wasperformed based on neighbor joining tree analysis of reference sequences (see Resultsand Additional file 1). The remaining sequences were binnedaccording to closest match assignments with reference sequences showing less than 2%dissimilarity merged (based on Figures 1 and 2). Results of both genes were combined and the entire butyratecommunity of each sample was used for ordination analysis. The nonmetricmultidimensional scaling based on Chao corrected Jaccard index distance was performedusing the vegan package in the R environment [21]. Both patients and time points were grouped for analysis.Diversity analysis (Shannon index) was calculated using the Biodiversity Rpackage.

Bottom Line: As a result, reliable information on this important bacterial group is often lacking in microbiome research.Most butyrate producers identified in previous studies were detected and the general patterns of taxa found were supported by 16S rRNA gene pyrotag analysis, but the gene-targeted approach provided more detail about the potential butyrate-producing members of the community.Furthermore, our analysis refines but and buk reference annotations found in central databases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA. colej@msu.edu.

ABSTRACT

Background: Butyrate, which is produced by the human microbiome, is essential for a well-functioning colon. Bacteria that produce butyrate are phylogenetically diverse, which hinders their accurate detection based on conventional phylogenetic markers. As a result, reliable information on this important bacterial group is often lacking in microbiome research.

Results: In this study we describe a gene-targeted approach for 454 pyrotag sequencing and quantitative polymerase chain reaction for the final genes in the two primary bacterial butyrate synthesis pathways, butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk). We monitored the establishment and early succession of butyrate-producing communities in four patients with ulcerative colitis who underwent a colectomy with ileal pouch anal anastomosis and compared it with three control samples from healthy colons. All patients established an abundant butyrate-producing community (approximately 5% to 26% of the total community) in the pouch within the 2-month study, but patterns were distinctive among individuals. Only one patient harbored a community profile similar to the healthy controls, in which there was a predominance of but genes that are similar to reference genes from Acidaminococcus sp., Eubacterium sp., Faecalibacterium prausnitzii and Roseburia sp., and an almost complete absence of buk genes. Two patients were greatly enriched in buk genes similar to those of Clostridium butyricum and C. perfringens, whereas a fourth patient displayed abundant communities containing both genes. Most butyrate producers identified in previous studies were detected and the general patterns of taxa found were supported by 16S rRNA gene pyrotag analysis, but the gene-targeted approach provided more detail about the potential butyrate-producing members of the community.

Conclusions: The presented approach provides quantitative and genotypic insights into butyrate-producing communities and facilitates a more specific functional characterization of the intestinal microbiome. Furthermore, our analysis refines but and buk reference annotations found in central databases.

No MeSH data available.


Related in: MedlinePlus