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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

Exploring the butyrate-producing bacterial community based on 16SrRNA gene analysis. (A) Candidates were split intobutyryl-CoA:acetate CoA-transferase (but; grey bars) and butyratekinase (buk; coarse white bars) containing groups. (B)Individual composition of but (Acidaminococcus sp. - olive,Anaerostipes sp. - dark green, Coprococcus sp. - dark red,Eubacterium sp. - black, Faecalibacterium sp. - darkpurple, Megasphaera sp. - light purple,Peptoniphilus sp. - blue, Oscillibacter sp. - grey andRoseburia sp. - orange) and buk (Anaerotruncus -coarse grey, C. beijerinckii - coarse white, C. butyricum -coarse light blue, C. perfringens - coarse light yellow,Enterococcus sp. - light green and Subdoligranulum sp. -coarse light red) communities are given. (C) Quantitative PCR datatargeting the 16S genes of Faecalibacterium sp. (purple bar),Roseburia sp./E. rectale (orange bar) and C.butyricum (white coarse bar). Note: Coprococcus sp. isconsidered to contain both but and buk genes. The error barsrepresent the range on duplicate measurements. All results are corrected formultiple 16S rRNA copy numbers of individual bacteria (see Methods). ?- Butyrate production was shown for one strain of Subdoligranulum sp.and it is unclear whether all members of this genus have the ability tosynthesize butyrate.
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Figure 6: Exploring the butyrate-producing bacterial community based on 16SrRNA gene analysis. (A) Candidates were split intobutyryl-CoA:acetate CoA-transferase (but; grey bars) and butyratekinase (buk; coarse white bars) containing groups. (B)Individual composition of but (Acidaminococcus sp. - olive,Anaerostipes sp. - dark green, Coprococcus sp. - dark red,Eubacterium sp. - black, Faecalibacterium sp. - darkpurple, Megasphaera sp. - light purple,Peptoniphilus sp. - blue, Oscillibacter sp. - grey andRoseburia sp. - orange) and buk (Anaerotruncus -coarse grey, C. beijerinckii - coarse white, C. butyricum -coarse light blue, C. perfringens - coarse light yellow,Enterococcus sp. - light green and Subdoligranulum sp. -coarse light red) communities are given. (C) Quantitative PCR datatargeting the 16S genes of Faecalibacterium sp. (purple bar),Roseburia sp./E. rectale (orange bar) and C.butyricum (white coarse bar). Note: Coprococcus sp. isconsidered to contain both but and buk genes. The error barsrepresent the range on duplicate measurements. All results are corrected formultiple 16S rRNA copy numbers of individual bacteria (see Methods). ?- Butyrate production was shown for one strain of Subdoligranulum sp.and it is unclear whether all members of this genus have the ability tosynthesize butyrate.

Mentions: We retrieved the major known butyrate-producing taxa from literature [5] and from the but and buk data andused this information to screen for those taxa in the total 16S 454 pyrotag analysispresented in the accompanying paper [14].Results are displayed in Figure 6A. Additionally, qPCRtargeting specific butyrate producers was performed (Figure 6C). 16S rRNA gene analysis supported the functional gene resultsin that similar overall patterns were detected by the two different techniques.Communities linked to buk were dominated by sequences similar to those ofC. butyricum and C. perfringens, whereas sequences similar toAcidaminococcus sp., F. prausnitzii and Roseburia sp.comprised the majority of but-associated bacteria in both methods(Figures 5 and 6, Additionalfile 1: Figures S3 and S4). Nevertheless, severaldifferences between 16S rRNA gene and functional gene analysis wereobserved. Only a minute fraction from 16S rRNA gene pyrotag data wasidentified as Eubacterium sp., whereas many but sequences wereassigned to strains of E. hallii and E. rectale. Other studies thatutilized fluorescence in-situ hybridization and clone libraries reportedhigh concentrations of those strains in the healthy colonic microbial flora[4,25], whichsuggests that 16S rRNA gene-based analysis could not reliably discriminatethem from other taxa. Furthermore, Subdoligranulum sp., which contain onebutyrate-producing isolate, S. variabile ([26] has the gene buk), were not detected in thefunctional gene data. But if this genus is considered to be butyrate-producing, thenthe 16S rRNA gene analysis suggests a considerable abundance of bukgenes in healthy control samples. Similarly, many more 16S rRNA genesequences were assigned to Acidaminococcus sp., Anaerostipes sp.,Coprococcus sp. and Peptoniphilus sp. in certain samplescompared with the results obtained from the functional gene analysis. These findingssupport earlier reports that butyrate synthesis is often not a homogenous feature ofall members of a genus [4,5] and strengthens the application of higher taxonomic resolutiontechniques to adequately assess the butyrate-producing potential of bacterialcommunities. Species resolution is also crucial for the functionally diverse genusClostridia. Several butyrate-producing members such asClostridium sp. SS2/1, Clostridium sp. M62/1, C.acetobutylicum, C. carboxidivorans and C. symbiosum were matched tonumerous functional gene sequences, but could not be detected in the 16SrRNA gene data.


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)

Exploring the butyrate-producing bacterial community based on 16SrRNA gene analysis. (A) Candidates were split intobutyryl-CoA:acetate CoA-transferase (but; grey bars) and butyratekinase (buk; coarse white bars) containing groups. (B)Individual composition of but (Acidaminococcus sp. - olive,Anaerostipes sp. - dark green, Coprococcus sp. - dark red,Eubacterium sp. - black, Faecalibacterium sp. - darkpurple, Megasphaera sp. - light purple,Peptoniphilus sp. - blue, Oscillibacter sp. - grey andRoseburia sp. - orange) and buk (Anaerotruncus -coarse grey, C. beijerinckii - coarse white, C. butyricum -coarse light blue, C. perfringens - coarse light yellow,Enterococcus sp. - light green and Subdoligranulum sp. -coarse light red) communities are given. (C) Quantitative PCR datatargeting the 16S genes of Faecalibacterium sp. (purple bar),Roseburia sp./E. rectale (orange bar) and C.butyricum (white coarse bar). Note: Coprococcus sp. isconsidered to contain both but and buk genes. The error barsrepresent the range on duplicate measurements. All results are corrected formultiple 16S rRNA copy numbers of individual bacteria (see Methods). ?- Butyrate production was shown for one strain of Subdoligranulum sp.and it is unclear whether all members of this genus have the ability tosynthesize butyrate.
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Related In: Results  -  Collection

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Figure 6: Exploring the butyrate-producing bacterial community based on 16SrRNA gene analysis. (A) Candidates were split intobutyryl-CoA:acetate CoA-transferase (but; grey bars) and butyratekinase (buk; coarse white bars) containing groups. (B)Individual composition of but (Acidaminococcus sp. - olive,Anaerostipes sp. - dark green, Coprococcus sp. - dark red,Eubacterium sp. - black, Faecalibacterium sp. - darkpurple, Megasphaera sp. - light purple,Peptoniphilus sp. - blue, Oscillibacter sp. - grey andRoseburia sp. - orange) and buk (Anaerotruncus -coarse grey, C. beijerinckii - coarse white, C. butyricum -coarse light blue, C. perfringens - coarse light yellow,Enterococcus sp. - light green and Subdoligranulum sp. -coarse light red) communities are given. (C) Quantitative PCR datatargeting the 16S genes of Faecalibacterium sp. (purple bar),Roseburia sp./E. rectale (orange bar) and C.butyricum (white coarse bar). Note: Coprococcus sp. isconsidered to contain both but and buk genes. The error barsrepresent the range on duplicate measurements. All results are corrected formultiple 16S rRNA copy numbers of individual bacteria (see Methods). ?- Butyrate production was shown for one strain of Subdoligranulum sp.and it is unclear whether all members of this genus have the ability tosynthesize butyrate.
Mentions: We retrieved the major known butyrate-producing taxa from literature [5] and from the but and buk data andused this information to screen for those taxa in the total 16S 454 pyrotag analysispresented in the accompanying paper [14].Results are displayed in Figure 6A. Additionally, qPCRtargeting specific butyrate producers was performed (Figure 6C). 16S rRNA gene analysis supported the functional gene resultsin that similar overall patterns were detected by the two different techniques.Communities linked to buk were dominated by sequences similar to those ofC. butyricum and C. perfringens, whereas sequences similar toAcidaminococcus sp., F. prausnitzii and Roseburia sp.comprised the majority of but-associated bacteria in both methods(Figures 5 and 6, Additionalfile 1: Figures S3 and S4). Nevertheless, severaldifferences between 16S rRNA gene and functional gene analysis wereobserved. Only a minute fraction from 16S rRNA gene pyrotag data wasidentified as Eubacterium sp., whereas many but sequences wereassigned to strains of E. hallii and E. rectale. Other studies thatutilized fluorescence in-situ hybridization and clone libraries reportedhigh concentrations of those strains in the healthy colonic microbial flora[4,25], whichsuggests that 16S rRNA gene-based analysis could not reliably discriminatethem from other taxa. Furthermore, Subdoligranulum sp., which contain onebutyrate-producing isolate, S. variabile ([26] has the gene buk), were not detected in thefunctional gene data. But if this genus is considered to be butyrate-producing, thenthe 16S rRNA gene analysis suggests a considerable abundance of bukgenes in healthy control samples. Similarly, many more 16S rRNA genesequences were assigned to Acidaminococcus sp., Anaerostipes sp.,Coprococcus sp. and Peptoniphilus sp. in certain samplescompared with the results obtained from the functional gene analysis. These findingssupport earlier reports that butyrate synthesis is often not a homogenous feature ofall members of a genus [4,5] and strengthens the application of higher taxonomic resolutiontechniques to adequately assess the butyrate-producing potential of bacterialcommunities. Species resolution is also crucial for the functionally diverse genusClostridia. Several butyrate-producing members such asClostridium sp. SS2/1, Clostridium sp. M62/1, C.acetobutylicum, C. carboxidivorans and C. symbiosum were matched tonumerous functional gene sequences, but could not be detected in the 16SrRNA gene data.

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