A gene-targeted approach to investigate the intestinal butyrate-producing bacterial community
- Marius Vital1,
- Christopher R Penton1,
- Qiong Wang1,
- Vincent B Young2,
- Dion A Antonopoulos3,
- Mitchell L Sogin4,
- Hilary G Morrison4,
- Laura Raffals5,
- Eugene B Chang6,
- Gary B Huffnagle2,
- Thomas M Schmidt1,
- James R Cole1Email author and
- James M Tiedje1Email author
© Vital et al; licensee BioMed Central Ltd. 2013
Received: 30 August 2012
Accepted: 8 January 2013
Published: 4 March 2013
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.
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.
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.
KeywordsButyrate Gene-targeted metagenomics Human microbiome project Pouchitis Ulcerative colitis
Basic Local Alignment Search Tool
Hidden Markov Model
Ileal pouch anal anastomosis
Polymerase chain reaction
Quantitative polymerase chain reaction
Ribosomal Database Project
Short chain fatty acid
Whole genome amplification
The relationship between a healthy functioning gut microbiome and overall human well-being is firmly established. Recently, large-scale projects in this field, namely the Human Microbiome Project and the Metagenomics of the Human Intestinal Tract framework program, have been launched, with the goal of developing a holistic understanding of the composition and functional properties of intestinal bacteria and their effects on the human host. Numerous host-microbiome interactions have been reported and microbial-derived metabolites such as vitamins or short chain fatty acids have been of specific interest in many studies (see [1, 2]). Among these, butyrate is considered as one of the most important metabolites as it serves as the major energy source of colonocytes; has anti-inflammatory properties; and regulates gene expression, differentiation and apoptosis in host cells .
Much of the information on the diversity of butyrate-producing bacteria has depended on culture-independent methods, however recent cultivation efforts for some of these strict anaerobes have been successful . The existing isolates within this functional group are phylogenetically diverse, with the two most abundant groups related to Eubacterium spp. and Roseburia spp. (Clostridium cluster XIVa) and Faecalibacterium prausnitzii (Clostridium cluster IV) . However, both clusters include additional non-butyrate-producing species. As such, 16S rRNA gene-targeted analysis often cannot distinguish the butyrate-producing from the non-producing community in a sample . Furthermore, it is increasingly recognized that horizontal gene transfer, which uncouples bacterial function from phylogeny, plays an important role in shaping the human microbiome . The shortcomings of relying only on traditional 16S rRNA gene-based phylogenetic analysis for functional inferences are now recognized in many other fields of microbial ecology. To resolve this, functional gene-targeted sequencing has emerged as the method of choice to investigate microbial functionality independent of phylogeny. This method has been used in several studies examining the nitrogen cycle , degradation of xenobiotic compounds  and antibiotic resistance of gut bacteria . These studies have demonstrated the value of obtaining a detailed insight into specific microbial processes.
In the human gut, butyrate is produced through two main pathways, the butyryl-CoA:acetate CoA-transferase pathway (but) and the butyrate kinase (buk), and previous studies on colonic isolates of healthy individuals have illustrated that the but pathway predominates . Consequently, Louis and Flint  developed a semi-quantitative PCR protocol targeting a selection of but sequences and used the same primers to construct clone libraries from fecal samples that revealed high gene diversity, including several unknown operational taxonomic units (based on a 98% DNA similarity ).
In this study, we present a novel approach that targets a broad range of but and buk genes based on both 454 pyrotag sequencing in combination with the Ribosomal Database Project’s (RDP) functional gene pipeline  and on quantitative PCR targeting selected groups of butyrate producers. The presented methods were applied on luminal samples from patients with ulcerative colitis (UC) who underwent a colectomy followed by ileal pouch anal anastomosis (IPAA) as described in the accompanying paper by Young et al.. In this procedure, the entire colon is resected, the terminal ileum is fashioned into a pouch and connected to the anal canal, and intestinal flow is re-established. Previous data indicate that approximately half of patients will develop pouchitis within 1 year, an inflammatory condition similar to UC . Because of the clinical similarity between pouchitis and UC, it is thought that studying the development of pouchitis can be used to reveal the etiology of UC. Several studies reported dysbiosis of the intestinal microbiome in patients with UC [16, 17]. However, it is unclear whether the observed microbiome changes are the cause or the consequence of UC. These difficulties make pouchitis an ideal model system as it allows for the clinical observation of individuals from “time zero”, when fecal flow is initiated through the newly established, disease-free pouch. In this study, we specifically monitored the initial establishment (first 2 months) of butyrate-producing microbial communities in four patients after IPAA and compared the results with healthy controls.
Processing of samples
Samples analyzed in this study
Number of days from ileostomy takedown
Primers, amplicon generation and 454 pyrotag sequencing
Primers designed for this study are illustrated
Functional genes - pyro-sequencing
Functional genes - quantitative PCR
16S genes - quantitative PCR
Quantitative real time PCR
Primers designed for quantitative PCR (qPCR; Table 2) targeting the but/buk genes were based on the Fungene database and were specific to all desired target genes with at least two mismatches in one or both primers for other non-target but/buk genes. BLAST analysis illustrated no significant matches to other unrelated sequences. The 16S rRNA gene primers (Rrec2 and Fprau) targeting butyrate producers are described in Ramirez-Farias et al.. Total 16S rRNA gene community qPCR primers were based on Leigh et al.. Additionally, primers for the 16S rRNA genes of C. butyricum were designed based on the RDP database. Specific amplification of targets was verified for all primers using the following pure cultures (amplification efficiency per nanogram of pure culture is given in brackets): Bacillus licheniformis ATCC 14580, Bacteroidetes thetaiotaomicron E50, C. acetobutylicum ATCC 824 (2.65 × 105), C. difficile ATCC 630, C. perfringens ATCC 13124 (4.88 × 105), Eubacterium hallii DSM(Z) 3353, E. rectale DSM(Z) 17629 (4.06 × 105), Faecalibacterium prausnitzii DSM(Z) 17677 (5.53 × 105), Roseburia intestinalis DSM(Z) 14610 (1.80 × 105) and R. inulinivorans DSM(Z) 16841 (4.73 × 105). Strains were purchased either from ATCC or DSM(Z) (as indicated in name). B. licheniformis and B. thetaiotaomicron E50 were provided by Daniel Clemens. For the primers targeting Acidaminococcus (but gene) and C. butyricum, (16S rRNA gene), instead of a pure culture, a patient sample containing many target bacteria (based on all methods presented here) served as a positive control.
Amplification was performed with the SYBR Green Master Mix (Life Technologies) with 10 ng template DNA per reaction (total volume of 15 μL; no WGA except for the healthy control samples) in 384-well plates (ABI Prism 7900 HT, Life Technologies). Annealing temperatures and final primer concentrations were as follows: G_buk (64°C; 0.83 μM), Cbuty (66°C; 0.67 μM), FPR/Fprau (60°C; 0.83 μM), G_Acida (67°C; 0.83 μM), G_Fprsn (70°C; 0.83 μM), G_Ros/Eub (62°C; 0.83 μM; G_Ros_R and G_Eub_R were mixed together at equal final concentrations of 0.42 μM), Rrec/Erec (60°C; 0.83 μM; the two forward primers were mixed together at equal final concentrations of 0.42 μM) and total 16S (60°C; 0.67 μM). Thermocycling was done as follows; 2 min at 50°C; 10 min at 95°C; 45 s at 95°C; 45 s at individual annealing temperature; and 45 s at 72°C (for total 16S rRNA, elongation at 72°C was omitted) (×40). Analysis was performed in duplicate samples. Genomic DNA of R. inulinivorans, F. prausnitzii and C. perfringens (for functional gene qPCRs) and cloned amplified products (for 16S qPCRs and G_Acida; TOPO cloning kit, Life Technologies) at concentrations of 102 to 107 copies (10-fold dilutions) were used for standard curves to determine target concentrations. Genomic DNA of Desulfotomaculum acetoxidans DSM 771 with 10 16S rRNA gene copy numbers was used for the standard curve (103 to 108) for total 16S rRNA gene quantification. The detection limit was set as 102 target sequences for all primers and results are expressed as a percentage of the total bacterial community based on total 16S rRNA gene qPCR. For 16S rRNA gene copy number normalizations of specific 16S rRNA targets see below (comparing functional gene results to 16S pyrotag data). Because but/buk target sequences are present as a single copy per genome, qPCR results of functional genes were multiplied by five to account for multiple 16S rRNA gene copies (five on average) of the intestinal bacterial flora).
Raw reads matching barcodes (106,708 for but and 84,222 for buk) were processed using the RDP pyro-sequencing pipeline , where 87% but and 94% buk sequences passed quality filtering. Subsequently, sequences were subjected to RDP FrameBot for frameshift corrections and closest match assignments. To develop a reference sequence set for FrameBot, we took the corresponding gene sequence sets from the Fungene database, developed through (Hidden Markov Model) HMM searches of the National Center for Biotechnology Information protein database, and removed partial sequences with less than 93% coverage (that is, last filled model position - first filled model position/model length) to the full gene length HMM model, giving 452 but and 422 buk reference sequences. For buk, 97% reads that passed the initial process passed FrameBot with minimum 30% identity to the closest match and 125 amino acids in length. On average, 1.6 frameshifts were corrected per sequence and 58% of the sequences contained at least one frameshift. For but, 59% reads that passed the initial process passed FrameBot with minimum 30% identity and 100 amino acids in length. The majority of non-passing sequences were identified as human origin. On average, 0.6 frameshifts were corrected per sequence, 30% of the sequences contained at least one frameshift. Sequences can be accessed at SRA062948.
Ordination and diversity analysis
Comparing functional gene results to 16S pyrotag data
For library generation of 16S rRNA gene analysis and pyro-sequencing see Young et al.. For the first healthy control, no data on luminal aspirate were available and shown results are based on a colon biopsy sample of the same individual. Data were analyzed for known butyrate producers in the human colon at the genus level (based on  and obtained but/buk gene sequences) except for Clostridia, where species discrimination was applied. All results were normalized to five 16S rRNA gene copy numbers, which represented the average for Firmicutes and Bacteroidetes, the two most abundant phyla in the gut. Average copy number of each genus was derived from rrnDB  and the Integrated Microbial Genome database . A list of taxa searched as well as individual 16S rRNA gene copy numbers is presented in Additional file 1: Table S3.
Investigating but gene diversity
Several short chain fatty acid (SCFA) transferases have been characterized that exhibit broad substrate specificities and show remarkable sequence similarities . Consequently, existing annotations in public databases are often unreliable and misleading. In our established Fungene database, many known but sequences are wrongly annotated (due to GenBank’s annotation) and SCFA transferases similar to but such as 4-hydroxybutyrate CoA transferases (4hbt) are present. A neighbor joining tree of all sequences from Fungene’s butyryl-CoA:acetate CoA-transferase (but) database (>93% coverage to model; to ensure only full-length sequences were considered) was constructed where all functionally verified but genes cluster together and apart from 4hbt genes (Additional file 1: Figure S1). Primers were designed to specifically target those but sequences. However, it is still likely that SCFA transferases related to but are amplified as well. In order to quality filter our obtained but sequences (in addition to the processing pipeline presented in the Methods section), only sequences located within the cluster identified in Additional file 1: Figure S1 were regarded as likely real but, whereas the remaining amplicons (<1%) matching 16 references outside the cluster were excluded from further analysis. We detected a broad diversity of but genes in our samples and they were linked to almost all described but carrying species (Figure 1A). Four closest FrameBot matches were assigned to 75% of all obtained sequences, namely R. intestinalis L1-82, R. inulinivorans A2-194, Acidaminococcus sp. D21 and E. rectale ATCC 33656. To verify the closest match assignments all amplicons were mapped on a tree together with full-length reference sequences using Pplacer (; Additional file 1: Figure S3). We observed minimal deep branching; nearly all amplicons diverged in the terminal branches to the reference sequences, and the numbers assigned correlated well with the FrameBot closest match assignments. An exception was Clostridium sp. SS3/4 where many more amplicons than expected, that FrameBot had originally assigned to C. symbiosum and Clostridium sp. M62/1, mapped to that reference sequence. The discrepancies were most likely due to the different underlying assignment methods used by FrameBot and Pplacer. The former compares blossum62-corrected pairwise distances, whereas the latter is based on maximum likelihood criteria. Conservation analysis of but showed a remarkably similar pattern between the reference and amplicon sequences, and several well-conserved amino acid sites (>95% conservation in both groups) were identified (Figure 1B).
Investigating buk gene diversity
A considerable diversity of buk sequences that included sequences similar to the majority of previously described butyrate producers were detected in our samples (Figure 2A). The Fungene database contains many sequences assigned to species not reported to produce butyrate, such as members of the phylum Bacteroidetes. Many of our amplicons closely matched sequences originating in Bacteroides and the established tree clusters them together with known butyrate producers and apart from acetate kinase, a closely related gene (Figure 2A; a neighbor joining tree of all Fungene sequences (93% cut-off) is shown in Additional file 1: Figure S2). Therefore, we included those sequences for analysis. Three quarters of all obtained buk amplicon sequences were assigned to four closest FrameBot matches; Bacteroides sp. D2, Bacteroides sp. 3_2_5, C. butyricum 5521 and C. perfringens. The resultant tree including the mapped amplicon sequences confirmed closest match assignments (Additional file 1: Figure S4). Sequence analysis revealed less similarity among buk genes than observed for but and fewer conserved amino acids could be detected (Figure 2B versus Figure 1B).
Ordination and diversity analysis of obtained data
Quantitative analysis of but/buk genes
Investigating the butyrate-producing community based on 16S rRNA gene analysis
In this study we show that functional gene-targeted analysis of the intestinal bacterial butyrate-producing community can overcome limitations imposed by relying solely on 16S rRNA gene targeted investigations. A combination of 454 pyrotag sequencing with qPCR analysis was essential to resolve the full differences among samples. Pyro-sequencing provided specific community profiles at great depth, whereas qPCR enabled the absolute quantification of genes. Ordination analysis based on pyrotag data revealed individual community patterns for each patient distinct from those of the healthy controls (Figure 3); however, only qPCR could demonstrate that overall gene concentrations differed over several orders of magnitude (Figure 5). Notably, the presented protocol for amplicon generation enabled amplification of genes for all samples, although actual abundance of individual targets was often below qPCR thresholds.
An abundant butyrate-producing community is essential for a well-functioning colon . Butyrate is also the preferred energy generating substrate for the pouch epithelium and it is believed that supply deficiencies could initiate or promote development of pouchitis . The pouch was aerobic before ileostomy takedown and only became anoxic after it was connected to the anal canal, which limits oxygen influx and promotes the establishment of anaerobes. Anoxic/oxic ratios of cultivars steadily increased over time after ileostomy takedown in all investigated patients . In this study, we could demonstrate that these environmental changes were accompanied by the development of butyrate-producing communities at abundances similar to healthy participants of other studies  and to the healthy controls of this study. However, only patient 210 displayed a community pattern comparable to healthy control samples, which was also the case in the companion global 16S rRNA community analysis . Patients 206 and 207 exhibited abnormal communities with buk genes predominating and only very few detectable but genes. Patient 200 displayed an ‘in-between’ community harboring both genes. Currently, the buk pathway is not considered to be important for butyrate production in healthy individuals , a finding further supported by this study. Whether the highly abundant buk-containing communities in patients 206 and 207 can compensate for low concentration of but is unclear. Unfortunately, no SCFA data are available to address this question. Enzyme assays on 17 butyrate-producing isolates demonstrated considerably higher activities for but than for buk, suggesting that the but pathway yields more butyrate in comparison to synthesis via buk. Interestingly, patient 210 is the only individual who did not show onset of inflammation 25 months post ileostomy takedown, whereas patients 200 (8 months), 206 (16 months) and 207 (17 months) all developed pouchitis. Although the patient number is low in this study, it does suggest that the initial establishment of a ‘healthy type’ butyrate-producing community is important to maintain a well-functioning pouch and to prevent the development of disease. The specific question of how butyrate production affects the development of disease will be addressed in a follow-up study where community profiles of patients undergoing IPAA will be monitored until the onset of inflammation and compared with those derived from asymptomatic individuals.
Our approach directly targets the genes coding for butyrate-synthesizing enzymes. We did observe some discrepancies between phylogeny and predicted function, which was especially true for the obtained buk gene sequences assigned to members of the genus Bacteroides. Bacteroides are currently not considered butyrate producers and several culture-based investigations point out their inability to synthesize butyrate (for examples, see [29, 30]). This also applies to many other sequences presented in Additional file 1: Figure S2. Interestingly, some early studies from the 1980s indicated butyrate production by closely related bacteria, namely certain Porphyromonas (former Bacteroides) strains [31, 32]. However, additional studies specifically investigating butyrate synthesis including more Bacteroides strains (and other candidates) under several different physiological conditions are needed to address this issue. Furthermore, even for known butyrate-synthesizing bacteria, gene detection does not automatically imply production of butyrate. Gene expression and a functioning pathway are determined by environmental conditions, with oxygen concentration as likely the most important factor . Most butyrate producers are considered to be strict anaerobes with their growth and function strongly coupled. However, it has been recently shown that certain butyrate producers, namely F. prausnitzii, can also grow under microaerophilic conditions using extracellular oxygen as the final electron acceptor . Butyrate production by this bacterium was still detected under these conditions but at a reduced rate.
The presented protocols provide a new approach to more specifically resolve the butyrate-producing community. We could clearly demonstrate that butyrate producers were established at high abundance (approximately 5% to 26% of total bacterial community) in the pouch of all patients undergoing an IPAA within the first 2 months after ileostomy takedown. Community profiles were distinctive among patients. Most important, one individual harbored a community profile similar to the healthy controls with but genes predominating and an almost complete absence of buk genes, whereas the other three patients had other variants. Only the former patient remained healthy 25 months later. 16S rRNA gene analysis showed similar overall patterns as the functional gene-targeted approach, but only the latter could reveal specific details on butyrate-producing taxa that were essential to assess the entire butyrogenic potential of the microbial communities analyzed. Furthermore, our analysis refines but and buk reference annotations found in central databases. In the near future, these methods will be complemented by metagenomic tools that will provide full-length gene sequences without prior amplification and will facilitate the investigation of not only individual genes of interest but also complete synthesis pathways.
Financial support was provided by NIH Human Microbiome Project Demonstration Project (UH3 DK083993) and the University of Chicago Digestive Disease Research Core Center (P30DK42086). Special thanks to Charles Falkiewicz, Jiarong Gao, Stephanie LaHaye, Craig McMullen, Derek St. Louis and Andrew Worden for technical assistance, and to Christopher Radek for his support during sequencing. The authors acknowledge Daniel Clemens for providing several strains.
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