Henning Seedorf

Methanogenic archaea: ecologically relevant differences in energy conservation

Most methanogenic archaea can reduce CO2 with H2 to methane, and it is generally assumed that the reactions and mechanisms of energy conservation that are involved are largely the same in all methanogens. However, this does not take into account the fact that methanogens with cytochromes have considerably higher growth yields and threshold concentrations for H2 than methanogens without cytochromes. These and other differences can be explained by the proposal outlined in this Review that in methanogens with cytochromes, the first and last steps in methanogenesis from CO2 are coupled chemiosmotically, whereas in methanogens without cytochromes, these steps are energetically coupled by a cytoplasmic enzyme complex that mediates flavin-based electron bifurcation.

The Long-Term Stability of the Human Gut Microbiota

Background Understanding the dynamics and stability of the human gut microbiota is important if its characterization is to play a role in the diagnosis, treatment, and prevention of disease. To characterize stability in related and unrelated individuals and its responsiveness to physiologic change (weight loss), we developed a method for bacterial 16S rRNA amplicon sequencing at high depth with high precision. We also sequenced the genomes of anaerobic bacteria represented in culture collections prepared from fecal samples collected from individuals over time. Methods Low-error amplicon sequencing (LEA-Seq) is a quantitative method based on redundant sequencing of bacterial 16S rRNA genes. A dilute, barcoded, oligonucleotide primer solution is used to create ~150,000 linear PCR extensions of the template DNA. The labeled, bottlenecked linear PCR pool is amplified with exponential PCR, using primers that specifically amplify only the linear PCR molecules. The exponential PCR pool is sequenced at sufficient depth to obtain ~20× coverage. Multiple reads enable the generation of an error-corrected consensus sequence for each barcoded template molecule. LEA-Seq can be used for a variety of other applications. Relationship among time, physiology, and microbiota stability. (A) Stability of fecal microbiota follows a power-law function (n = 37 females sampled over time; >1 week to <5 years). Dashed lines show 95% confidence bounds over 10- and 50-year extrapolations (inset). (B) Microbiota stability is inversely related to the stability of each individual’s body mass index. Results and Discussion LEA-Seq of fecal samples from 37 healthy U.S. adults sampled 2 to 13 times up to 296 weeks apart revealed that they harbored 195 ± 48 bacterial strains, representing 101 ± 27 species. On average, their individual microbiota was remarkably stable, with 60% of strains remaining over the course of 5 years. Stability followed a power law, which, when extrapolated, suggests that most strains in an individual’s intestine are residents for decades (figure, panel A). Members of Bacteroidetes and Actinobacteria are significantly more stable components than the population average. LEA-Seq of four individuals sampled during an 8- to 32-week period during a calorie-restricted dietary study showed that weight stability is a significantly better predictor of microbiota stability than the time interval between samples (figure, panel B). After generating clonally arrayed collections of anaerobic bacteria from frozen fecal samples collected from six weight-stable individuals sampled 7 to 69 weeks apart, we produced draft genome sequences for 534 isolates representing 188 strains and 75 species. A targeted approach focused on Methanobrevibacter smithii isolates from two sets of twin pairs and their mothers and Bacteroides thetaiotaomicron strains from nine donors including sister-sister and mother-daughter pairs. Strains, defined as isolates sharing >96% of their genome content, were maintained over time within an individual and between family members but not between unrelated individuals. Thus, early gut colonizers, such as those acquired from our parents and siblings, have the potential to exert their physiologic, metabolic, and immunologic effects for most, and perhaps all, of our lives. Inheritance Guts We know little about the stability of the constituent microbiota in the human gut or the extent to which the gut microbiota are a potential target for long-term health interventions. Faith et al. (p. 10.1126/science.1237439) analyzed the fecal microbiota of 37 individuals and found that, on average, 60% of bacterial strains remained stable for up to 5 years and many were estimated to remain stable for decades. Low-error sequencing data suggest that initial microbial colonizers of infant guts could persist over the life span of an individual. A low-error 16S ribosomal RNA amplicon sequencing method, in combination with whole-genome sequencing of >500 cultured isolates, was used to characterize bacterial strain composition in the fecal microbiota of 37 U.S. adults sampled for up to 5 years. Microbiota stability followed a power-law function, which when extrapolated suggests that most strains in an individual are residents for decades. Shared strains were recovered from family members but not from unrelated individuals. Sampling of individuals who consumed a monotonous liquid diet for up to 32 weeks indicated that changes in strain composition were better predicted by changes in weight than by differences in sampling interval. This combination of stability and responsiveness to physiologic change confirms the potential of the gut microbiota as a diagnostic tool and therapeutic target.

Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla

The adult human distal gut microbial community is typically dominated by 2 bacterial phyla (divisions), the Firmicutes and the Bacteroidetes. Little is known about the factors that govern the interactions between their members. Here, we examine the niches of representatives of both phyla in vivo. Finished genome sequences were generated from Eubacterium rectale and E. eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades. Comparison of these and 25 other gut Firmicutes and Bacteroidetes indicated that the Firmicutes possess smaller genomes and a disproportionately smaller number of glycan-degrading enzymes. Germ-free mice were then colonized with E. rectale and/or a prominent human gut Bacteroidetes, Bacteroides thetaiotaomicron, followed by whole-genome transcriptional profiling, high-resolution proteomic analysis, and biochemical assays of microbial–microbial and microbial–host interactions. B. thetaiotaomicron adapts to E. rectale by up-regulating expression of a variety of polysaccharide utilization loci encoding numerous glycoside hydrolases, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is used by the gut epithelium. This simplified model of the human gut microbiota illustrates niche specialization and functional redundancy within members of its major bacterial phyla, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability.

The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features

Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions.

The Genome Sequence of Methanosphaera stadtmanae Reveals Why This Human Intestinal Archaeon Is Restricted to Methanol and H2 for Methane Formation and ATP Synthesis

Methanosphaera stadtmanae has the most restricted energy metabolism of all methanogenic archaea. This human intestinal inhabitant can generate methane only by reduction of methanol with H2 and is dependent on acetate as a carbon source. We report here the genome sequence of M. stadtmanae, which was found to be composed of 1,767,403 bp with an average G+C content of 28% and to harbor only 1,534 protein-encoding sequences (CDS). The genome lacks 37 CDS present in the genomes of all other methanogens. Among these are the CDS for synthesis of molybdopterin and for synthesis of the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex, which explains why M. stadtmanae cannot reduce CO2 to methane or oxidize methanol to CO2 and why this archaeon is dependent on acetate for biosynthesis of cell components. Four sets of mtaABC genes coding for methanol:coenzyme M methyltransferases were found in the genome of M. stadtmanae. These genes exhibit homology to mta genes previously identified in Methanosarcina species. The M. stadtmanae genome also contains at least 323 CDS not present in the genomes of all other archaea. Seventy-three of these CDS exhibit high levels of homology to CDS in genomes of bacteria and eukaryotes. These 73 CDS include 12 CDS which are unusually long (>2,400 bp) with conspicuous repetitive sequence elements, 13 CDS which exhibit sequence similarity on the protein level to CDS encoding enzymes involved in the biosynthesis of cell surface antigens in bacteria, and 5 CDS which exhibit sequence similarity to the subunits of bacterial type I and III restriction-modification systems.

Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial, Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities

Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the communitys bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.

Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome

Ruminant livestock represent the single largest anthropogenic source of the potent greenhouse gas methane, which is generated by methanogenic archaea residing in ruminant digestive tracts. While differences between individual animals of the same breed in the amount of methane produced have been observed, the basis for this variation remains to be elucidated. To explore the mechanistic basis of this methane production, we measured methane yields from 22 sheep, which revealed that methane yields are a reproducible, quantitative trait. Deep metagenomic and metatranscriptomic sequencing demonstrated a similar abundance of methanogens and methanogenesis pathway genes in high and low methane emitters. However, transcription of methanogenesis pathway genes was substantially increased in sheep with high methane yields. These results identify a discrete set of rumen methanogens whose methanogenesis pathway transcription profiles correlate with methane yields and provide new targets for CH4 mitigation at the levels of microbiota composition and transcriptional regulation.

Two different bacterial community types are linked with the low-methane emission trait in sheep.

The potent greenhouse gas methane (CH4) is produced in the rumens of ruminant animals from hydrogen produced during microbial degradation of ingested feed. The natural animal-to-animal variation in the amount of CH4 emitted and the heritability of this trait offer a means for reducing CH4 emissions by selecting low-CH4 emitting animals for breeding. We demonstrate that differences in rumen microbial community structure are linked to high and low CH4 emissions in sheep. Bacterial community structures in 236 rumen samples from 118 high- and low-CH4 emitting sheep formed gradual transitions between three ruminotypes. Two of these (Q and S) were linked to significantly lower CH4 yields (14.4 and 13.6 g CH4/kg dry matter intake [DMI], respectively) than the third type (H; 15.9 g CH4/kg DMI; p<0.001). Low-CH4 ruminotype Q was associated with a significantly lower ruminal acetate to propionate ratio (3.7±0.4) than S (4.4±0.7; p<0.001) and H (4.3±0.5; p<0.001), and harbored high relative abundances of the propionate-producing Quinella ovalis. Low-CH4 ruminotype S was characterized by lactate- and succinate-producing Fibrobacter spp., Kandleria vitulina, Olsenella spp., Prevotella bryantii, and Sharpea azabuensis. High-CH4 ruminotype H had higher relative abundances of species belonging to Ruminococcus, other Ruminococcaceae, Lachnospiraceae, Catabacteriaceae, Coprococcus, other Clostridiales, Prevotella, other Bacteroidales, and Alphaproteobacteria, many of which are known to form significant amounts of hydrogen. We hypothesize that lower CH4 yields are the result of bacterial communities that ferment ingested feed to relatively less hydrogen, which results in less CH4 being formed.

More than 200 genes required for methane formation from H₂ and CO₂ and energy conservation are present in Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus

The hydrogenotrophic methanogens Methanothermobacter marburgensis and Methanothermobacter thermautotrophicus can easily be mass cultured. They have therefore been used almost exclusively to study the biochemistry of methanogenesis from H2 and CO2, and the genomes of these two model organisms have been sequenced. The close relationship of the two organisms is reflected in their genomic architecture and coding potential. Within the 1,607 protein coding sequences (CDS) in common, we identified approximately 200 CDS required for the synthesis of the enzymes, coenzymes, and prosthetic groups involved in CO2 reduction to methane and in coupling this process with the phosphorylation of ADP. Approximately 20 additional genes, such as those for the biosynthesis of F430 and methanofuran and for the posttranslational modifications of the two methyl-coenzyme M reductases, remain to be identified.

RIM-DB: a taxonomic framework for community structure analysis of methanogenic archaea from the rumen and other intestinal environments

Methane is formed by methanogenic archaea in the rumen as one of the end products of feed fermentation in the ruminant digestive tract. To develop strategies to mitigate anthropogenic methane emissions due to ruminant farming, and to understand rumen microbial differences in animal feed conversion efficiency, it is essential that methanogens can be identified and taxonomically classified with high accuracy. Currently available taxonomic frameworks offer only limited resolution beyond the genus level for taxonomic assignments of sequence data stemming from high throughput sequencing technologies. Therefore, we have developed a QIIME-compatible database (DB) designed for species-level taxonomic assignment of 16S rRNA gene amplicon data targeting methanogenic archaea from the rumen, and from animal and human intestinal tracts. Called RIM-DB (Rumen and Intestinal Methanogen-DB), it contains a set of 2,379 almost full-length chimera-checked 16S rRNA gene sequences, including 20 previously unpublished sequences from isolates from three different orders. The taxonomy encompasses the recently-proposed seventh order of methanogens, the Methanomassiliicoccales, and allows differentiation between defined groups within this order. Sequence reads from rumen contents from a range of ruminant-diet combinations were taxonomically assigned using RIM-DB, Greengenes and SILVA. This comparison clearly showed that taxonomic assignments with RIM-DB resulted in the most detailed assignment, and only RIM-DB taxonomic assignments allowed methanogens to be distinguished taxonomically at the species level. RIM-DB complements the use of comprehensive databases such as Greengenes and SILVA for community structure analysis of methanogens from the rumen and other intestinal environments, and allows identification of target species for methane mitigation strategies.