Crystal Structures of Bacterial Pectin Methylesterases Pme8A and PmeC2 from Rumen Butyrivibrio.

Pectin is a complex polysaccharide that forms a substantial proportion of the plant's middle lamella of forage ingested by grazing ruminants. Methanol in the rumen is derived mainly from methoxy groups released from pectin by the action of pectin methylesterase (PME) and is subsequently used by rumen methylotrophic methanogens that reduce methanol to produce methane (CH4). Members of the genus Butyrivibrio are key pectin-degrading rumen bacteria that contribute to methanol formation and have important roles in fibre breakdown, protein digestion, and the biohydrogenation of fatty acids. Therefore, methanol release from pectin degradation in the rumen is a potential target for CH4 mitigation technologies. Here, we present the crystal structures of PMEs belonging to the carbohydrate esterase family 8 (CE8) from Butyrivibrio proteoclasticus and Butyrivibrio fibrisolvens, determined to a resolution of 2.30 Å. These enzymes, like other PMEs, are right-handed ß-helical proteins with a well-defined catalytic site and reaction mechanisms previously defined in insect, plant, and other bacterial pectin methylesterases. Potential substrate binding domains are also defined for the enzymes.

Structural determination of archaeal UDP-N-acetylglucosamine 4-epimerase from Methanobrevibacter ruminantium M1 in complex with the bacterial cell wall intermediate UDP-N-acetylmuramic acid.

The crystal structure of UDP-N-acetylglucosamine 4-epimerase (UDP-GlcNAc 4-epimerase; WbpP; EC 5.1.3.7), from the archaeal methanogen Methanobrevibacter ruminantium strain M1, was determined to a resolution of 1.65 Å. The structure, with a single monomer in the crystallographic asymmetric unit, contained a conserved N-terminal Rossmann-fold for nucleotide binding and an active site positioned in the C-terminus. UDP-GlcNAc 4-epimerase is a member of the short-chain dehydrogenases/reductases superfamily, sharing sequence motifs and structural elements characteristic of this family of oxidoreductases and bacterial 4-epimerases. The protein was co-crystallized with coenzyme NADH and UDP-N-acetylmuramic acid, the latter an unintended inclusion and well known product of the bacterial enzyme MurB and a critical intermediate for bacterial cell wall synthesis. This is a non-native UDP sugar amongst archaea and was most likely incorporated from the E. coli expression host during purification of the recombinant enzyme.

Expression, Purification, and Characterization of (R)-Sulfolactate Dehydrogenase (ComC) from the Rumen Methanogen Methanobrevibacter millerae SM9.

(R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC from Methanobrevibacter millerae SM9 was cloned and expressed in Escherichia coli and biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen for α-ketoglutarate. Optimal activity was observed at pH 6.5. The apparent KM for coenzyme (NADH) was 55.1 µM, and for sulfopyruvate, it was 196 µM (for sulfopyruvate the Vmax was 93.9 µmol min-1 mg-1 and kcat was 62.8 s-1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.

Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase.

One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn(2+) cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn(2+) cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH.

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.

Transposition of Tn916 in the four replicons of the Butyrivibrio proteoclasticus B316(T) genome.

The rumen bacterium Butyrivibrio proteoclasticus B316(T) has a 4.4-Mb genome composed of four replicons (approximately 3.55 Mb, 361, 302 and 186 kb). Mutagenesis of B316(T) was performed with the broad host-range conjugative transposon Tn916 to screen for functionally important characteristics. The insertion sites of 123 mutants containing a single copy of Tn916 were identified and corresponded to 53 different insertion points, of which 18 (34.0%), representing 39 mutants (31.7%), were in ORFs and 12 were where transposition occurred in both directions (top and bottom DNA strand). Up to eight mutants from several independent conjugation experiments were found to have the same integration site. Although transposition occurred in all four replicons, the number of specific insertion sites, transposition frequency and the average intertransposon distance between insertions varied between the four replicons. In silico analysis of the 53 insertion sites was used to model a target consensus sequence for Tn916 integration into B316(T) . A search of the B316(T) genome using the modelled target consensus sequence (up to two mismatches) identified 39 theoretical Tn916 insertion sites (19 coding, 20 noncoding), of which nine corresponded to Tn916 insertions identified in B316(T) mutants during our conjugation experiments.

The genome sequence of the rumen methanogen Methanobrevibacter ruminantium reveals new possibilities for controlling ruminant methane emissions.

BACKGROUND: Methane (CH(4)) is a potent greenhouse gas (GHG), having a global warming potential 21 times that of carbon dioxide (CO(2)). Methane emissions from agriculture represent around 40% of the emissions produced by human-related activities, the single largest source being enteric fermentation, mainly in ruminant livestock. Technologies to reduce these emissions are lacking. Ruminant methane is formed by the action of methanogenic archaea typified by Methanobrevibacter ruminantium, which is present in ruminants fed a wide variety of diets worldwide. To gain more insight into the lifestyle of a rumen methanogen, and to identify genes and proteins that can be targeted to reduce methane production, we have sequenced the 2.93 Mb genome of M. ruminantium M1, the first rumen methanogen genome to be completed. METHODOLOGY/PRINCIPAL FINDINGS: The M1 genome was sequenced, annotated and subjected to comparative genomic and metabolic pathway analyses. Conserved and methanogen-specific gene sets suitable as targets for vaccine development or chemogenomic-based inhibition of rumen methanogens were identified. The feasibility of using a synthetic peptide-directed vaccinology approach to target epitopes of methanogen surface proteins was demonstrated. A prophage genome was described and its lytic enzyme, endoisopeptidase PeiR, was shown to lyse M1 cells in pure culture. A predicted stimulation of M1 growth by alcohols was demonstrated and microarray analyses indicated up-regulation of methanogenesis genes during co-culture with a hydrogen (H(2)) producing rumen bacterium. We also report the discovery of non-ribosomal peptide synthetases in M. ruminantium M1, the first reported in archaeal species. CONCLUSIONS/SIGNIFICANCE: The M1 genome sequence provides new insights into the lifestyle and cellular processes of this important rumen methanogen. It also defines vaccine and chemogenomic targets for broad inhibition of rumen methanogens and represents a significant contribution to worldwide efforts to mitigate ruminant methane emissions and reduce production of anthropogenic greenhouse gases.