Complete genome sequence of Methanosphaera sp. ISO3-F5, a rumen methylotrophic methanogen.

Methanosphaera spp. are methylotrophic methanogenic archaea and members of the order Methanobacteriales with few cultured representatives. Methanosphaera sp. ISO3-F5 was isolated from sheep rumen contents in New Zealand. Here, we report its complete genome, consisting of a large chromosome and a megaplasmid (GenBank accession numbers CP118753 and CP118754, respectively).

Genomic insights into the physiology of Quinella, an iconic uncultured rumen bacterium.

Quinella is a genus of iconic rumen bacteria first reported in 1913. There are no cultures of these bacteria, and information on their physiology is scarce and contradictory. Increased abundance of Quinella was previously found in the rumens of some sheep that emit low amounts of methane (CH4) relative to their feed intake, but whether Quinella contributes to low CH4 emissions is not known. Here, we concentrate Quinella cells from sheep rumen contents, extract and sequence DNA, and reconstruct Quinella genomes that are >90% complete with as little as 0.20% contamination. Bioinformatic analyses of the encoded proteins indicate that lactate and propionate formation are major fermentation pathways. The presence of a gene encoding a potential uptake hydrogenase suggests that Quinella might be able to use free hydrogen (H2). None of the inferred metabolic pathways is predicted to produce H2, a major precursor of CH4, which is consistent with the lower CH4 emissions from those sheep with high abundances of this bacterium.

Machine learning predicts and provides insights into milk acidification rates of Lactococcus lactis.

Lactococcus lactis strains are important components in industrial starter cultures for cheese manufacturing. They have many strain-dependent properties, which affect the final product. Here, we explored the use of machine learning to create systematic, high-throughput screening methods for these properties. Fast acidification of milk is such a strain-dependent property. To predict the maximum hourly acidification rate (Vmax), we trained Random Forest (RF) models on four different genomic representations: Presence/absence of gene families, counts of Pfam domains, the 8 nucleotide long subsequences of their DNA (8-mers), and the 9 nucleotide long subsequences of their DNA (9-mers). Vmax was measured at different temperatures, volumes, and in the presence or absence of yeast extract. These conditions were added as features in each RF model. The four models were trained on 257 strains, and the correlation between the measured Vmax and the predicted Vmax was evaluated with Pearson Correlation Coefficients (PC) on a separate dataset of 85 strains. The models all had high PC scores: 0.83 (gene presence/absence model), 0.84 (Pfam domain model), 0.76 (8-mer model), and 0.85 (9-mer model). The models all based their predictions on relevant genetic features and showed consensus on systems for lactose metabolism, degradation of casein, and pH stress response. Each model also predicted a set of features not found by the other models.

Improved taxonomic assignment of rumen bacterial 16S rRNA sequences using a revised SILVA taxonomic framework.

The taxonomy and associated nomenclature of many taxa of rumen bacteria are poorly defined within databases of 16S rRNA genes. This lack of resolution results in inadequate definition of microbial community structures, with large parts of the community designated as incertae sedis, unclassified, or uncultured within families, orders, or even classes. We have begun resolving these poorly-defined groups of rumen bacteria, based on our desire to name these for use in microbial community profiling. We used the previously-reported global rumen census (GRC) dataset consisting of >4.5 million partial bacterial 16S rRNA gene sequences amplified from 684 rumen samples and representing a wide range of animal hosts and diets. Representative sequences from the 8,985 largest operational units (groups of sequence sharing >97% sequence similarity, and covering 97.8% of all sequences in the GRC dataset) were used to identify 241 pre-defined clusters (mainly at genus or family level) of abundant rumen bacteria in the ARB SILVA 119 framework. A total of 99 of these clusters (containing 63.8% of all GRC sequences) had no unique or had inadequate taxonomic identifiers, and each was given a unique nomenclature. We assessed this improved framework by comparing taxonomic assignments of bacterial 16S rRNA gene sequence data in the GRC dataset with those made using the original SILVA 119 framework, and three other frameworks. The two SILVA frameworks performed best at assigning sequences to genus-level taxa. The SILVA 119 framework allowed 55.4% of the sequence data to be assigned to 751 uniquely identifiable genus-level groups. The improved framework increased this to 87.1% of all sequences being assigned to one of 871 uniquely identifiable genus-level groups. The new designations were included in the SILVA 123 release (https://www.arb-silva.de/documentation/release-123/) and will be perpetuated in future releases.

Sharpea and Kandleria are lactic acid producing rumen bacteria that do not change their fermentation products when co-cultured with a methanogen.

Sharpea and Kandleria are associated with rumen samples from low-methane-emitting sheep. Four strains of each genus were studied in culture, and the genomes of nine strains were analysed, to understand the physiology of these bacteria. All eight cultures grew equally well with d-glucose, d-fructose, d-galactose, cellobiose, and sucrose supplementation. d-Lactate was the major end product, with small amounts of the mixed acid fermentation products formate, acetate and ethanol. Genes encoding the enzymes necessary for this fermentation pattern were found in the genomes of four strains of Sharpea and five of Kandleria. Strains of Sharpea produced traces of hydrogen gas in pure culture, but strains of Kandleria did not. This was consistent with finding that Sharpea, but not Kandleria, genomes contained genes coding for hydrogenases. It was speculated that, in co-culture with a methanogen, Sharpea and Kandleria might change their fermentation pattern from a predominately homolactic to a predominately mixed acid fermentation, which would result in a decrease in lactate production and an increase in formation of acetate and perhaps ethanol. However, Sharpea and Kandleria did not change their fermentation products when co-cultured with Methanobrevibacter olleyae, a methanogen that can use both hydrogen and formate, and lactate remained the major end product. The results of this study therefore support a hypothesis that explains the link between lower methane yields and larger populations of Sharpea and Kandleria in the rumens of sheep.

Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection.

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ∼75% of the genus-level bacterial and archaeal taxa present in the rumen.

Correction: The bacterial community associated with the sheep gastrointestinal nematode parasite Haemonchus contortus.

[This corrects the article DOI: 10.1371/journal.pone.0192164.].

The bacterial community associated with the sheep gastrointestinal nematode parasite Haemonchus contortus.

Culture-independent methods were used to study the microbiota of adult worms, third-stage larvae and eggs, both in faeces and laid in vitro, of Haemonchus contortus, a nematode parasite of the abomasa of ruminants which is a major cause of production losses and ill-health. Bacteria were identified in eggs, the female reproductive tract and the gut of adult and third-stage larvae (L3). PCR amplification of 16S rRNA sequences, denaturing gradient gel electrophoresis (DGGE) and clone libraries were used to compare the composition of the microbial communities of the different life-cycle stages of the parasites, as well as parasites and their natural environments. The microbiomes of adult worms and L3 were different from those in the abomasum or faeces respectively. The H. contortus microbiota was mainly comprised of members of the phyla Proteobacteria, Firmicutes and Bacteroidetes. Bacteria were localised in the gut, inside eggs and within the uterus of adult female worms using the universal FISH Eub338 probe, which targets most bacteria, and were also seen in these tissues by light and transmission electron microscopy. Streptococcus/Lactococcus sp. were identified within the distal uterus with the probe Strc493. Sequences from the genera Weissella and Leuconostoc were found in all life-cycle stages, except eggs collected from faeces, in which most sequences belonged to Clostridium sp. Bacteria affiliated with Weissella/Leuconostoc were identified in both PCR-DGGE short sequences and clone libraries of nearly full length 16S rRNA bacterial sequences in all life-cycle stages and subsequently visualised in eggs by fluorescent in situ hybridisation (FISH) with group-specific probes. This strongly suggests they are vertically transmitted endosymbionts. As this study was carried out on a parasite strain which has been maintained in the laboratory, other field isolates will need to be examined to establish whether these bacteria are more widely dispersed and have potential as targets to control H. contortus infections.

Diversity of formyltetrahydrofolate synthetase genes in the rumens of roe deer (Capreolus pygargus) and sika deer (Cervus nippon) fed different diets.

Reductive acetogenesis by homoacetogens represents an alternative pathway to methanogenesis to remove metabolic hydrogen during rumen fermentation. In this study, we investigated the occurrence of homoacetogen in the rumens of pasture-fed roe deer (Capreolus pygargus) and sika deer (Cervus nippon) fed either oak-leaf-based (tannin-rich, 100 mg/kg dried matter), corn-stover-based, or corn-silage-based diets, by using formyltetrahydrofolate synthetase (FTHFS) gene sequences as a marker. The diversity and richness of FTHFS sequences was lowest in animals fed oak leaf, indicating that tannin-containing plants may affect rumen homoacetogen diversity. FTHFS amino acid sequences in the rumen of roe deer significantly differed from those of sika deer. The phylogenetic analyses showed that 44.8% of sequences in pasture-fed roe deer, and 72.1%, 81.1%, and 37.5% of sequences in sika deer fed oak-leaf-, corn-stover-, and corn-silage-based diets, respectively, may represent novel bacteria that have not yet been cultured. These results demonstrate that the rumens of roe deer and sika deer harbor potentially novel homoacetogens and that diet may influence homoacetogen community structure.

The complete genome sequence of the methanogenic archaeon ISO4-H5 provides insights into the methylotrophic lifestyle of a ruminal representative of the Methanomassiliicoccales.

Methane emissions from agriculture represent around 9 % of global anthropogenic greenhouse emissions. The single largest source of this methane is animal enteric fermentation, predominantly from ruminant livestock where it is produced mainly in their fermentative forestomach (or reticulo-rumen) by a group of archaea known as methanogens. In order to reduce methane emissions from ruminants, it is necessary to understand the role of methanogenic archaea in the rumen, and to identify their distinguishing characteristics that can be used to develop methane mitigation technologies. To gain insights into the role of methylotrophic methanogens in the rumen environment, the genome of a methanogenic archaeon has been sequenced. This isolate, strain ISO4-H5, was isolated from the ovine rumen and belongs to the order Methanomassiliicoccales. Genomic analysis suggests ISO4-H5 is an obligate hydrogen-dependent methylotrophic methanogen, able to use methanol and methylamines as substrates for methanogenesis. Like other organisms within this order, ISO4-H5 does not possess genes required for the first six steps of hydrogenotrophic methanogenesis. Comparison between the genomes of different members of the order Methanomassiliicoccales revealed strong conservation in energy metabolism, particularly in genes of the methylotrophic methanogenesis pathway, as well as in the biosynthesis and use of pyrrolysine. Unlike members of Methanomassiliicoccales from human sources, ISO4-H5 does not contain the genes required for production of coenzyme M, and so likely requires external coenzyme M to survive.

Taxonomic Assessment of Rumen Microbiota Using Total RNA and Targeted Amplicon Sequencing Approaches.

Taxonomic characterization of active gastrointestinal microbiota is essential to detect shifts in microbial communities and functions under various conditions. This study aimed to identify and quantify potentially active rumen microbiota using total RNA sequencing and to compare the outcomes of this approach with the widely used targeted RNA/DNA amplicon sequencing technique. Total RNA isolated from rumen digesta samples from five beef steers was subjected to Illumina paired-end sequencing (RNA-seq), and bacterial and archaeal amplicons of partial 16S rRNA/rDNA were subjected to 454 pyrosequencing (RNA/DNA Amplicon-seq). Taxonomic assessments of the RNA-seq, RNA Amplicon-seq, and DNA Amplicon-seq datasets were performed using a pipeline developed in house. The detected major microbial phylotypes were common among the three datasets, with seven bacterial phyla, fifteen bacterial families, and five archaeal taxa commonly identified across all datasets. There were also unique microbial taxa detected in each dataset. Elusimicrobia and Verrucomicrobia phyla; Desulfovibrionaceae, Elusimicrobiaceae, and Sphaerochaetaceae families; and Methanobrevibacter woesei were only detected in the RNA-Seq and RNA Amplicon-seq datasets, whereas Streptococcaceae was only detected in the DNA Amplicon-seq dataset. In addition, the relative abundances of four bacterial phyla, eight bacterial families and one archaeal taxon were different among the three datasets. This is the first study to compare the outcomes of rumen microbiota profiling between RNA-seq and RNA/DNA Amplicon-seq datasets. Our results illustrate the differences between these methods in characterizing microbiota both qualitatively and quantitatively for the same sample, and so caution must be exercised when comparing data.

The complete genome sequence of Eubacterium limosum SA11, a metabolically versatile rumen acetogen.

Acetogens are a specialized group of anaerobic bacteria able to produce acetate from CO2 and H2 via the Wood-Ljungdahl pathway. In some gut environments acetogens can compete with methanogens for H2, and as a result rumen acetogens are of interest in the development of microbial approaches for methane mitigation. The acetogen Eubacterium limosum SA11 was isolated from the rumen of a New Zealand sheep and its genome has been sequenced to examine its potential application in methane mitigation strategies, particularly in situations where hydrogenotrophic methanogens are inhibited resulting in increased H2 levels in the rumen. The 4.15 Mb chromosome of SA11 has an average G + C content of 47 %, and encodes 3805 protein-coding genes. There is a single prophage inserted in the chromosome, and several other gene clusters appear to have been acquired by horizontal transfer. These include genes for cell wall glycopolymers, a type VII secretion system, cell surface proteins and chemotaxis. SA11 is able to use a variety of organic substrates in addition to H2/CO2, with acetate and butyrate as the principal fermentation end-products, and genes involved in these metabolic pathways have been identified. An unusual feature is the presence of 39 genes encoding trimethylamine methyltransferase family proteins, more than any other bacterial genome. Overall, SA11 is a metabolically versatile organism, but its ability to grow on such a wide range of substrates suggests it may not be a suitable candidate to take the place of hydrogen-utilizing methanogens in the rumen.

Human oral isolate Lactobacillus fermentum AGR1487 induces a pro-inflammatory response in germ-free rat colons.

Lactobacilli are thought to be beneficial for human health, with lactobacilli-associated infections being confined to immune-compromised individuals. However, Lactobacillus fermentum AGR1487 negatively affects barrier integrity in vitro so we hypothesized that it caused a pro-inflammatory response in the host. We compared germ-free rats inoculated with AGR1487 to those inoculated with another L. fermentum strain, AGR1485, which does not affect in vitro barrier integrity. We showed that rats inoculated with AGR1487 had more inflammatory cells in their colon, higher levels of inflammatory biomarkers, and increased colonic gene expression of pro-inflammatory pathways. In addition, our in vitro studies showed that AGR1487 had a greater capacity to activate TLR signaling and induce pro-inflammatory cytokines in immune cells. This study indicates the potential of strains of the same species to differentially elicit inflammatory responses in the host and highlights the importance of strain characterization in probiotic approaches to treat inflammatory disorders.

Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range.

Ruminant livestock are important sources of human food and global greenhouse gas emissions. Feed degradation and methane formation by ruminants rely on metabolic interactions between rumen microbes and affect ruminant productivity. Rumen and camelid foregut microbial community composition was determined in 742 samples from 32 animal species and 35 countries, to estimate if this was influenced by diet, host species, or geography. Similar bacteria and archaea dominated in nearly all samples, while protozoal communities were more variable. The dominant bacteria are poorly characterised, but the methanogenic archaea are better known and highly conserved across the world. This universality and limited diversity could make it possible to mitigate methane emissions by developing strategies that target the few dominant methanogens. Differences in microbial community compositions were predominantly attributable to diet, with the host being less influential. There were few strong co-occurrence patterns between microbes, suggesting that major metabolic interactions are non-selective rather than specific.

Considerations in the use of fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy to characterize rumen methanogens and define their spatial distributions.

In this study, methanogen-specific coenzyme F420 autofluorescence and confocal laser scanning microscopy were used to identify rumen methanogens and define their spatial distribution in free-living, biofilm-, and protozoa-associated microenvironments. Fluorescence in situ hybridization (FISH) with temperature-controlled hybridization was used in an attempt to describe methanogen diversity. A heat pretreatment (65 °C, 1 h) was found to be a noninvasive method to increase probe access to methanogen RNA targets. Despite efforts to optimize FISH, 16S rRNA methanogen-specific probes, including Arch915, bound to some cells that lacked F420, possibly identifying uncharacterized Methanomassiliicoccales or reflecting nonspecific binding to other members of the rumen bacterial community. A probe targeting RNA from the methanogenesis-specific methyl coenzyme M reductase (mcr) gene was shown to detect cultured Methanosarcina cells with signal intensities comparable to those of 16S rRNA probes. However, the probe failed to hybridize with the majority of F420-emitting rumen methanogens, possibly because of differences in cell wall permeability among methanogen species. Methanogens were shown to integrate into microbial biofilms and to exist as ecto- and endosymbionts with rumen protozoa. Characterizing rumen methanogens and defining their spatial distribution may provide insight into mitigation strategies for ruminal methanogenesis.

Lambs fed fresh winter forage rape (Brassica napus L.) emit less methane than those fed perennial ryegrass (Lolium perenne L.), and possible mechanisms behind the difference.

The objectives of this study were to examine long-term effects of feeding forage rape (Brassica napus L.) on methane yields (g methane per kg of feed dry matter intake), and to propose mechanisms that may be responsible for lower emissions from lambs fed forage rape compared to perennial ryegrass (Lolium perenne L.). The lambs were fed fresh winter forage rape or ryegrass as their sole diet for 15 weeks. Methane yields were measured using open circuit respiration chambers, and were 22-30% smaller from forage rape than from ryegrass (averages of 13.6 g versus 19.5 g after 7 weeks, and 17.8 g versus 22.9 g after 15 weeks). The difference therefore persisted consistently for at least 3 months. The smaller methane yields from forage rape were not related to nitrate or sulfate in the feed, which might act as alternative electron acceptors, or to the levels of the potential inhibitors glucosinolates and S-methyl L-cysteine sulfoxide. Ruminal microbial communities in forage rape-fed lambs were different from those in ryegrass-fed lambs, with greater proportions of potentially propionate-forming bacteria, and were consistent with less hydrogen and hence less methane being produced during fermentation. The molar proportions of ruminal acetate were smaller and those of propionate were greater in forage rape-fed lambs, consistent with the larger propionate-forming populations and less hydrogen production. Forage rape contained more readily fermentable carbohydrates and less structural carbohydrates than ryegrass, and was more rapidly degraded in the rumen, which might favour this fermentation profile. The ruminal pH was lower in forage rape-fed lambs, which might inhibit methanogenic activity, shifting the rumen fermentation to more propionate and less hydrogen and methane. The significance of these two mechanisms remains to be investigated. The results suggest that forage rape is a potential methane mitigation tool in pastoral-based sheep production systems.

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.

Determining the culturability of the rumen bacterial microbiome.

The goal of the Hungate1000 project is to generate a reference set of rumen microbial genome sequences. Toward this goal we have carried out a meta-analysis using information from culture collections, scientific literature, and the NCBI and RDP databases and linked this with a comparative study of several rumen 16S rRNA gene-based surveys. In this way we have attempted to capture a snapshot of rumen bacterial diversity to examine the culturable fraction of the rumen bacterial microbiome. Our analyses have revealed that for cultured rumen bacteria, there are many genera without a reference genome sequence. Our examination of culture-independent studies highlights that there are few novel but many uncultured taxa within the rumen bacterial microbiome. Taken together these results have allowed us to compile a list of cultured rumen isolates that are representative of abundant, novel and core bacterial species in the rumen. In addition, we have identified taxa, particularly within the phylum Bacteroidetes, where further cultivation efforts are clearly required. This information is being used to guide the isolation efforts and selection of bacteria from the rumen microbiota for sequencing through the Hungate1000.

Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.