Chordicoccus furentiruminis, gen. nov., sp. nov., a novel succinic acid producing bacterium isolated from a steer on a high grain diet.

This study presents MP1D12T (=NRRL B-67553T=NCTC 14480T), an isolate from the ruminal content of an Angus steer fed a high grain diet. Phenotypic and genotypic traits of the isolate were explored. MP1D12T was found to be a strictly anaerobic, catalase-negative, oxidase-negative, coccoid bacterium that frequently grows in chains. Analysis of metabolic products as a result of carbohydrate fermentation showed succinic acid as the major organic acid produced with lactic acid and acetic acid as minor products. Phylogenetic analysis of MP1D12T based on 16S rRNA nucleotide sequence and amino acid sequences from the whole genome presents a divergent lineage from other members in the family Lachnospiraceae. 16S rRNA sequence comparison, whole genome average nucleotide identity digital DNA-DNA hybridization and average amino acid identity results suggest that MP1D12T represents a novel species in a novel genus within the family Lachnospiraceae. We propose the creation of the genus Chordicoccus in which MP1D12T represents the type strain for the novel species Chordicoccus furentiruminis.

Ruminococcus bovis sp. nov., a novel species of amylolytic Ruminococcus isolated from the rumen of a dairy cow.

This study describes JE7A12T (=ATCC TSD-225T=NCTC 14479T), an isolate from the ruminal content of a dairy cow. Phenotypic and genotypic traits of the isolate were explored. JE7A12T was found to be a strictly anaerobic, catalase-negative, oxidase-negative, coccoid bacterium that grows in chains. The API 50 CH carbon source assay detected fermentation of d-glucose, d-fructose, d-galactose, glycogen and starch. HPLC showed acetate to be the major fermentation product as a result of carbohydrate fermentation. Phylogenetic analysis of JE7A12T based on 16S rRNA nucleotide sequence and amino acid sequences from the whole genome indicated a divergent lineage from the closest neighbours in the genus Ruminococcus. The results of 16S rRNA sequence comparison, whole genome average nucleotide identity (ANI) and DNA G+C content data indicate that JE7A12T represents a novel species which we propose the name Ruminococcus bovis with JE7A12T as the type strain.

Rumen fluid metabolomics of beef steers differing in feed efficiency.

INTRODUCTION: Beef is the most consumed red meat in the United States, and the US is the largest producer and consumer of beef cattle globally. Feed is one of the largest input costs for the beef cattle industry, accounting for 40-60% of the total input costs. Identifying methods for improving feed efficiency in beef cattle herds could result in decreased cost to both producers and consumers, as well as increased animal protein available for global consumption. METHODS: In this study, rumen fluid was collected from low- (n = 14) and high-RFI (n = 15) steers. Rumen fluid was filtered through a 0.22 µM syringe filter, extracted using 0.1% formic acid in acetonitrile:water:methanol (2:2:1) and injected into the Dionex UltiMate 3000 UHPLC system with an Exactive Plus Orbitrap MS. Peaks were identified using MAVEN and analyzed using MetaboAnalyst 4.0 and SAS. Significance was determined using an α ≤ 0.05. RESULTS: Eight metabolites were greater in low-RFI steers compared to high-RFI steers, including 3,4-dihydroxyphenylacetate, 4-pyridoxate, citraconate, hypoxanthine, succinate/methylmalonate, thymine, uracil, and xylose (P ≤ 0.05). These metabolites were predominantly involved in amino acid and lipid metabolism. CONCLUSIONS: Rumen fluid metabolomes differ in steers of varying feed efficiencies. These metabolites may be used as biomarkers of feed efficiency, and may provide insight as to factors contributing to differences in feed efficiency that may be exploited to improve feed efficiency in beef cattle herds.

Networks of energetic and metabolic interactions define dynamics in microbial communities.

Microorganisms form diverse communities that have a profound impact on the environment and human health. Recent technological advances have enabled elucidation of community diversity at high resolution. Investigation of microbial communities has revealed that they often contain multiple members with complementing and seemingly redundant metabolic capabilities. An understanding of the communal impacts of redundant metabolic capabilities is currently lacking; specifically, it is not known whether metabolic redundancy will foster competition or motivate cooperation. By investigating methanogenic populations, we identified the multidimensional interspecies interactions that define composition and dynamics within syntrophic communities that play a key role in the global carbon cycle. Species-specific genomes were extracted from metagenomic data using differential coverage binning. We used metabolic modeling leveraging metatranscriptomic information to reveal and quantify a complex intertwined system of syntrophic relationships. Our results show that amino acid auxotrophies create additional interdependencies that define community composition and control carbon and energy flux through the system while simultaneously contributing to overall community robustness. Strategic use of antimicrobials further reinforces this intricate interspecies network. Collectively, our study reveals the multidimensional interactions in syntrophic communities that promote high species richness and bolster community stability during environmental perturbations.

Supplementation of saturated long-chain fatty acids maintains intestinal eubiosis and reduces ethanol-induced liver injury in mice.

BACKGROUND & AIMS: Alcoholic liver disease is a leading cause of mortality. Chronic alcohol consumption is accompanied by intestinal dysbiosis, and development of alcoholic liver disease requires gut-derived bacterial products. However, little is known about how alterations to the microbiome contribute to pathogenesis of alcoholic liver disease. METHODS: We used the Tsukamoto-French mouse model, which involves continuous intragastric feeding of isocaloric diet or alcohol for 3 weeks. Bacterial DNA from the cecum was extracted for deep metagenomic sequencing. Targeted metabolomics assessed concentrations of saturated fatty acids in cecal contents. To maintain intestinal metabolic homeostasis, diets of ethanol-fed and control mice were supplemented with saturated long-chain fatty acids (LCFA). Bacterial genes involved in fatty acid biosynthesis, amounts of lactobacilli, and saturated LCFA were measured in fecal samples of nonalcoholic individuals and patients with active alcohol abuse. RESULTS: Analyses of intestinal contents from mice revealed alcohol-associated changes to the intestinal metagenome and metabolome, characterized by reduced synthesis of saturated LCFA. Maintaining intestinal levels of saturated fatty acids in mice resulted in eubiosis, stabilized the intestinal gut barrier, and reduced ethanol-induced liver injury. Saturated LCFA are metabolized by commensal Lactobacillus and promote their growth. Proportions of bacterial genes involved in fatty acid biosynthesis were lower in feces from patients with active alcohol abuse than controls. Total levels of LCFA correlated with those of lactobacilli in fecal samples from patients with active alcohol abuse but not in controls. CONCLUSIONS: In humans and mice, alcohol causes intestinal dysbiosis, reducing the capacity of the microbiome to synthesize saturated LCFA and the proportion of Lactobacillus species. Dietary approaches to restore levels of saturated fatty acids in the intestine might reduce ethanol-induced liver injury in patients with alcoholic liver disease.

The iron stimulon and fur regulon of Geobacter sulfurreducens and their role in energy metabolism.

Iron plays a critical role in the physiology of Geobacter species. It serves as both an essential component for proteins and cofactors and an electron acceptor during anaerobic respiration. Here, we investigated the iron stimulon and ferric uptake regulator (Fur) regulon of Geobacter sulfurreducens to examine the coordination between uptake of Fe(II) and the reduction of Fe(III) at the transcriptional level. Gene expression studies across a variety of different iron concentrations in both the wild type and a Δfur mutant strain were used to determine the iron stimulon. The stimulon consists of a broad range of gene products, ranging from iron-utilizing to central metabolism and iron reduction proteins. Integration of gene expression and chromatin immunoprecipitation (ChIP) data sets assisted in the identification of the Fur transcriptional regulatory network and Fur's role as a regulator of the iron stimulon. Additional physiological and transcriptional analyses of G. sulfurreducens grown with various Fe(II) concentrations revealed the depth of Fur's involvement in energy metabolism and the existence of redundancy within the iron-regulatory network represented by IdeR, an alternative iron transcriptional regulator. These characteristics enable G. sulfurreducens to thrive in environments with fluctuating iron concentrations by providing it with a robust mechanism to maintain tight and deliberate control over intracellular iron homeostasis.

Sulfide-driven microbial electrosynthesis.

Microbial electrosynthesis, the conversion of carbon dioxide to organic molecules using electricity, has recently been demonstrated for acetogenic microorganisms, such as Sporomusa ovata. The energy for reduction of carbon dioxide originates from the hydrolysis of water on the anode, requiring a sufficiently low potential. Here we evaluate the use of sulfide as an electron source for microbial electrosynthesis. Abiotically oxidation of sulfide on the anode yields two electrons. The oxidation product, elemental sulfur, can be further oxidized to sulfate by Desulfobulbus propionicus, generating six additional electrons in the process. The eight electrons generated from the combined abiotic and biotic steps were used to reduce carbon dioxide to acetate on a graphite cathode by Sporomusa ovata at a rate of 24.8 mmol/day · m(2). Using a strain of Desulfuromonas as biocatalyst on the anode resulted in an acetate production rate of 49.9 mmol/day · m(2), with a Coulombic efficiency of over 90%. These results demonstrate that sulfide can serve effectively as an alternative electron donor for microbial electrosynthesis.

The PurR regulon in Escherichia coli K-12 MG1655.

The PurR transcription factor plays a critical role in transcriptional regulation of purine metabolism in enterobacteria. Here, we elucidate the role of PurR under exogenous adenine stimulation at the genome-scale using high-resolution chromatin immunoprecipitation (ChIP)-chip and gene expression data obtained under in vivo conditions. Analysis of microarray data revealed that adenine stimulation led to changes in transcript level of about 10% of Escherichia coli genes, including the purine biosynthesis pathway. The E. coli strain lacking the purR gene showed that a total of 56 genes are affected by the deletion. From the ChIP-chip analysis, we determined that over 73% of genes directly regulated by PurR were enriched in the biosynthesis, utilization and transport of purine and pyrimidine nucleotides, and 20% of them were functionally unknown. Compared to the functional diversity of the regulon of the other general transcription factors in E. coli, the functions and size of the PurR regulon are limited.

The effect of supplementing native rumen microbes on milk production of dairy cows.

We evaluated the effects of 2 direct-fed microbial (DFM) supplements containing 4 native rumen microorganisms on the production of dairy cows. Ninety Holstein cows (43% primiparous) were fed a common diet. Mean days in milk, milk yield, and body weight at the beginning of the study (mean ± standard deviation) were 92 ± 23 d, 45 ± 10 kg/d, and 659 ± 86 kg, respectively. After 14 d, they were blocked by parity, days in milk, and energy-corrected milk (ECM) per unit of metabolic body weight. Within block, cows were randomly assigned to treatments, which were top-dressed daily for the next 112 d. Treatments were 150 g of ground corn mixed with (1) no live DFM (CON), (2) 5 g of a live DFM (Galaxis 2.0; G2), and (3) 5 g of a live DFM (Galaxis 2.0 Plus; G2P). G2 and G2P were products of Native Microbials Inc. (San Diego, CA) and contained the same organisms but in different concentrations. Supplementation with DFM did not alter yield of total milk, protein, or fat, but slightly decreased body weight gain and body condition score gain with no difference between G2 and G2P. The DFM tended to decrease dry matter intake (DMI) and tended to improve ECM/DMI. The DFM did not alter digestibility of fiber, starch, or protein and did not alter concentrations of glucose or nonesterified fatty acids, but tended to decrease concentration of insulin in plasma. Direct-fed microbials decreased somatic cell counts in milk with no difference between G2 and G2P. In conclusion, supplementation with native DFM had little impact on animal production and efficiency.

Effect of supplementation with ruminal probiotics on growth performance, carcass characteristics, plasma metabolites, methane emissions, and the associated rumen microbiome changes in beef cattle.

To evaluate the effect of supplementing beef cattle with a ruminal probiotic consisting of native rumen microbes (NRM; Chordicoccus furentiruminis, Prevotella albensis, and Succinivibrio dextrinosolvens) on methane (CH4) emissions, growth performance, carcass characteristics, and plasma metabolites, Angus × SimAngus-crossbred steers (n = 32; 8 per pen) and heifers (n = 48; 12 per pen) with an initial body weight (BW) of 353 ± 64 kg were used in randomized complete block design. Cattle were blocked by sex and BW and randomly assigned to 1 of 2 treatments (2 pens per treatment). Treatments consisted of diets offered for ad libitum intake with (NRM) or without (CON) the inclusion of the ruminal probiotic. Cattle were fed a growing diet for 49 d followed by a ground corn-based diet for 124 ± 27 d until reaching the targeted final BW (635 kg for steers and 590 kg for heifers). Methane emissions were estimated using the GreenFeed system (n = 12 per treatment) prior to trial commencement (baseline; period 1), and on three (2, 3, and 4), and two (5 and 6) different sampling periods throughout the growing and finishing stage, respectively. All data were analyzed using the PROC MIXED procedure of SAS. For CH4 production (g/d), there was a tendency for an NRM supplementation × period interaction (P = 0.07) where cattle-fed diets with NRM had lower production of methane in periods 3 and 4. Including NRM in the diet decreased CH4 yield (g/kg of dry matter intake (DMI)) by 20%. For CH4 emission intensity (g/kg of average daily gain (ADG)), an interaction (P < 0.01) of NRM supplementation × period occurred. In periods 2 and 3, cattle-fed diets with NRM inclusion had lower CH4 emission intensity than CON cattle. During the 84-d period when all cattle were still on the finishing diet, feeding NRM increased (P = 0.02) ADG and tended to increase (P = 0.10) DMI. At the end of the 84-d period, cattle-fed NRM tended to be heavier (P = 0.06) than CON cattle. Cattle supplemented with NRM required less (P = 0.04) days on feed to reach the targeted final BW. No differences (P ≤ 0.11) were detected for gain-to-feed ratio and carcass characteristics. Cattle-fed NRM had greater abundance of uncultured rumen bacteria that may improve rumen digestion when fed a high grain diet and potentially promote the reduction of enteric CH4 production. Results from this study suggest that daily administration of NRM may be a strategy to mitigate methanogenesis and improve the growth performance of beef cattle.

Greenhouse gas emissions are a major concern in the beef industry. This study examined the effects of supplementation with ruminal probiotics consisting of three native ruminal microbes (NRM) for their influence on methane reduction and growth performance of beef cattle. Eighty Angus × SimAngus-crossbred cattle were grouped by sex and weight, randomly assigned to a treatment group, control or NRM supplementation, and subsequently fed commercially relevant diets for at least 134 d with or without NRM supplementation until they reached a target finishing weight. Methane emissions and growth performance metrics were recorded at regular intervals. Cattle-fed diets with NRM had a greater average daily gain during most part of the experimental period, required fewer days to reach the finishing weight, and emitted less methane than cattle in the control treatment. Supplementing NRM can be a viable method to reduce greenhouse gas emissions while improving the performance of beef cattle-fed concentrates-based diets.

Effects of rumen-native microbial feed supplementation on milk yield, composition, and feed efficiency in lactating dairy cows.

The objective of this study was to evaluate the effects of two rumen-native microbial feed supplements (MFS) on milk production, milk composition, and feed efficiency. A total of 90 multiparous cows between 40 and 60 d in milk were enrolled in a randomized block design study. Within each block (baseline milk yield), cows were randomly assigned to: control (no microbial feed supplementation), MFS1 (0.33 g/kg total mixed ration [TMR] of an MFS containing a minimum of Clostridium beijerinckii at 2 × 106 CFU/g and Pichia kudriavzevii at 2 × 107 CFU/g), or MFS2 (0.33 g/kg TMR of a MFS containing a minimum of C. beijerinckii at 2 × 106 CFU/g, P. kudriavzevii at 2 × 107 CFU/g, Ruminococcus bovis at 2 × 107 CFU/g, and Butyrivibrio fibrisolvens at 2 × 107 CFU/g). Cows were housed in a single group and fed the study diets ad libitum for 270 d. Individual milk yield was recorded using electronic milk meters, and milk fat and protein were measured using optical in-line analyzers at each of two daily milkings. Treatment and treatment by time effects were assessed through multiple linear regression analyses. Treatment effects were observed for milk and energy-corrected milk (ECM) yields, milk fat and protein yields and concentrations, dry matter intake (DMI), and feed efficiency; those effects were conditional to time for milk yield, DMI, and feed efficiency. Overall, milk, ECM, fat, and protein yields were higher for MFS2 compared with control cows (+3.0, 3.7, 0.12, and 0.12 kg/d, respectively). Compared with MFS1, milk yield was higher and protein yield tended to be higher for MFS2 cows (+2.9 and 0.09 kg/d, respectively). In contrast, MFS1 cows produced 0.17 and 0.08 units of percentage per day more fat and protein than MFS2 cows, and 0.07 units of percentage per day more protein than control cows. Dry matter intake and feed efficiency were higher for MFS2 cows compared with MFS1 cows (+1.3 kg/d and 0.06, respectively), and feed efficiency was higher for MFS2 cows compared with control cows (+0.04). Where observed, treatment by time effects suggest that the effects of MFS2 were more evident as time progressed after supplementation was initiated. No effects of microbial supplementation were observed on body weight, body condition score, somatic cell count, or clinical mastitis case incidence. In conclusion, the supplementation of MFS2 effectively improved economically important outcomes such as milk yield, solids, and feed efficiency.

This study evaluates the effects of two rumen-native microbial feed supplements (MFS) on milk yield, composition, and feed efficiency in lactating dairy cows. Ninety multiparous Holstein cows between 40 and 60 d in milk were assigned to control (no microbial feed supplementation), MFS1 (Clostridium beijerinckii and Pichia kudriavzevii), or MFS2 (C. beijerinckii, P. kudriavzevii, Ruminococcus bovis, and Butyrivibrio fibrisolvens) total mixed ration supplementation. Overall, MFS2 cows had higher milk and milk component yields than control and MFS1, while MFS1 cows had higher milk component concentrations than control and MFS2. Feed efficiency was higher for MFS2 compared with control and MFS1 cows. Microbial feed supplementation improved economically important outcomes such as milk yield, solids, and feed efficiency.

Acetate reprograms gut microbiota during alcohol consumption.

Liver damage due to chronic alcohol use is among the most prevalent liver diseases. Alcohol consumption frequency is a strong factor of microbiota variance. Here we use isotope labeled [1-13C] ethanol, metagenomics, and metatranscriptomics in ethanol-feeding and intragastric mouse models to investigate the metabolic impacts of alcohol consumption on the gut microbiota. First, we show that although stable isotope labeled [1-13C] ethanol contributes to fatty acid pools in the liver, plasma, and cecum contents of mice, there is no evidence of ethanol metabolism by gut microbiota ex vivo under anaerobic conditions. Next, we observe through metatranscriptomics that the gut microbiota responds to ethanol-feeding by activating acetate dissimilation, not by metabolizing ethanol directly. We demonstrate that blood acetate concentrations are elevated during ethanol consumption. Finally, by increasing systemic acetate levels with glyceryl triacetate supplementation, we do not observe any impact on liver disease, but do induce similar gut microbiota alterations as chronic ethanol-feeding in mice. Our results show that ethanol is not directly metabolized by the gut microbiota, and changes in the gut microbiota linked to ethanol are a side effect of elevated acetate levels. De-trending for these acetate effects may be critical for understanding gut microbiota changes that cause alcohol-related liver disease.

Ruminal Protozoal Populations of Angus Steers Differing in Feed Efficiency.

Feed accounts for as much as 70% of beef production costs, and improvement of the efficiency with which animals convert feed to product has the potential to have substantial financial impact on the beef industry. The rumen microbiome plays a key role in determining feed efficiency; however, previous studies of rumen microbiota have not focused on protozoal communities despite the estimation that these organisms represent approximately 50% of rumen content biomass. Protozoal communities participate in the regulation of bacterial populations and nitrogen cycling-key aspects of microbiome dynamics. The present study focused on identifying potential associations of protozoal community profiles with feed efficiency. Weaned steers (n = 50) 7 months of age weighing approximately 260 kg were adapted to a growing ration and GrowSafe for 2 weeks prior to a 70-day feed efficiency trial. The GrowSafe system is a feeding system that monitors feed intake in real time. Body weights were collected on the first day and then every 7 days of the feed efficiency trial, and on the final day, approximately 50 mL of rumen content were collected via orogastric tubing and frozen at -80 °C. Body weight and feed intake were used to calculate residual feed intake (RFI) as a measure of feed efficiency, and steers were categorized as high (n = 14) or low (n = 10) RFI based on ±0.5 standard deviations about the mean RFI. Microbial DNA was extracted, and the eukaryotic component profiled by amplification and sequencing of 18S genes using degenerate primers that can amplify this locus across a range of protists. The taxonomy of protozoal sequences was assigned using QIIME 1.9 and analyzed using QIIME and SAS 9.4 with significance determined at α ≤ 0.05. Greater abundances of unassigned taxa were associated with high-RFI steers (p = 0.03), indicating a need for further study to identify component protozoal species. Differences were observed between low- and high-RFI steers in protozoal community phylogenetic diversity, including weighted beta-diversity (p = 0.04), Faith's phylogenetic diversity (p = 0.03), and observed Operational taxonomic unit (OTU) (p = 0.03). The unassigned taxa and differences in phylogenetic diversity of protozoal communities may contribute to divergences observed in feed efficiency phenotypes in beef steers.

Temporal Stability of the Ruminal Bacterial Communities in Beef Steers.

Nutritional studies involving ruminants have traditionally relied on relatively short transition or wash-out periods between dietary treatments, typically two to four weeks. However, little is known about adequate adaptation periods required to reach stabilization of the rumen microbiome that could provide more accurate results from nutritional studies in ruminants. This study determined the rumen bacterial communities and rumen environment parameters over ten weeks following transition from a forage-based to concentrate-based diet. Several α-diversity metrics, including observed OTUs and Simpson's Evenness fluctuated throughout the trial, but were typically either greatest (observed OTUs) or lowest (Simpson's) at week 5 of the trial contrasted from weeks 1 and 10 (P < 0.05). At week 4, several orders associated with the shift to the final bacterial community composition, including Pasteurellales, Aeromonadales, and Bacteroidales. At week 5, rumen pH was correlated with α-diversity (P = 0.005) and predictive of the rumen microbiome signature at week 10 (R2 = 0.48; P = 0.04). Rumen microbiome stability did not occur until approximately 9 weeks following adaptation to the diet and was associated with changes in specific bacterial populations and rumen environment. The results of this study suggest that adaptation and wash-out periods must be re-evaluated in order to accommodate necessary rumen microbiome acclimation.

Rumen Bacteria and Serum Metabolites Predictive of Feed Efficiency Phenotypes in Beef Cattle.

The rumen microbiome is critical to nutrient utilization and feed efficiency in cattle. Consequently, the objective of this study was to identify microbial and biochemical factors in Angus steers affecting divergences in feed efficiency using 16S amplicon sequencing and untargeted metabolomics. Based on calculated average residual feed intake (RFI), steers were divided into high- and low-RFI groups. Features were ranked in relation to RFI through supervised machine learning on microbial and metabolite compositions. Residual feed intake was associated with several features of the bacterial community in the rumen. Decreased bacterial α- (P = 0.03) and ß- diversity (P < 0.001) was associated with Low-RFI steers. RFI was associated with several serum metabolites. Low-RFI steers had greater abundances of pantothenate (P = 0.02) based on fold change (high/low RFI). Machine learning on RFI was predictive of both rumen bacterial composition and serum metabolomic signature (AUC ≥ 0.7). Log-ratio proportions of the bacterial classes Flavobacteriia over Fusobacteriia were enriched in low-RFI steers (F = 6.8, P = 0.01). Reductions in Fusobacteriia and/or greater proportions of pantothenate-producing bacteria, such as Flavobacteriia, may result in improved nutrient utilization in low-RFI steers. Flavobacteriia and Pantothenate may potentially serve as novel biomarkers to predict or evaluate feed efficiency in Angus steers.

Efficient Synergistic Single-Cell Genome Assembly.

As the vast majority of all microbes are unculturable, single-cell sequencing has become a significant method to gain insight into microbial physiology. Single-cell sequencing methods, currently powered by multiple displacement genome amplification (MDA), have passed important milestones such as finishing and closing the genome of a prokaryote. However, the quality and reliability of genome assemblies from single cells are still unsatisfactory due to uneven coverage depth and the absence of scattered chunks of the genome in the final collection of reads caused by MDA bias. In this work, our new algorithm Hybrid De novo Assembler (HyDA) demonstrates the power of coassembly of multiple single-cell genomic data sets through significant improvement of the assembly quality in terms of predicted functional elements and length statistics. Coassemblies contain significantly more base pairs and protein coding genes, cover more subsystems, and consist of longer contigs compared to individual assemblies by the same algorithm as well as state-of-the-art single-cell assemblers SPAdes and IDBA-UD. Hybrid De novo Assembler (HyDA) is also able to avoid chimeric assemblies by detecting and separating shared and exclusive pieces of sequence for input data sets. By replacing one deep single-cell sequencing experiment with a few single-cell sequencing experiments of lower depth, the coassembly method can hedge against the risk of failure and loss of the sample, without significantly increasing sequencing cost. Application of the single-cell coassembler HyDA to the study of three uncultured members of an alkane-degrading methanogenic community validated the usefulness of the coassembly concept. HyDA is open source and publicly available at http://chitsazlab.org/software.html, and the raw reads are available at http://chitsazlab.org/research.html.

Single-cell genome and metatranscriptome sequencing reveal metabolic interactions of an alkane-degrading methanogenic community.

Microbial interactions have a key role in global geochemical cycles. Although we possess significant knowledge about the general biochemical processes occurring in microbial communities, we are often unable to decipher key functions of individual microorganisms within the environment in part owing to the inability to cultivate or study them in isolation. Here, we circumvent this shortcoming through the use of single-cell genome sequencing and a novel low-input metatranscriptomics protocol to reveal the intricate metabolic capabilities and microbial interactions of an alkane-degrading methanogenic community. This methanogenic consortium oxidizes saturated hydrocarbons under anoxic conditions through a thus-far-uncharacterized biochemical process. The genome sequence of a dominant bacterial member of this community, belonging to the genus Smithella, was sequenced and served as the basis for subsequent analysis through metabolic reconstruction. Metatranscriptomic data generated from less than 500 pg of mRNA highlighted metabolically active genes during anaerobic alkane oxidation in comparison with growth on fatty acids. These data sets suggest that Smithella is not activating hexadecane by fumarate addition. Differential expression assisted in the identification of hypothetical proteins with no known homology that may be involved in hexadecane activation. Additionally, the combination of 16S rDNA sequence and metatranscriptomic data enabled the study of other prevalent organisms within the consortium and their interactions with Smithella, thus yielding a comprehensive characterization of individual constituents at the genome scale during methanogenic alkane oxidation.

Characterization and modelling of interspecies electron transfer mechanisms and microbial community dynamics of a syntrophic association.

Syntrophic associations are central to microbial communities and thus have a fundamental role in the global carbon cycle. Despite biochemical approaches describing the physiological activity of these communities, there has been a lack of a mechanistic understanding of the relationship between complex nutritional and energetic dependencies and their functioning. Here we apply a multi-omic modelling workflow that combines genomic, transcriptomic and physiological data with genome-scale models to investigate dynamics and electron flow mechanisms in the syntrophic association of Geobacter metallireducens and Geobacter sulfurreducens. Genome-scale modelling of direct interspecies electron transfer reveals insights into the energetics of electron transfer mechanisms. While G. sulfurreducens adapts to rapid syntrophic growth by changes at the genomic and transcriptomic level, G. metallireducens responds only at the transcriptomic level. This multi-omic approach enhances our understanding of adaptive responses and factors that shape the evolution of syntrophic communities.

Characterizing the interplay between multiple levels of organization within bacterial sigma factor regulatory networks.

Bacteria contain multiple sigma factors, each targeting diverse, but often overlapping sets of promoters, thereby forming a complex network. The layout and deployment of such a sigma factor network directly impacts global transcriptional regulation and ultimately dictates the phenotype. Here we integrate multi-omic data sets to determine the topology, the operational, and functional states of the sigma factor network in Geobacter sulfurreducens, revealing a unique network topology of interacting sigma factors. Analysis of the operational state of the sigma factor network shows a highly modular structure with σ(N) being the major regulator of energy metabolism. Surprisingly, the functional state of the network during the two most divergent growth conditions is nearly static, with sigma factor binding profiles almost invariant to environmental stimuli. This first comprehensive elucidation of the interplay between different levels of the sigma factor network organization is fundamental to characterize transcriptional regulatory mechanisms in bacteria.