Rumen Lachnospiraceae isolate NK3A20 exhibits metabolic flexibility in response to substrate and coculture with a methanogen.

Hydrogen (H2) is the primary electron donor for methane formation in ruminants, but the H2-producing organisms involved are largely uncharacterized. This work integrated studies of microbial physiology and genomics to characterize rumen bacterial isolate NK3A20 of the family Lachnospiraceae. Isolate NK3A20 was the first recognized isolate of the NK3A20 group, which is among the ten most abundant bacterial genera in 16S rRNA gene surveys of rumen microbiota. NK3A20 produced acetate, butyrate, H2, and formate from glucose. The end product ratios varied when grown with different substrates and at different H2 partial pressures. NK3A20 produced butyrate as a major product using glucose or under high H2 partial pressures and switched to mainly acetate in the presence of galacturonic acid (an oxidized sugar) or in coculture with a methanogen. Growth with galacturonic acid was faster at elevated H2 concentrations, while elevated H2 slowed growth with glucose. Genome analyses revealed the presence of multiple hydrogenases including a membrane-bound Ech hydrogenase, an electron bifurcating butyryl-CoA dehydrogenase (Bcd-Etf), and an Rnf complex that may be involved in modulating the observed metabolic pathway changes, providing insight into H2 formation in the rumen. IMPORTANCE The genus-level NK3A20 group is one of the ten most abundant genera of rumen bacteria. Like most of the rumen bacteria that produce the hydrogen that is converted to methane in the rumen, it is understudied, without any previously characterized isolates. We investigated isolate NK3A20, a cultured member of this genus, and showed that it modulates hydrogen production in response to its growth substrates and the hydrogen concentration in its environment. Low-hydrogen concentrations stimulated hydrogen formation, while high concentrations inhibited its formation and shifted the fermentation to more reduced organic acid products. We found that growth on uronic acids, components of certain plant polymers, resulted in low hydrogen yields compared to glucose, which could aid in the selection of low-methane feeds. A better understanding of the major genera that produce hydrogen in the rumen is part of developing strategies to mitigate biogenic methane emitted by livestock agriculture.

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.

Glucocorticoids and immune checkpoint inhibitors in glioblastoma.

PURPOSE: Immunotherapy, activation of the immune system to target tumor cells, represents a paradigm shift in the treatment of cancer. Immune checkpoint therapies, which target immunomodulatory molecules expressed on T-lymphocytes, have demonstrated improved survival in a variety of malignancies. However, benefit in glioblastoma, the most common and devastating malignant brain tumor, remains to be seen. With several recent clinical trials failing to show efficacy of immunotherapy, concerns have been raised regarding the impact of glucocorticoid use in this patient population that may impair the ability for immune checkpoint inhibitors to affect a response. METHODS: For this article we examined the mechanism by which immune checkpoint inhibitors activate, and glucocorticoids impair, T-lymphocyte function. RESULTS: In this context, we review the clinical data of immune checkpoint inhibitors in glioblastoma as well as the impact glucocorticoids have on immune checkpoint inhibitor efficacy. Finally, we highlight key questions that remain in the field, and the potential benefit of further research for central nervous system tumors. CONCLUSION: More information on the extent, character and duration of glucocorticoids on patients treated with PD-(L)1 will better inform both clinical management and novel therapeutic development of immunotherapy in patients with CNS malignancies.

Investigation of the Impact from IL-2, IL-7, and IL-15 on the Growth and Signaling of Activated CD4+ T Cells.

While CAR-T therapy is a growing and promising area of cancer research, it is limited by high cost and the difficulty of consistently culturing T-cells to therapeutically relevant concentrations ex-vivo. Cytokines IL-2, IL-7 and IL-15 have been found to stimulate the growth of T cells, however, the optimized combination of these three cytokines for T cell proliferation is unknown. In this study, we designed an integrated experimental and modeling approach to optimize cytokine supplementation for rapid expansion in clinical applications. We assessed the growth data for statistical improvements over no cytokine supplementation and used a systems biology approach to identify genes with the highest magnitude of expression change from control at several time points. Further, we developed a predictive mathematical model to project the growth rate for various cytokine combinations, and investigate genes and reactions regulated by cytokines in activated CD4+ T cells. The most favorable conditions from the T cell growth study and from the predictive model align to include the full range of IL-2 and IL-7 studied, and at lower levels of IL-15 (6 ng/mL or 36 ng/mL). The highest growth rates were observed where either IL-2 or IL-7 was at the highest concentration tested (15 ng/mL for IL-2 and 80 ng/mL for IL-7) while the other was at the lowest (1 ng/mL for IL-2 and 6 ng/mL for IL-7), or where both IL-2 and IL-7 concentrations are moderate-corresponding to condition keys 200, 020, and 110 respectively. This suggests a synergistic interaction of IL-2 and IL-7 with regards to promoting optimal proliferation and survival of the activated CD4+ T cells. Transcriptomic data analysis identified the genes and transcriptional regulators up/down-regulated by each of the cytokines IL-2, IL-7, and IL-15. It was found that the genes with persistent expressing changes were associated with major pathways involved in cell growth and proliferation. In addition to influencing T cell metabolism, the three cytokines were found to regulate specific genes involved in TCR, JAK/STAT, MAPK, AKT and PI3K-AKT signaling. The developed Fuzzy model that can predict the growth rate of activated CD4+ T cells for various combinations of cytokines, along with identified optimal cytokine cocktails and important genes found in transcriptomic data, can pave the way for optimizing activated CD4 T cells by regulating cytokines in the clinical setting.

Butyrivibrio hungatei MB2003 Competes Effectively for Soluble Sugars Released by Butyrivibrio proteoclasticus B316T during Growth on Xylan or Pectin.

Rumen bacterial species belonging to the genus Butyrivibrio are important degraders of plant polysaccharides, particularly hemicelluloses (arabinoxylans) and pectin. Currently, four species are recognized; they have very similar substrate utilization profiles, but little is known about how these microorganisms are able to coexist in the rumen. To investigate this question, Butyrivibrio hungatei MB2003 and Butyrivibrio proteoclasticus B316T were grown alone or in coculture on xylan or pectin, and their growth, release of sugars, fermentation end products, and transcriptomes were examined. In monocultures, B316T was able to grow well on xylan and pectin, while MB2003 was unable to utilize either of these insoluble substrates to support significant growth. Cocultures of B316T grown with MB2003 revealed that MB2003 showed growth almost equivalent to that of B316T when either xylan or pectin was supplied as the substrate. The effect of coculture on the transcriptomes of B316T and MB2003 was assessed; B316T transcription was largely unaffected by the presence of MB2003, but MB2003 expressed a wide range of genes encoding proteins for carbohydrate degradation, central metabolism, oligosaccharide transport, and substrate assimilation, in order to compete with B316T for the released sugars. These results suggest that B316T has a role as an initiator of primary solubilization of xylan and pectin, while MB2003 competes effectively for the released soluble sugars to enable its growth and maintenance in the rumen.IMPORTANCE Feeding a future global population of 9 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Butyrivibrio species are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings suggest that closely related species of Butyrivibrio have developed unique strategies for the degradation of plant fiber and the subsequent assimilation of carbohydrates in order to coexist in the competitive rumen environment. The identification of genes expressed during these competitive interactions gives further insight into the enzymatic machinery used by these bacteria as they degrade the xylan and pectin components of plant fiber.

Comparative Genomics of Rumen Butyrivibrio spp. Uncovers a Continuum of Polysaccharide-Degrading Capabilities.

Plant polysaccharide breakdown by microbes in the rumen is fundamental to digestion in ruminant livestock. Bacterial species belonging to the rumen genera Butyrivibrio and Pseudobutyrivibrio are important degraders and utilizers of lignocellulosic plant material. These bacteria degrade polysaccharides and ferment the released monosaccharides to yield short-chain fatty acids that are used by the ruminant for growth and the production of meat, milk, and fiber products. Although rumen Butyrivibrio and Pseudobutyrivibrio species are regarded as common rumen inhabitants, their polysaccharide-degrading and carbohydrate-utilizing enzymes are not well understood. In this study, we analyzed the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent fraction of New Zealand dairy cows to explore the polysaccharide-degrading potential of these important rumen bacteria. Comparative genome analyses combined with phylogenetic analysis of their 16S rRNA genes and short-chain fatty acid production patterns provide insight into the genomic diversity and physiology of these bacteria and divide Butyrivibrio into 3 species clusters. Rumen Butyrivibrio bacteria were found to encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) and binding proteins. In total, 4,421 glycoside hydrolases (GHs), 1,283 carbohydrate esterases (CEs), 110 polysaccharide lyases (PLs), 3,605 glycosyltransferases (GTs), and 1,706 carbohydrate-binding protein modules (CBM) with predicted activities involved in the depolymerization and transport of the insoluble plant polysaccharides were identified. Butyrivibrio genomes had similar patterns of CAZyme families but varied greatly in the number of genes within each category in the Carbohydrate-Active Enzymes database (CAZy), suggesting some level of functional redundancy. These results suggest that rumen Butyrivibrio species occupy similar niches but apply different degradation strategies to be able to coexist in the rumen.IMPORTANCE Feeding a global population of 8 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Members of the genera Butyrivibrio and Pseudobutyrivibrio are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings have highlighted the immense enzymatic machinery of Butyrivibrio and Pseudobutyrivibrio species for the degradation of plant fiber, suggesting that these bacteria occupy similar niches but apply different degradation strategies in order to coexist in the competitive rumen environment.

Bifidobacterium pseudolongum in the Ceca of Rats Fed Hi-Maize Starch Has Characteristics of a Keystone Species in Bifidobacterial Blooms.

Starches resistant to mammalian digestion are present in foods and pass to the large bowel, where they may be degraded and fermented by the microbiota. Increases in relative abundances of bifidobacteria (blooms) have been reported in rats whose diet was supplemented with Hi-Maize resistant starch. We determined that the bifidobacterial species present in the rat cecum under these circumstances mostly belonged to Bifidobacterium animalis However, cultures of B. animalis isolated from the rats failed to degrade Hi-Maize starch to any extent. In contrast, Bifidobacterium pseudolongum also detected in the rat microbiota had high starch-degrading ability. Transcriptional comparisons showed increased expression of a type 1 pullulanase, alpha-amylase, and glycogen debranching enzyme by B. pseudolongum when cultured in medium containing Hi-Maize starch. Maltose was released into the culture medium, and B. animalis cultures had shorter doubling times in maltose medium than did B. pseudolongum Thus, B. pseudolongum, which was present at a consistently low abundance in the microbiota, but which has extensive enzymatic capacity to degrade resistant starch, showed the attributes of a keystone species associated with the bifidobacterial bloom.IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the rat gut) using DNA-based observations and in vitro experimentation. The microbial community of the large bowel of animals, including humans, has been studied extensively through the use of high-throughput DNA sequencing methods and advanced bioinformatics analysis. These studies reveal the compositions and genetic capacities of microbiotas but not the intricacies of how microbial communities function. Our work, combining DNA sequence analysis and laboratory experiments with cultured strains of bacteria, revealed that the increased abundance of bifidobacteria in the rat gut, induced by feeding indigestible starch, involved a species that cannot itself degrade the starch (Bifidobacterium animalis) but cohabits with a species that can (Bifidobacterium pseudolongum). B. pseudolongum has the characteristics of a keystone species in the community because it had low abundance but high ability to perform a critical function, the hydrolysis of resistant starch.

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.

Monoglobus pectinilyticus gen. nov., sp. nov., a pectinolytic bacterium isolated from human faeces.

A novel anaerobic pectinolytic bacterium (strain 14T) was isolated from human faeces. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 14T belonged to the family Ruminococcaceae, but was located separately from known clostridial clusters within the taxon. The closest cultured relative of strain 14T was Acetivibrio cellulolyticus (89.7 % sequence similarity). Strain 14T shared ~99 % sequence similarity with cloned 16S rRNA gene sequences from uncultured bacteria derived from the human gut. Cells were Gram-stain-positive, non-motile cocci approximately 0.6 µm in diameter. Strain 14T fermented pectins from citrus peel, apple, and kiwifruit as well as carbohydrates that are constituents of pectins and hemicellulose, such as galacturonic acid, xylose, and arabinose. TEM images of strain 14T, cultured in association with plant tissues, suggested extracellular fibrolytic activity associated with the bacterial cells, forming zones of degradation in the pectin-rich regions of middle lamella. Phylogenetic and phenotypic analysis supported the differentiation of strain 14T as a novel genus in the family Ruminococcaceae. The name Monoglobus pectinilyticus gen. nov., sp. nov. is proposed; the type strain is 14T (JCM 31914T=DSM 104782T).

Complete genome sequence of the rumen bacterium Butyrivibrio fibrisolvens D1T.

Butyrivibrio are anaerobic bacteria and members of the family Lachnospiraceae with important roles in fiber digestion in both animals and humans. This report describes the complete genome of Butyrivibrio fibrisolvens type strain D1T (DSM 3071) consisting of a chromosome (CP146963), megaplasmid (pNP243), and small plasmid (pNP21).

P53 loss of heterozygosity (LOH) in formalin-fixed paraffin-embedded leiomyosarcoma (LMS): a novel report.

BACKGROUND: The occurrence of p53 loss of heterozygosity (LOH) is a common genetic event in malignancy. LOH occurs when a heterozygous locus loses one of its two parental alleles, becoming homozygous at that locus, by either copy number loss (CNL-LOH) or by becoming copy number neutral (CNN-LOH). A role for CNL-LOH (cnLOH) has been postulated in cancer aetiology. Loss of heterozygosity (LOH) results in irreversible genetic loss. AIMS: LOH was determined in DNA extracted from formalin-fixed paraffin-embedded (FFPE) leiomyosarcoma (LMS) specimens in a retrospective study from 30 patients, to assess the prognostic significance of LOH. The findings were analysed and their validity assessed. LOH was an adverse prognostic factor in LMS. Prospective uniform standardisation of formalin-fixation techniques is required. METHODS: DNA was extracted from 169 formalin-fixed paraffin blocks of 30 patients with LMS, following extensive tissue microdissection. Genomic DNA was amplified using the polymerase chain reaction (PCR) technique. Fluorescence-based microsatellite PCR was used to detect and quantitate heterozygosity loss. RESULTS: LOH was detected at gene locus 17p13 in 16 LMS (Four grade 2 and 12 grade 3 LMS). LOH was not detected in 14 LMS cases (one grade 1, five grade 2 and eight grade 3 LMS). LOH was associated with shorter patient survival. CONCLUSIONS: The results reported herein endorse the value of utilizing FFPE DNA in identifying LOH as a prognostic factor in LMS. The results have implications for tumour biobanking and precision medicine in patients with sarcomas.

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).

Methanobrevibacter boviskoreani JH1T growth on alcohols allows development of a high throughput bioassay to detect methanogen inhibition.

Rumen methanogenic archaea use by-products of fermentation to carry out methanogenesis for energy generation. A key fermentation by-product is hydrogen (H2), which acts as the source of reducing potential for methane (CH4) formation in hydrogenotrophic methanogens. The in vitro cultivation of hydrogenotrophic rumen methanogens requires pressurised H2 which limits the ability to conduct high-throughput screening experiments with these organisms. The genome of the hydrogenotrophic methanogen Methanobrevibacter boviskoreani JH1T harbors genes encoding an NADP-dependent alcohol dehydrogenase and a F420-dependent NADP reductase, which may facilitate the transfer of reducing potential from ethanol to F420 via NADP. The aim of this study was to explore the anaerobic culturing of JH1T without pressurised H2, using a variety of short chain alcohols. The results demonstrate that in the absence of H2, JHIT can use ethanol, 1-propanol, and 1-butanol but not methanol, as a source of reducing potential for methanogenesis. The ability to use ethanol to drive CH4 formation in JH1T makes it possible to develop a high throughput culture-based bioassay enabling screening of potential anti-methanogen compounds. The development of this resource will help researchers globally to accelerate the search for methane mitigation technologies for ruminant animals. Global emissions pathways that are consistent with the temperature goal of the Paris Agreement, rely on substantial reductions of agricultural greenhouse gasses, particularly from ruminant animals.

Extracellular polysaccharide-degrading proteome of Butyrivibrio proteoclasticus.

Plant polysaccharide-degrading rumen microbes are fundamental to the health and productivity of ruminant animals. Butyrivibrio proteoclasticus B316(T) is a gram-positive, butyrate-producing anaerobic bacterium with a key role in hemicellulose degradation in the rumen. Gel-based proteomics was used to examine the growth-phase-dependent abundance patterns of secreted proteins recovered from cells grown in vitro with xylan or xylose provided as the sole supplementary carbon source. Five polysaccharidases and two carbohydrate-binding proteins (CBPs) were among 30 identified secreted proteins. The endo-1,4-ß-xylanase Xyn10B was 17.5-fold more abundant in the culture medium of xylan-grown cells, which suggests it plays an important role in hemicellulose degradation. The secretion of three nonxylanolytic enzymes and two CBPs implies they augment hemicellulose degradation by hydrolysis or disruption of associated structural polysaccharides. Sixteen ATP-binding cassette (ABC) transporter substrate-binding proteins were identified, several of which had altered relative abundance levels between growth conditions, which suggests they are important for oligosaccharide uptake. This study demonstrates that B. proteoclasticus modulates the secretion of hemicellulose-degrading enzymes and ATP-dependent sugar uptake systems in response to growth substrate and supports the notion that this organism makes an important contribution to polysaccharide degradation in the rumen.

An In-Vitro Study of the Expansion and Transcriptomics of CD4+ and CD8+ Naïve and Memory T Cells Stimulated by IL-2, IL-7 and IL-15.

The growth of T cells ex vivo for the purpose of T cell therapies is a rate-limiting step in the overall process for cancer patients to achieve remission. Growing T cells is a fiscally-, time-, and resource-intensive process. Cytokines have been shown to accelerate the growth of T cells, specifically IL-2, IL-7, and IL-15. Here a design of experiments was conducted to optimize the growth rate of different naïve and memory T cell subsets using combinations of cytokines. Mathematical models were developed to study the impact of IL-2, IL-7, and IL-15 on the growth of T cells. The results show that CD4+ and CD8+ naïve T cells grew effectively using moderate IL-2 and IL-7 in combination, and IL-7, respectively. CD4+ and CD8+ memory cells favored moderate IL-2 and IL-15 in combination and moderate IL-7 and IL-15 in combination, respectively. A statistically significant interaction was observed between IL-2 and IL-7 in the growth data of CD4+ naïve T cells, while the interaction between IL-7 and IL-15 was found for CD8+ naïve T cells. The important genes and related signaling pathways and metabolic reactions were identified from the RNA sequencing data for each of the four subsets stimulated by each of the three cytokines. This systematic investigation lays the groundwork for studying other T cell subsets.

Hydrogen and formate production and utilisation in the rumen and the human colon.

Molecular hydrogen (H2) and formate (HCOO-) are metabolic end products of many primary fermenters in the mammalian gut. Both play a vital role in fermentation where they are electron sinks for individual microbes in an anaerobic environment that lacks external electron acceptors. If H2 and/or formate accumulate within the gut ecosystem, the ability of primary fermenters to regenerate electron carriers may be inhibited and microbial metabolism and growth disrupted. Consequently, H2- and/or formate-consuming microbes such as methanogens and homoacetogens play a key role in maintaining the metabolic efficiency of primary fermenters. There is increasing interest in identifying approaches to manipulate mammalian gut environments for the benefit of the host and the environment. As H2 and formate are important mediators of interspecies interactions, an understanding of their production and utilisation could be a significant entry point for the development of successful interventions. Ruminant methane mitigation approaches are discussed as a model to help understand the fate of H2 and formate in gut systems.

Electron flow: key to mitigating ruminant methanogenesis.

Disposal of electrons generated during the fermentation of ingested feed is a fundamental feature of anaerobic microbial gut ecosystems. Here, we focus on the well-studied rumen environment to highlight how electrons are transferred through anaerobic fermentation pathways and how manipulating this electron flow is important to reducing methane emissions from ruminants. Priorities for research that can accelerate understanding in this area are highlighted.

The large episomes of Butyrivibrio proteoclasticus B316T have arisen through intragenomic gene shuttling from the chromosome to smaller Butyrivibrio-specific plasmids.

The genome of Butyrivibrio proteoclasticus B316(T) contains three large episomes including a 302 kb chromid (BPc2) and two large plasmids of 361 (pCY360) and 186 kb (pCY186). The two plasmids are largely cryptic and it is therefore difficult to gauge their contributions or importance to the biology of B. proteoclasticus. Here, we provide evidence that at least BPc2 and pCY360 are essential as neither could be cured using several previously described curing techniques. We show that BPc2 exists at a copy number of 1, while pCY360 and pCY186 exist at copy numbers of 4 and 0.9, respectively. Yet the transcriptional activities of each episome are much less than that of the 3.5 Mb chromosome. Codon usage analyses did not support the hypothesis that the genes of all three episomes were acquired horizontally. Instead our analyses suggest that the vast majority of genes on each episome were transferred from the 3.5 Mb B. proteoclasticus chromosome. Analysis of their replication origins, however, suggests the plasmid backbones share an evolutionary lineage with the smaller Butyrivibrio specific plasmids, pRJF1 and pRJF2. A survey of 13 species of the Butyrivibrio/Pseudobutyrivibrio assemblage identified similar large episomes in nine strains. DNA hybridization experiments revealed none contained an rRNA operon and only a 145 kb episome from Pseudobutyrivibrioruminis possessed an ortholog of the pCY360 plasmid replication initiation protein. The size and distribution of episomes within the nine strains of Butyrivibrio/Pseudobutyrivibrio showed no correlation with 16S rRNA based phylogeny, leading to a hypothesis that the large episomes of Butyrivibrio spp., have arisen through intragenomic gene transfer events from the chromosome to small horizontally acquired elements.

Mixing and oxygen transfer characteristics of a microplate bioreactor with surface-attached microposts.

Bioprocess optimization for cell-based therapies is a resource heavy activity. To reduce the associated cost and time, process development may be carried out in small volume systems, with the caveat that such systems be predictive for process scale-up. The transport of oxygen from the gas phase into the culture medium, characterized using the volumetric mass transfer coefficient, kL a, has been identified as a critical parameter for predictive process scale-up. Here, we describe the development of a 96-well microplate with integrated Redbud Posts to provide mixing and enhanced kL a. Mixing in the microplate is characterized by observation of dyes and analyzed using the relative mixing index (RMI). The kL a is measured via dynamic gassing out method. Actuating Redbud Posts are shown to increase rate of planar homogeneity (2 min) verse diffusion alone (120 min) and increase oxygenation, with increasing stirrer speed (3500-9000 rpm) and decreasing fill volume (150-350 µL) leading to an increase in kL a (4-88 h-1 ). Significant increase in Chinese Hamster Ovary growth in Redbud Labs vessel (580,000 cells mL-1 ) versus the control (420,000 cells mL-1 ); t(12.814) = 8.3678, p ≤ .001), and CD4+ Naïve cell growth in the microbioreactor indicates the potential for this technology in early stage bioprocess development and optimization.

Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium Butyrivibrio proteoclasticus.

The release of polysaccharide from the plant cell wall is a key process to release the stored energy from plant biomass. Within the ruminant digestive system, a host of commensal microorganisms speed the breakdown of plant cell matter releasing fermentable sugars. The presence of phenolic compounds, most notably ferulic acid (FA), esterified within the cell wall is thought to pose a significant impediment to the degradation of the plant cell wall. The structure of a FA esterase from the ruminant bacterium Butyrivibrio proteoclasticus has been determined in two different space groups, in both the apo-form, and the ligand bound form with FA located in the active site. The structure reveals a new lid domain that has no structural homologues in the PDB. The flexibility of the lid domain is evident by the presence of three different conformations adopted by different molecules in the crystals. In the FA-bound structures, these conformations show sequential binding and closing of the lid domain over the substrate. Enzymatic activity assays demonstrate a broad activity against plant-derived hemicellulose, releasing at least four aromatic compounds including FA, coumaric acid, coumarin-3-carboxylic acid, and cinnamic acid. The rumen is a complex ecosystem that efficiently degrades plant biomass and the genome of B. proteoclasticus contains greater than 130 enzymes, which are potentially involved in this process of which Est1E is the first to be well characterized.