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

Identication and Elucidation of Mechanisms Involved in Microbial Community Dynamics using Systems Biology /

Microbial communities play a key role in the natural environment. However, the study of these communities if often limited by the inability to cultivate individual community members. Although culture-independent meta-level analyses are useful for establishing the basic structure of the community, they often mask the role individual species fulfill within the community, stymieing our ability to understand and thus manipulate the community phenotype. Within this dissertation, microbial communities involved in global carbon cycling are studied from a species-centric perspective. First, the ferric uptake regulator (Fur) is studied in Fe(III)-reducing Geobacter sulfurreducens, a common member of subsurface soil environments. Chromatin immunopreciptation was utilized to establish the genome-wide binding profile of Fur. This dataset coupled with gene expression datasets derived from a variety of iron conditions was used to elucidate the iron stimulon and Fur regulon of G.sulfurreducens. Additional physiological and transcriptional analyses of G. sulfurreducens grown with various Fe(II) concentrations revealed the depth of Fur's involvement in energy metabolism. Next, single cell de novo sequencing and metatranscriptomics was used to reveal the intricate metabolic interactions of a 15-year-old alkane-degrading methanogenic consortium. The methanogenic consortium oxidizes saturated hydrocarbons under anoxic conditions through a thus far unknown biochemical process. The genome sequence of a dominant bacterial member belonging to the family of Syntrophace was sequenced and served as the basis for metabolic reconstruction. Metatranscriptomic data highlighted metabolically active genes of this bacterium. Subsequent genome analysis assisted in the identification of critical genes and pathways involved in anaerobic alkane metabolism. When perturbed by butyric or caprylic acid, the species composition of the alkane- degrading community shifts to accommodate the change. Although some species persist and remain active despite the shift, major ecological succession by these dormant microbes leads to a new, stable community optimized to digest the newly added substrate. The final chapter of this thesis describes the study of these poorly characterized genera from the species perspective using metagenomic binning, metatranscriptomics, and metabolic modeling. Subsequent community analysis revealed that energetic requirements, amino acid auxotrophies, and strategic antimicrobial usage all contribute to the robustness of the community. Collectively, these results suggest that the multidimensional interactions utilized by natural communities bolster their stability during environmental perturbation and promote the maintenance of high species richnes

Ruminococcus bovis Gaffney & Embree & Gilmore & Embree 2021, SP. NOV.

DESCRIPTION OF RUMINOCOCCUS BOVIS SP. NOV. Ruminococcus bovis (bo'vis. L. gen. n. bovis of the cow) Ruminococcus bovis is an obligately anaerobic, catalasenegative and oxidase-negative bacterium. It is Gram-stainpositive and forms chains of small cocci when cultured in liquid medium. When cultured on TSB+FAC solid medium, it forms small, slightly opaque, off-white, circular colonies with even margins. Fermentation of D-galactose,D-glucose, D-fructose, maltose, glycogen, aesculin/ferric citrate and starch is indicated by API CH 50. The major fermentation product is acetate, with ethanol and glycerol as minor products. No lactate, butyrate, butanol, propionate, succinate or pyruvate is produced. The type strain is JE7A12 T (=ATCC TSD-225 T =NCTC 14479 T) and was originally isolated from rumen content of a healthy, Holstein cow from Tulare, California, USA. The genomic DNA G+C content of the type strain is 34.6 mol%.Published as part of Gaffney, James, Embree, Jordan, Gilmore, Sean & Embree, Mallory, 2021, RUMiNOCOCCUS BOViS sp. nov., a novel species of amylolytic RUMiNOCOCCUS isolated from the rumen of a dairy cow, pp. 1-7 in International Journal of Systematic and Evolutionary Microbiology (004924) (004924) 71 (8) on page 6, DOI: 10.1099/ijsem.0.004924, http://zenodo.org/record/622413

Extracellular matrix turnover and inflammation in chemically-induced TMJ arthritis mouse models.

The temporomandibular joint (TMJ) is a fibrocartilaginous tissue critical for chewing and speaking. In patients with temporomandibular disorders (TMDs), permanent tissue loss can occur. Recapitulating the complexity of TMDs in animal models is difficult, yet critical for the advent of new therapies. Synovial fluid from diseased human samples revealed elevated levels of tumor necrosis factor alpha (TNF-alpha). Here, we propose to recapitulate these findings in mice by subjecting murine TMJs with TNF-alpha or CFA (Complete Freund's Adjuvant) in mandibular condyle explant cultures and by local delivery in vivo using TMJ intra-articular injections. Both TNF-alpha and CFA delivery to whole mandibular explants and in vivo increased extracellular matrix deposition and increased cartilage thickness, while TNF-alpha treated explants had increased expression of inflammatory cytokines and degradative enzymes. Moreover, the application of TNF-alpha or CFA in both models reduced cell number. CFA delivery in vivo caused soft tissue inflammation, including pannus formation. Our work provides two methods of chemically induced TMJ inflammatory arthritis through a condyle explant model and intra-articular injection model that replicate findings seen in synovial fluid of human patients, which can be used for further studies delineating the mechanisms underlying TMJ pathology

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

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

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

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 <i>Sporomusa ovata</i>. 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 <i>Desulfobulbus propionicus</i>, 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 <i>Sporomusa ovata</i> at a rate of 24.8 mmol/day·m². Using a strain of <i>Desulfuromonas</i> as biocatalyst on the anode resulted in an acetate production rate of 49.9 mmol/day·m², with a Coulombic efficiency of over 90%. These results demonstrate that sulfide can serve effectively as an alternative electron donor for microbial electrosynthesis

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