Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Animal Science
Non Technical Summary
Considering the global abundance of cellulosic biomass and the fact that it cannot be efficiently metabolized and assimilated by humans, ruminant livestock production represents a sustainable and critical mechanism to convert cellulose into animal protein for human populations worldwide. Since the genomes of animals do not encode the necessary enzymes to digest plant fiber material, ruminants rely on symbiotic microorganisms to transform the feed they ingest into nutrients they can assimilate for growth and maintenance.Since ruminal microorganisms directly affect the efficiency with which ruminants can transform feed into animal products, modulating their activity is critical to improving the feed efficiency of ruminant livestock. However, as the function and metabolic potential of most ruminal microorganisms remain poorly characterized, one of the main challenges in pursuing effective strategies to manipulate ruminal microbial activities is to overcome our limited knowledge of how their metabolic functions are regulated.On an individual basis, ruminal microorganisms are metabolic specialists (i.e. that they are limited in the number and types of metabolic functions that they can perform). In order to overcome their limitations, they assemble into very diverse and complex communities that consist of thousands of different microbial species. This allows them to benefit from the complementary metabolic capabilities of other community members. In the context of the nutrient complexity of a ruminant diet, the ability of the rumen to metabolize the different components of feed is the result of the complementary metabolic activities of a wide array of microbial specialists.Based on current available knowledge, most bacterial cells tend to follow similar basic rules. For instance, bacterial cells that can metabolize more than one nutrient have intrinsic mechanisms that prioritize the utilization of certain substrates over the others. In the context of the rumen environment, many different bacterial species are likely to preferably utilize cellulose because of its high abundance in plant fibers, but they are likely to differ in the other types of substrates that they can metabolize because they are present in lower abundance in particular feed ingredients. As bacterial species with the same substrate preferences compete for the same resources, their shared substrate needs may be a cause of reduced ruminal efficiency. One possible strategy to increase ruminal productivity could then be to promote conditions that favor the activity of the most beneficial or efficient bacterial species while minimizing the activity of their competitors.Regulation of gene expression (i.e. control of the synthesis of enzymes and other proteins required for metabolic pathways) is a primary mechanism used to control metabolic pathways in bacterial cells. Control of microbial metabolism is thought to function mainly as a simple "on/off" switch. In its simplest form, a sensor triggers a signal to induce production of the enzymes and proteins that are required to utilize a nutrient when it is present. Depletion of the nutrient then results in disappearance of the signal, followed by the breakdown of enzymes that are no longer necessary, which allows the cell to reuse the newly available amino acids to synthesize the next set of enzymes that are needed. When two or more substrates are available at the same time, the signal triggered by the preferred substrate will inhibit the signals induced from the other substrates until it becomes depleted.In bacterial cells, the production of enzymes and other proteins is mainly controlled at the level of transcription (i.e. by controlling the synthesis of RNA copies from the respective genes that encode the enzymes or other proteins that are needed). Transcription is a cellular process that is typically regulated by a group of proteins referred to 'transcriptional regulators' or 'transcription factors'. In order to perform their duties, transcriptional regulators typically need to bind directly to specific DNA recognition sites that are located in close proximity to where RNA synthesis of their target gene is initiated. Since each type of transcription factor can be characterized by the specific DNA sequence that it recognizes, one effective strategy to determine how genes are regulated is to search for the presence of specific DNA sequence patterns in bacterial genomic sequences.In the context of this research project, we wish to identify and map DNA recognition sites in available genomic sequences from ruminal bacteria, with a primary focus on comparing genes that encode enzymes and proteins involved in the utilization of nutrients. In addition, we propose to use that information to also identify other genes with the same recognition sites, as they would be expected to be co-expressed (i.e. expressed under the same conditions). Based on these comparisons, we will be able to identify metabolic networks (sets of metabolic pathways that would be active at the same time and induced by the same signal). Together, these data will provide valuable insights on how the metabolism of ruminal bacterial cells is controlled.In the short term, potential applications of this information would include improved prebiotic formulations to promote the proliferation and activity of favorable bacterial species or consortia. Prebiotics are nutrients that can be metabolized by microbial symbionts, but not by their animal host. Through mapping of metabolic pathways that are co-regulated or cross-regulated with the utilization of specific prebiotic substrates, these can be used individually or in combinations as part of prebiotic formulations to target the metabolism of particular symbionts.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Ruminant livestock species are crucial to sustainable animal production strategies because of their ability to transform cellulosic biomass into animal products. As animal genomes do not encode the necessary enzymes to digest the structural polysaccharide components of plant fibers (i.e. cellulose and hemicellulose), ruminants rely on communities of symbiotic gut microorganisms to metabolize feed into nutrients that they can assimilate. Thus, the metabolic activities of symbiotic gut microorganisms directly affect the efficiency with which ruminant livestock species can transform feed into animal products. The metabolic potential of most ruminal microorganisms remains poorly characterized, especially for uncultured microbial species. Thus the main challenge in pursuing effective strategies to manipulate ruminal microbial activities is overcoming our limited knowledge of the metabolic functions of these uncharacterized microorganisms and how they are controlled.As part of my previous Hatch project, a metagenomics approach was used to explore the metabolic potential of uncharacterized ruminal bacterial species that we could culture when ruminal fluid was supplemented with specific substrates such as starch or cellulose. In addition to genes encoding for enzymes involved in starch or cellulose utilization, respectively, we also identified genes encoding enzymes involved in metabolic pathways for the utilization of other nutrients or substrates. For instance, one enriched bacterial species was predicted to be able to metabolize mannose, as well as peptidoglycans, in addition to cellulose.In the context of the ruminal habitat where thousands of microbial species coexist, this highlighted the importance of taking into account other traits besides utilization of a major substrate when defining ecological niches for individual organisms. It also highlighted the importance of gaining a better understanding of the mechanisms involved in regulating the respective pathways involved in the utilization of different nutrients. Better insight into cellular decision-making mechanisms that prioritize certain substrates over others would greatly benefit the improvement of current strategies or the development of new approaches for increased efficiency of ruminal function by manipulating the activity of its resident communities of microorganisms.General hypothesisSince transcriptional regulation is the main regulatory mechanism used by bacteria to control the production of enzymes and proteins in response to their metabolic needs, we hypothesize that the expression of the genes involved in the utilization of one substrate is regulated by the same transcriptional regulators, while genes involved in metabolizing other substrates are regulated by different sets of transcriptional regulators.Overall strategyAs transcription is controlled by DNA-binding proteins that physically interact with specific DNA recognition sequences in the promoter of their target genes, we aim to identify genes regulated by the same signals through the identification of common or shared DNA motifs in their respective promoters. In other words, genes that share the same sequence motifs in their respective promoters would be expected to be co-expressed, while genes with other sequence motifs in their respective promoters would be controlled by other transcriptional regulators.In this context, we propose the following main objectives for this project:Objective 1. Identification of DNA recognition sites potentially involved in regulating the transcription of genes that participate in the same metabolic functions.The search for DNA motifs will focus on intergenic regions (i.e. nucleotide sequences between coding sequences). Since bacterial genomes tend to be very compact, intergenic regions are typically of limited length (i.e. in the range of a few hundred nucleotides). For this reason, they are generally considered as promoter regions. Identification of candidate DNA recognition sites will include targeted searches for motifs known to be recognized by transcription factors, as well as identification of other nucleotide patterns.Objective 2. Identification of metabolic pathways regulated by common transcription regulators. To gain further insight on metabolic networks regulated by the same transcriptional regulators, the enzymes and proteins encoded by sets of genes that share DNA nucleotide recognition motifs will be organized into pathways. A comparative analysis of metabolic networks regulated by different transcriptional regulators will provide further insight on how different pathways are prioritized in ruminal bacterial cells.Objective 3.Confirmation of regulatory pathway using in vitro cultures of ruminal fluid. Following up on the previous objective that will result in the identification of pathways predicted to be expressed under the same conditions, the goal of this final objective will be to experimentally test these predictions using ruminal bacterial cells maintained in culture.
Project Methods
Overall strategyTranscription is controlled by DNA-binding proteins that physically interact with specific DNA recognition sequences in the promoter of their target genes. We aim to identify genes regulated by the same signals by identifying common or shared DNA motifs in their respective promoters. The majority of the proposed work will be conducted using bioinformatics.Objective 1. Identification of DNA recognition sites potentially involved in regulating the transcription of genes that participate in the same metabolic functions.The genomic sequences from ruminal bacteria to be used for this objective will include the metagenomics sets that have so far been assembled by my group, as well as others available from publicly accessible databases such as those maintained by NCBI. The search for DNA motifs will focus on intergenic regions (i.e. nucleotide sequences between coding sequences). Coding regions will first be identified using publicly available web-based tools, such as ORFfinder (NCBI), or RAST (http://rast.nmpdr.org), then subsequently removed from this analysis in order to focus the search for DNA motifs in the intergenic regions. Since bacterial genomes tend to be very compact, intergenic regions are typically of limited length (i.e. in the range of a few hundred nucleotides), and are thus generally considered as promoter regions. Identification of candidate DNA recognition sites will include targeted searches for motifs known to be recognized by transcription factors, as well as identification of other nucleotide patterns which may represent cis-regulatory sites that have yet to be characterized. This analysis will make use of publicly available software such as MEME (http://meme-suite.org/tools/meme), PredictRegulon (http://prodoric.tu-bs.de/), and BioProspector (http://robotics.stanford.edu/~xsliu/BioProspector/). Custom written Perl script developed by my research group will also be utilized.Objective 2. Identification of metabolic pathways regulated by common transcription regulators. To gain further insight on metabolic networks regulated by the same transcriptional regulators, the enzymes and proteins encoded by sets of genes that share DNA nucleotide recognition motifs will be organized into pathways. Publicly available web-based tools such as RAST (http://rast.nmpdr.org) and KEGG will be used for this analysis. A comparative analysis of metabolic networks regulated by different transcriptional regulators will provide further insight on how different pathways are prioritized in ruminal bacterial cells.Objective 3.Confirmation of regulatory pathway using in vitro cultures of ruminal fluid. My previous Hatch project resulted in the successful cultivation of bacteria from rumen fluid, which allowed us to identify candidate starch and cellulose utilizers. Thus we have established protocols to reproducibly enrich for different types of ruminal bacterial species. Using the results from objective 2 that will identify pathways predicted to be expressed under the same conditions, the goal of this final objective will be to confirm these predictions in cultured cells. Triplicate cultures will be established using cellulose, starch, or other relevant substrates/nutrients based on the pathways being analyzed. From samples collected at different time points, DNA and RNA will be extracted and purified to perform qPCR using custom oligonucleotide primers designed to specifically target genomic sequences predicted to be either expressed under the conditions tested or not expressed (negative control). For genes predicted to be expressed, qPCR will be expected to detect the target sequence in both DNA (positive control - detection of genomic sequences as a result of enrichment of its corresponding species) and RNA (expression of the gene under the experimental culture conditions tested). For control genes of an enriched species that would not be expected to be expressed under the conditions tested, qPCR would detect its targets in the DNA sample (positive control), but not in the RNA sample (negative control).