Source: UNIVERSITY OF ILLINOIS submitted to NRP
GENOMCE-ENABLED ANALYSIS OF CARBON AND NITROGEN METABOLISM IN PREVOTELLA RUMINICOLA TO ENHANCE RUMEN FUNCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0212911
Grant No.
2008-35206-18784
Cumulative Award Amt.
$326,000.00
Proposal No.
2007-04250
Multistate No.
(N/A)
Project Start Date
Jan 1, 2008
Project End Date
Dec 31, 2012
Grant Year
2008
Program Code
[42.0]- (N/A)
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
ANIMAL SCIENCES
Non Technical Summary
Improving the release and capture of energy and protein-yielding nutrients from feedstuffs remain high priority goals for all ruminant production systems. In North America the need to reduce our dependence on foreign oil imports has increased the demand for cornstarch for ethanol production and concomittantly the supply of co-products arising from these processes. Further improvements in ruminal function and digestive capacity will be required if these co-products, as well as any other fibrous feedstuff, are to substitute for corn, and the capture of nitrogen and energy-yielding nutrients is to be improved. Thus, the long-term goal of our research group is to elucidate the biochemical, genetic and molecular mechanisms underpinning the microbial biology of plant cell wall and protein degradation, and the subsequent utilization of these products by Prevotella ruminicola, one of the most prevalent and metabolically versatile rumen bacteria. Our multiphasic approach involves examining how P. ruminicola responds, in a global or whole genome manner, to the supply of different energy and protein sources, followed by detailed biochemical characterization of the proteins encoded by genes identified using the microarrays as well as from bioinformatic mining of the genome sequence. We will also develop gene transfer and mutagenesis protocols in order to overcome barriers to the creation and evaluation of recombinant Prevotella spp. Advances in our understanding of the biology of ruminal Prevotellas should ultimately result in productive alterations in ruminant growth and nutrient utilization. More efficient nutrient utilization will also reduce the environmental footprint associated with ruminant productions systems in North America, particularly with respect to nitrogen metabolism.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023910100050%
3023910110050%
Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3910 - Cross-commodity research--multiple animal species;

Field Of Science
1100 - Bacteriology; 1000 - Biochemistry and biophysics;
Goals / Objectives
The long-term goal of our research group is to elucidate the biochemical, genetic and molecular mechanisms underpinning the microbial biology of plant cell wall and protein degradation, and the subsequent utilization of these products by rumen bacteria. This proposal seeks to capitalize on the availability of closed genome sequence for P. ruminicola strain 23 as an outcome of the funding provided by the USDA-NSF to the North American Consortium for the Genomics of Fibrolytic Ruminal Bacteria (NAC-GFRB). P. ruminicola was selected for genome sequencing by USDA because of its dominant role in ruminal fiber and protein degradation as well as fundamental scientific interest. Our overarching hypothesis is that the genome of Prevotella ruminicola encodes a model for one of the most metabolically versatile, successful and numerically dominant ruminal anaerobes with a prodigious capacity for digesting indigestible dietary polysaccharides and proteins. We propose to use a functional genomics approach to reveal, for the first time, the genetic, enzymatic and cellular mechanisms employed by P. ruminicola 23 following the specific objectives outlined below: Specific Objective 1. Global expression profiles using a whole genome microarray approach for identification and interactions of genetic regulatory networks involved in carbon and nitrogen metabolism. Specific Objective 2. Characterization of key proteins and regulators of peptide hydrolysis and utilization, ammonia assimilation and arabinoxylan degradation using a directed genome mining approach for future structural and catalytic studies to engineer optimized proteins for reintroduction to the rumen. Specific Objective 3. Development and application of gene transfer and mutagenesis methods for use with P. ruminicola and P. bryantii to overcome barriers to the creation and evaluation of recombinant Prevotella spp. that might improve ruminal function and performance.
Project Methods
Specific Objective 1. Global expression profiles of genes involved in carbon and nitrogen metabolism using a whole genome microarray approach. Total genome arrays of ca. 3,000 ORFs will be fabricated and replicated reverse-dye experiments performed. P. ruminicola 23 will be grown in chemostat to ensure identical growth rates and thus similar rrn expression levels. In addition, environmental conditions are constant with the exception of nutrient conditions imposed. Batch grown cultures will also be hybridized to the microarrays to examine temporal expression patterns and clarify induction or repression responses when grown on xylan. RNA isolation, purification and labeling, as well as hybridization will be carried out using optimized protocols. A number of genes (20-25) differentially expressed in the microarray experiments will be selected for real-time QRT-PCR experiments to validate the induction levels measured. Specific Objective 2. Characterization of key proteins and regulators of ammonia assimilation, peptidase activity and arabinoxylan degradation at the DNA, RNA and protein level using a directed genome mining approach. Computer analyses were used to search the P. ruminicola 23 genome for genes encoding proteins expected to play a role in ammonia assimilation, peptidase activity and arabinoxylan degradation. This objective is based on a genome mining, bioinformatic search for homologs of each of the proteins involved in ammonia assimilation, peptidase activity and arabinoxylan degradation. Genes of interest are amplified and cloned into an overexpression vector in order to make large quantities of proteins for biochemical characterization. In addition, selected highly expressed genes identified in Specific Objective 1 involved in ammonia assimilation, and plant cell wall degradation and their regulation will be amplified, cloned, overexpressed and biochemically characterized using well documented and standard protocols in our laboratory. Specific Objective 3: Development and application of gene transfer and mutagenesis methods for use with P. ruminicola and P. bryantii. Our principal goal for this objective is to maximize the transformation efficiency in both Prevotella spp., and to employ these optimized methods to generate mutants by gene displacement. Our first approach with P. ruminicola 23 is to use the pB8-51 series of plasmid shuttle vectors which are stably replicated in Bacteroides spp. and P. bryantii; and the pBI143 series of plasmid shuttle vectors, which are known to replicate in human colonic Bacteroides but were not conjugatively transferred to P. bryantii. We will also use the pBI191-type plasmid vector that was shown to be stably maintained in ruminal Bacteroides isolates. In each instance, the plasmid DNA will first be methylated. We anticipate that the systematic approaches described here will result in a high efficiency gene transfer system for use with both species. Our approach to mutational analysis of P. ruminicola and P. bryantii will be to produce gene replacement mutants using suicide-plasmid delivery vectors.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Changes in the global gene expression profiles of P. ruminicola 23 in response to excess (10 mM) or growth-limiting (0.7 mM) concentrations of ammonia were analyzed by microarray and related to changes in enzymatic activity. The results demonstrated dramatic changes in gene expression and enzymatic activity between the two conditions. In total, 166 genes (5.8% of the genome) were transcriptionally up-regulated during growth on an excess concentration of ammonia, while 287 genes (10.0% of the genome) were transcriptionally up-regulated during growth on a limiting concentration of ammonia. Specifically, growth in excess concentrations of ammonia induced genes involved in amino acid biosynthesis, while ammonia-limiting conditions caused the induced genes involved in DNA metabolism, protein fate and the manipulation of the cell envelope. Ammonia limiting conditions also led to the induction of 20 genes involved in the transport of various substrates including amino acids, peptides, amines and carbohydrates, organic alcohols and acids transporters. We observed links between carbohydrate and nitrogen metabolism through glutamate biosynthesis and reverse TCA cycle up-regulation when ammonia was in excess. Interestingly, the NADPH-GDH and GS-GOGAT pathways were also up-regulated under these conditions. Our results provide a whole genome overview of P. ruminicola responses to growth in limiting and excess concentrations of ammonia. Improving the current understanding of regulatory mechanisms we used by this important rumen bacterium to adapt to both excess and limiting environmental concentrations of ammonia. In addition, changes in the global gene expression profile of P. ruminicola 23 in response to variations in the available nitrogen source (ammonia or peptides) were analyzed by microarray and related to changes in enzymatic activity and the proteome. In total, 110 genes (3.8% of the genome) were transcriptionally upregulated during growth on ammonia, while 120 genes (4.2 % of the genome) were transcriptionally upregulated during growth on peptides. P. ruminicola 23 grown on ammonia induced genes which are predicted to be involved in amino acid biosynthesis, molecular transport and several that affect the cell envelope. Growth on peptides induced genes whose products are involved in DNA metabolism, protein fate, protein synthesis and transcription. GS-GOGAT pathways were also upregulated when grown on ammonia. The greatest transcriptional up-regulation was observed for the ammonium transporter, amt (47.0 fold) and the nitrogen regulatory protein PII (46.5 fold) during growth with ammonia. In contrast, growth on peptides resulted in the up-regulation of more than 17 ribosomal proteins but no up-regulation of other nitrogen metabolism pathways. Our results provide a whole genome transcriptional overview of the responses by P. ruminicola 23 to two different nitrogen sources and collectively lead to an overall improvement in the understanding of the genetic responses and biochemical pathways used by P. ruminicola 23 to obtain and utilize different nitrogen sources. PARTICIPANTS: Dr. Jong-Nam Kim who completed this work for his PhD obtained a Postdoctoral position at the Division of Microbiology, National Center for Toxicological Research/U.S. FDA, Jefferson, Arkansas 72079, USA. He is working on completing these manuscripts for journal submission. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The goal of this study was to gain a combined biochemical and genetic understanding of the processes ruminal bacteria undertake for the utilization of different hemicellulose and nitrogen sources. Firstly, this study analyzes the regulation of gene expression and enzyme activities involved in ammonia assimilation by one of the major cellulolytic bacterium R. albus 8, which may be applied as a model for ammonia assimilation in the Firmicutes phylum. Secondary, this study investigates the global gene expressions of the numerically-dominant hemicellulolytic bacterium P. ruminicola 23 in the Bacteroidetes phylum enabled by closed genome sequence. Further, we characterize hemicellulase, carbohydrate esterase, as well as ammonia assimilation enzymes and analyze enzyme activities. Collectively these analyses enable the characterization of the transcriptional regulation pathway and corresponding expression of genes whose enzymatic products respond to the different nitrogen sources. Genomic and postgenomic analyses of hemicelluose and nitrogen metabolism of the numerically-dominant hemicellulolytic P. ruminicola 23 and predominant cellulolytic R. albus 8 therefore fill the present gaps in knowledge regarding the regulation of important hemicellulose and nitrogen metabolism enzymes, and their responses to the different carbon and nitrogen sources.

Publications

  • No publications reported this period


Progress 01/01/08 to 12/31/12

Outputs
OUTPUTS: Changes in the global gene expression profile of P. ruminicola 23 in response to variations in the available nitrogen source (ammonia or peptides) were analyzed by microarray and related to changes in enzymatic activity and the proteome. The results demonstrated dramatic changes in gene expression and enzymatic activity between the two conditions. In total, 110 genes (3.7% of the genome) were transcriptionally upregulated during growth on ammonia, while 120 genes (4.1% of the genome) were transcriptionally upregulated during growth on peptides. Specifically, growth on ammonia induced genes whose products are predicted to be involved in amino acid biosynthesis, molecular transport and several other factors that affect the cell envelope. Growth on peptides induced genes whose products are involved in DNA metabolism, protein fate, protein synthesis and transcription. We observed that links between carbohydrate and nitrogen metabolism were evident from glutamate biosynthesis and reverse TCA cycle upregulation when ammonia was on ammonia. Interestingly, the GS-GOGAT pathway was also upregulated under these conditions. The greatest transcriptional up-regulations were observed for the ammonium transporter, amt and the nitrogen regulatory protein PII during growth with ammonia. Our results lead to an overall improvement in the understanding of the genetic responses and biochemical pathways used by P. ruminicola to obtain and utilize different nitrogen sources. Next, using genomic and transcriptomic analyses we set out to interrogate the regulatory networks and genomic organization of the P. ruminicola genes whose enzymes and associated proteins facilitate the degradation of xylan. We analyzed changes in the transcriptome during growth on four enriched xylan sources and then placed these results in the context of complete natural substrates. Growth dynamics of P. ruminicola demonstrated a marked preference for soluble xylans over the insoluble substrates. Transcriptional analysis identified ten separate xylan utilization clusters, which each appear to be independently regulated as distinct operons. This unique genomic and regulatory organization provides significant insight into the metabolic strategy that has evolved in P. ruminicola and make this organism so successful within and important to the rumen environment. We also measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium P. ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide from corn relative to a non-esterified fraction of corn oligosaccharides. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The goal of this study was to gain a combined biochemical and genetic understanding of the processes ruminal bacteria undertake for the utilization of different hemicellulose and nitrogen sources. Firstly, this study analyzes the regulation of gene expression and enzyme activities involved in ammonia assimilation by one of the major cellulolytic bacterium R. albus 8, which may be applied as a model for ammonia assimilation in the Firmicutes phylum. Secondary, this study investigates the global gene expressions of the numerically dominant, hemicellulolytic bacterium P. ruminicola 23 in the Bacteroidetes phylum enabled by closed genome sequence. Further, we characterize hemicellulase, carbohydrate esterase, as well as ammonia assimilation enzymes and analyze enzyme activities. Collectively these analyses enable the characterization of the transcriptional regulation pathway and corresponding expression of genes whose enzymatic products respond to the different nitrogen sources. Genomic and postgenomic analyses of hemicelluose and nitrogen metabolism of the numerically-dominant hemicellulolytic P. ruminicola 23 and predominant cellulolytic R. albus 8 therefore fill the present gaps in knowledge regarding the regulation of important hemicellulose and nitrogen metabolism enzymes, and their responses to the different carbon and nitrogen sources.

Publications

  • Dodd, D., Kocherginskaya, S.A., Spies, M.A., Beery, K.E., Abbas, C.A., Mackie, R.I. and Cann, I.K. 2009. Biochemical analysis of a beta-D-xylosidase and a bifunctional xylanase-ferulic acid esterase from a xylanolytic gene cluster in Prevotella ruminicola 23. J Bacteriol. 191:3328-3338.
  • Purushe, J., Fouts, D.E., Morrison, M., White, B.A., Mackie, R.I. and the North American Consortium for Rumen Bacteria (Coutinho, P.M., Henrissat, B. and Nelson, K.E.). 2010. Comparative genome analysis of Prevotella ruminicola and Prevotella bryantii: Insights into their environmental niche. Microb. Ecol. 60:721-729.

  • Dodd, D., Kiyonari, S., Mackie, R.I. and Cann, I.K.O. 2010. Functional diversity of four glycoside hydrolase family 3 enzymes from the rumen bacterium Prevotella bryantii B14. J. Bacteriol. 192:2335-2345.

  • Dodd, D., Moon, Y.H., Swaminathan, K., Mackie, R.I. and Cann, I.K. 2010. Transcriptomic analyses of xylan degradation by Prevotella bryantii and insights into energy acquisition by xylanolytic bacteroidetes. J. Biol. Chem. 285:30261-30273.
  • Kabel, M.A., Yeoman, C.J., Han, Y., Dodd, D., Abbas, C.A., de Bont, J.A., Morrison, M., Cann, I.K. and Mackie, R.I. 2011. Biochemical characterization and relative expression levels of multiple carbohydrate esterases of the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate. Appl. Environ. Microbiol. 77:5671-581.

  • Dodd, D., Mackie, R.I. and Cann, I.K. 2011. Xylan degradation, a metabolic property shared by rumen and human colonic Bacteroidetes. Mol. Microbiol. 79:292-304.

  • Kim, J.N., Cann, I.K. and Mackie, R.I. 2012. Purification, characterization, and expression of multiple Glutamine Synthetases from Prevotella ruminicola 23. J. Bacteriol. 194:176-84.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOS(FA,Ac)) from corn relative to a non-esterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40 degC) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOS(FA,Ac), a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly non-polymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The goal of this study was to gain a combined biochemical and genetic understanding of the processes ruminal bacteria undertake for the utilization of different hemicellulose and nitrogen sources. Firstly, this study analyzes the regulation of gene expression and enzyme activities involved in ammonia assimilation by one of the major cellulolytic bacterium R. albus 8, which may be applied as a model for ammonia assimilation in the Firmicutes phylum. Secondary, this study investigates the global gene expressions of the numerically-dominant hemicellulolytic bacterium P. ruminicola 23 in the Bacteroidetes phylum enabled by closed genome sequence. Further, we characterize hemicellulase, carbohydrate esterase, as well as ammonia assimilation enzymes and analyze enzyme activities. Collectively these analyses enable the characterization of the transcriptional regulation pathway and corresponding expression of genes whose enzymatic products respond to the different nitrogen sources. Genomic and postgenomic analyses of hemicelluose and nitrogen metabolism of the numerically-dominant hemicellulolytic P. ruminicola 23 and predominant cellulolytic R. albus 8 therefore fill the present gaps in knowledge regarding the regulation of important hemicellulose and nitrogen metabolism enzymes, and their responses to the different carbon and nitrogen sources.

Publications

  • Kabel, M.A., Yeoman, C.J., Han, Y., Dodd, D., Abbas, C.A., de Bont, J.A., Morrison, M., Cann, I.K. and Mackie, R.I. 2011. Biochemical characterization and relative expression levels of multiple carbohydrate esterases of the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate. Appl. Environ. Microbiol. 77:5671-581.
  • Dodd, D., Mackie, R.I. and Cann, I.K. 2011. Xylan degradation, a metabolic property shared by rumen and human colonic Bacteroidetes. Mol. Microbiol. 79:292-304.
  • Kim, J.N., Cann, I.K. and Mackie, R.I. 2012. Purification, characterization, and expression of multiple Glutamine Synthetases from Prevotella ruminicola 23. J. Bacteriol. 194:176-84.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The genus Prevotella is one of the more numerically dominant bacteria in the rumen, making significant contributions to both carbohydrate and nitrogen metabolism. In addition it plays a major role in proteolysis. Previous research has shown P. ruminicola 23 is able to efficiently utilize both ammonia and peptides as a nitrogen source for growth. Changes in the global gene expression profile of P. ruminicola 23 in response to variations in the available nitrogen source (ammonia or peptides) were analyzed by microarray and related to changes in enzymatic activity and the proteome. The results demonstrated dramatic changes in gene expression and enzymatic activity between the two conditions. In total, 110 genes (3.7% of the genome) were transcriptionally upregulated during growth on ammonia, while 120 genes (4.1% of the genome) were transcriptionally upregulated during growth on peptides. Specifically, growth on ammonia induced genes whose products are predicted to be involved in amino acid biosynthesis, molecular transport and several other factors that affect the cell envelope. Growth on peptides induced genes whose products are involved in DNA metabolism, protein fate, protein synthesis and transcription. We observed that links between carbohydrate and nitrogen metabolism were evident from glutamate biosynthesis and reverse TCA cycle upregulation when ammonia was on ammonia. Interestingly, the GS-GOGAT (GSIII-2: 22.5, GOGAT large subunit: 26.3, and small subunit: 22.4 fold) pathway was also upregulated under these conditions. The greatest transcriptional up-regulations were observed for the ammonium transporter, amt (47.0 fold) and the nitrogen regulatory protein PII (46.5 fold) during growth with ammonia. Our results provide a whole genome transcriptional overview of the responses by P. ruminicola 23 to two different nitrogen sources and are supported by proteomic and biochemical evidence, collectively leading to an overall improvement in the understanding of the genetic responses and biochemical pathways used by P. ruminicola to obtain and utilize different nitrogen sources. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The goal of this study was to gain a combined biochemical and genetic understanding of the processes ruminal bacteria undertake for the utilization of different nitrogen sources. Firstly this study analyzes the regulation of gene expression and enzyme activities involved in ammonia assimilation by one of the major cellulolytic bacterium R. albus 8, which may be applied as a model for ammonia assimilation in the Firmicutes phylum. Secondary, this study investigates the global gene expressions of the numerically dominant hemicellulytic bacterium P. ruminicola 23 in the Bacteroidetes phylum enabled by closed genome sequence. Further, we characterize ammonia assimilation enzymes and analysze enzyme activities. Collectively these analyses enable the characterization of the transcriptional regulation pathway and corresponding expression of genes whose enzymatic products respond to the different nitrogen sources. Genomic and postgenomic analyses of nitrogen metabolism of the numerically-dominant hemicallulytic P. ruminicola 23 and predominant cellulolytic R. albus 8 therefore fill the present gaps in knowledge regarding the regulation of important nitrogen metabolism enzymes, and their responses to the different nitrogen sources.

Publications

  • Purushe, J., Fouts, D.E., Morrison, M., White, B.A., Mackie, R.I. and the North American Consortium for Rumen Bacteria (Coutinho, P.M., Henrissat, B. and Nelson, K.E.) 2010. Comparative genome analysis of Prevotella ruminicola and Prevotella bryantii: Insights into their environmental niche. Microb. Ecol. 60:721-729.
  • Dodd, D., Kiyonari, S., Mackie, R.I. and Cann, I.K.O. 2010. Functional diversity of four glycoside hydrolase family 3 enzymes from the rumen bacterium Prevotella bryantii B14. J. Bacteriol. 192:2335-2345.
  • Dodd, D., Moon, Y.H., Swaminathan, K., Mackie, R.I. and Cann, I.K. 2010. Transcriptomic analyses of xylan degradation by Prevotella bryantii and insights into energy acquisition by xylanolytic bacteroidetes. J. Biol. Chem. 285:30261-30273.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Recently the genome of Prevotella ruminicola 23 has been sequenced by the North American Consortium for Genomics of Fibrolytic Rumen Bacteria and this has enabled the construction of a whole genome microarray. Through genomic and transcriptomic analyses we set out to interrogate the regulatory networks and genomic organization of the P. ruminicola genes whose enzymes and associated proteins facilitate the degradation of xylan. As the xylan substrates P. ruminicola encounters within the rumen vary dramatically with respect to their composition and degree of polymerization, we analyzed changes in the organisms transcriptome during growth on four enriched xylan sources (oat spelts xylan, soluble corn xylan and soluble and insoluble forms of wheat arabinoxylan) and then placed these results in the context of complete natural substrates (switch grass and rye grass). The genic requirements and growth dynamics of P. ruminicola demonstrated a marked preference for soluble xylans over the insoluble substrates. This was most evident in the biphasic growth pattern observed on oat spelts xylan, which comprises a mixture of soluble and insoluble xylans. Transcriptional analysis identified ten separate xylan utilization clusters, which each appear to be independently regulated as distinct operons. The expression of these operons relative to one another varies by substrate demonstrating the ability of this organism to fine tune its metabolic response to match the structure of the xylan it encounters. This unique genomic and regulatory organization provides significant insight into the metabolic strategy that has evolved in P. ruminicola and make this organism so successful within and important to the rumen environment. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Overall these findings demonstrate a unique genomic and regulatory adaptation of P. ruminicola which enable this organism to match its response to the structural heterogeneities inherent in the xylan source it is presented. There is also evidence P. ruminicola may preferentially use soluble forms of xylan over the more recalcitrant insoluble forms. Collectively these features indicate the success of P. ruminicola is confered by the organisms ability to rapidly adapt to heterogeneities in xylan sources, illicit substrate specific responses and be energetically efficient in its catabolism.

Publications

  • Dodd, D., Kocherginskaya, S.A., Spies, M.A., Beery, K.E., Abbas, C.A., Mackie, R.I. and Cann, I.K. 2009. Biochemical analysis of a beta-D-xylosidase and a bifunctional xylanase-ferulic acid esterase from a xylanolytic gene cluster in Prevotella ruminicola 23. J Bacteriol. 191:3328-3338.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Three different types of glutamine synthetase (GS) were identified from the closed genome sequence of P. ruminicola strain 23. GSI (ORFB02151), an ORF 1506 bp in length, was identified and encodes a polypeptide of 501 amino acids with a molecular weight of 56 kDa. GSIII-1 (ORFB01459) is 2193 bp in length and encodes a polypeptide of 730 amino acids with molecular weight of 83 kDa. GSIII-2 (ORFB02034) is 2214 bp in length and encodes a polypeptide of 737 amino acids with molecular weight of 83 kDa. The predicted P. ruminicola 23 GS sequences were compared with those of biochemically and hypothetical GS type I and type III proteins in the available databases. GSI of P. ruminicola 23 has 77% identity with GSI of B. fragilis YCH46. Amino acid sequence alignment of GS III-1 showed 81% similarity with GSIII of P. bryantii and GSIII-2 showed with 77% similarity with GSIII of P. bryantii. Alignment of sequences revealed strong conservation of the conserved motifs I, II, III, IV, and V in our GS sequences. After expression and purification of GSI, GSIII-1, and GSIII-2 proteins, enzymatic characterization was carried out to detect the optimal conditions for the enzymatic activity. The optimal pH and temperatures were for GSI at pH 5.6 and 35 to 45 degrees C, GSIII-1 at pH 6.0 -7.0 and 37-60 degrees C, and GS III-2 at pH 6.0-6.8 and 37-60 degrees C. The proteins were strongly stabilized in the presence of divalent Mn. Km values for glutamate (8.6 mM for GSIII-1 and 1.7 mM for GSIII-2) are higher than glutamine (1.9 mM for GSI, 1.3 mM for GSIII-1, and 0.6 mM for GSIII-2), indicating that GSI, GSIII-1, and GSIII-2 have higher affinity for glutamine than glutamate. ATP hydrolysis is an important component of the biosynthetic reaction, and GSI, GSIII-1, and GSIII-2 were shown to have high ATPase activity. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Biochemical characterization indicates that GSIII-1 and GSIII-2 of Prevotella ruminicola strain 23 are biologically functional proteins and may play important roles in ammonia assimilation and nitrogen metabolism. Gene expression levels will be evaualted under different nitrogen sources and concentrations in future experiments.

Publications

  • No publications reported this period