North American Consortium for Genomics of Fibrolytic Ruminal Bacteria

Transcription

1 North American Consortium for Genomics of Fibrolytic Ruminal Bacteria Ohio State University (Lead Institution) Mark Morrison The Institute for Genomics Research (TIGR) Karen Nelson, Sean Daugherty, Bill Nelson, Emmanuel Mongodin Cornell University David Wilson, Jim Russell University of Guelph Cecil Forsberg University of Illinois Isaac Cann,, Rod Mackie,, Bryan White

2 North American Consortium for Genomics of Fibrolytic Ruminal Bacteria Funds awarded by USDA-IFAFS, the USDA/NSF Microbe Sequencing Program, and USDA-NRICGP. Sequence the Fibrobacter succinogenes (strain S85) and Ruminococcus albus (strain 8) genomes to closure (TIGR). Sequence the Prevotella ruminicola (strain 23) genome to 8x coverage (TIGR). Low coverage sequencing of Ruminococcus flavefaciens (strain FD-1, Keck Center, Univ. of Illinois). These bacteria were selected because of their presumed roles in governing the rate and extent of plant fiber degradation. Improving ruminal fiber digestibility remains a universal research priority for both all ruminant enterprises, especially developing countries.

3 An overview of ruminal microbiology: Enzymatic Degradation of Polymers in the Diet Glycerol and Fatty Acids Sugars Amino acids Urea in saliva FERMENTATION Ammonia Residues Hydrogenation VFA + H2 + CO2 MICROBIAL BIOMASS Abomasum Absorbed by the animal CH4 Digestion and Absorption

4 Analysis of any habitat requires elaboration of... the kinds and numbers of microorganisms present (ecology). the activities of those microorganisms (enzymology). the extent to which these activities are expressed (regulation). R. E. Hungate

5 A (devil s advocate) overview of ruminal microbiology: Ecology: Relatively good, but our knowledge base is largely derived from cultivation-dependent studies. Enzymology: Relatively poor, because no structural data are available, and a focus on enzymes with high activity against artificial substrates. Regulation: Superficial at best, largely based on a comparative analysis with the eubacterial paradigm derived by the study of gram-negative enterics.

6 So, why continue and why -omics approaches? Ruminal function remains sub-optimal, from microbiological, economic and environmental contexts. Ecology: Whole genome sequencing facilitates cultivation-independent approaches (genomics) to understanding microbial biology. Enzymology: Mutational analysis and proteomics allow broader, more quantitative approaches. Regulation: Microarray and quantitative RT-PCR techniques expands the examination of microbial physiology (transcriptomics).

7 From discovery to knowledge: Transcriptomic s Whole genome and (or) gene family arrays Genomic s Genome sequencing, SSH and RDA analysis, Proteomics 2D-PAGE, chromatography, and MS methods Bioinformatics Fundamental understanding of microbial biology Increasing ruminal fiber degradation

8 Genome Sequence Data: Ability to construct a genetic blueprint that defines a microbe s lifestyle at a specific point in evolutionary time. Inventory of degradative and metabolic schemes, and their regulation. Provides evidence for highly conserved hypothetical genes in functionally similar microbes, new insights into physiology. Vaccine development for pathogens based on genome sequences providing novel, highly specific targets.

9 Sequencing Strategies: TIGR s random shotgun strategy and vectors will be used, which have been successful for all of their microbial genome projects to date. These methods have effectively accommodated variations in genome size (560 kb to 6.2 Mb), base compositions ranging from 19% to 67% G+C, and the presence of various repeat elements, IS elements, and multiple chromosomes and plasmids.

10 Fibrobacter succinogenes: A Gram-negative, obligate anaerobe, originally classified as a member of the Bacteroides genus. Now separated into their own distinct phylum, located between the Cytophaga-Flexibacter-Bacteroides group and the Spirochaetes. Possesses a cellulase system that is very active against crystalline cellulose, but is thought to be different from the cellulosomal- and free-form cellulase systems. However, the genetics and supramolecular structure of the complex have not been fully elucidated.

11 Ruminococcus albus: Cellulose degradation by this bacterium is greatly enhanced by phenylacetic and phenylpropionic acids (PAA/PPA), both of which elicit changes in cell wall ultrastructure, cellulase complex assembly, and adhesion. Cells with PAA/PPA Cells without PAA/PPA (Stack and Hungate 1984)

12 From discovery to knowledge: Transcriptomic s Whole genome and (or) gene family arrays Genomic s Genome sequencing, SSH and RDA analysis, Proteomics 2D-PAGE, chromatography, and MS methods Bioinformatics Fundamental understanding of microbial biology Increasing ruminal fiber degradation

13 Identification of Cel9B and Cel48A: Proteins from wild-type and mutant strains are separated by 2D-PAGE Excision of the spots or the band of interest N terminal sequence (Edman degradation) Peptide mass fingerprint (trypsin + MALDI-TOF) BLAST against genome (TIGR) Contigs and ORFs Protein identification Theoretical tryptic digestion

14 WT subproteome Adm subproteome Cel9B and Cel48A SP CBM6 Ig-like domain GH9 Fn3 SP GH48 Fn3 E. Devillard et al. (2003) in press Mutants have lesser amounts of Cel9B and Cel48A: Identification of two new genes essential for forage utilization in livestock. Can forage utilization be improved, by ensuring maximal production of these gene products?

15 From discovery to knowledge: Transcriptomic s Whole genome and (or) gene family arrays Genomic s Genome sequencing, SSH and RDA analysis, Proteomics 2D-PAGE, chromatography, and MS methods Bioinformatics Fundamental understanding of microbial biology Increasing ruminal fiber degradation

16 The FibRumBa database: Subtractive hybribization Contain annotated and searchable sequence data. Also contain comparative genome hybridization data. Genomes Including draft and complete genome sequences produced by other groups. A subset of tools available via CMR will be adapted for use with this database. Additional Modules Reference maps of bacterial proteomes. 16S rrna gene sequence banks, arising from biodiversity studies. Microarrays Include tabular and graphical display of microarray results. Provide details of experimental protocols (MIAME).

17 Ramifications of High Throughput DNA Sequencing Technologies:

18 Ramifications Creation of the -omics terminology: Genomics (functional, comparative, structural) Proteomics, transcriptomics, metabolomics Business as usual, or not? Bioinformatics: Specialist microbes require consistent and informative annotation, best provided by coordinated efforts of specialist scientists. How/what is the best way to provide this? Conserved hypotheticals and FUN genes: May be as much as 50% genomic content Should efforts to establish gene transfer methods be reinitiated?

19 Ramifications What microbes are missing? How many more should be considered? Fresh isolates vs. lab-trained isolates? Why stop at one genome: metagenomics technologies? New lines of investigation in cellulases and cellulosomes? Protein secretion, glycosylation, and other post-translational modifications? Regulation of gene expression and mrna stability To what extent is the dockerin-cohesin interaction employed in nature?

20 We now have the opportunity to see all of the iceberg...to what extent will that below the surface be explored and captured?