North American Consortium forGenomics of Fibrolytic Ruminal Bacteria

Ohio State University (Lead Institution)Mark Morrison Mark Morrison

The Institute for Genomics Research (TIGR)Karen Nelson, Sean Daugherty, Bill Nelson, Karen Nelson, Sean Daugherty, Bill Nelson, Emmanuel Emmanuel MongodinMongodin

Cornell UniversityDavid Wilson, Jim RussellDavid Wilson, Jim Russell

University of GuelphCecilCecil ForsbergForsberg

University of IllinoisIsaac Isaac CannCann, Rod , Rod MackieMackie, Bryan White, Bryan White

North American Consortium forGenomics 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.

An overview of ruminal microbiology:Enzymatic Degradation of Polymers in the Diet

Glycerol andFatty Acids

Sugars Amino acidsUrea in saliva

FERMENTATION Ammonia Residues

HydrogenationVFA + H2 + CO2

MICROBIALBIOMASS

CH4Absorbed by

the animal

Abomasum

Digestion andAbsorption

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

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.

So, why continue and why “-omics” approaches?RuminalRuminal function remains subfunction remains sub--optimal, from optimal, from microbiological, economic and environmental contexts.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).

From discovery to knowledge:

Bioinformatics

GenomicsTranscriptomic

sProteomicsGenome sequencing,

SSH and RDA analysis, 2D-PAGE, chromatography,

and MS methodsWhole genome and (or)

gene family arrays

Fundamental understanding of microbial biology

Increasing ruminal fiber degradation

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.

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.

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-Bacteroidesgroup 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 andsupramolecular structure of the complex have not been fully elucidated.

Ruminococcus albus:Cellulose degradation by this bacterium is greatly enhanced by phenylacetic and phenylpropionic acids (PAA/PPA), both of which elicit changes in cell wallultrastructure, cellulase complex assembly, and adhesion.

Cells with PAA/PPA Cells without PAA/PPA(Stack and Hungate 1984)

From discovery to knowledge:

Bioinformatics

GenomicsTranscriptomic

sProteomicsGenome sequencing,

SSH and RDA analysis, 2D-PAGE, chromatography,

and MS methodsWhole genome and (or)

gene family arrays

Fundamental understanding of microbial biology

Increasing ruminal fiber degradation

Identification of Cel9B and Cel48A:

Proteins from wild-type and mutant strains areseparated by 2D-PAGE

Excision of the spots or the band of interest

Peptide mass fingerprint(trypsin + MALDI-TOF)

N terminal sequence(Edman degradation)

BLAST against genome (TIGR)

Theoretical tryptic digestion

ProteinidentificationContigs and ORFs

Adm subproteome

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 ensuringmaximal production of these gene products?

Cel9B and Cel48A

WT subproteome

SP CBM6 Ig-like domain GH9 Fn3

SP GH48 Fn3

E. Devillard et al. (2003) in press

From discovery to knowledge:

Bioinformatics

GenomicsTranscriptomic

sProteomicsGenome sequencing,

SSH and RDA analysis, 2D-PAGE, chromatography,

and MS methodsWhole genome and (or)

gene family arrays

Fundamental understanding of microbial biology

Increasing ruminal fiber degradation

The FibRumBa database: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.Microarrays

Include tabular and graphical display of microarray results.

Provide details of experimental protocols (MIAME).

Subtractive hybribizationContain annotated and

searchable sequence data.

Also contain comparative genome hybridization data.

Additional ModulesReference maps of bacterial

proteomes.

16S rRNA gene sequence banks, arising from biodiversity studies.

Ramifications of High Throughput DNA Sequencing Technologies:

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 contentShould efforts to establish gene transfer methods be reinitiated?

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 stabilityTo what extent is the dockerin-cohesin interaction employed in nature?

We now have the opportunity to see all of the iceberg……..

….to what extent will that below the surface be explored and captured?