Progress 12/15/01 to 12/31/05
Outputs
The Ruminococcus flavefaciens FD-1 sequencing project is at 4x coverage (4.4 Mb genome). The 1,143 assembled contigs have an average size of 3,686 bp and range from 205 to 31,132 bp. Cellulosome organization in strain FD-1 appears similar to that of strain 17. Analysis of the assembly revealed a cluster of genes encoding for the putative scaffoldin proteins, ScaA, ScaB and ScaC. Like in strain 17, the FD1 scaB is situated downstream of scaA and the scaC equivalent is located upstream of ScaA. Compared to R. flavefaciens 17, the architecture of the FD1 proteins is very similar, except for the number of cohesins in ScaA and ScaB. In silico analysis for identification of putative dockerin modules, novel glycoside hydrolases and putative scaffoldins was conducted. Dockerin motifs were used to search for dockerin containing ORFs, revealing an unprecedented number (171) of putative dockerin modules. This is more than double the number so far detected in C. thermocellum-the
bacterial standard for comparison of cellulosome systems. Phylogenetic analysis indicated five major clusters of dockerin groups; EndB dockerins in 52 ORFs, CesA (or type-3) dockerins in 25 ORFs, XynE dockerins in 54 ORFs, a ScaA dockerin in one ORF, and ScaB dockerins in 3 ORFs. These dockerin-containing ORFs have associated protein families including xylanases, cellulases, proteinases, serpins, surface-anchoring proteins and unknown activities. Catalytic domains and CBMs implicated in plant cell wall breakdown are found associated with EndB, XynE, type-3, and ScaB groups. Proteinases are found in the XynE and type-3 groups. A ScaB X-module-dockerin (XDoc) pair has been discovered in an orf that is situated between scaB and scaE in the sca gene cluster of both strains 17 and FD-1. The expressed ORF protein binds to cellulose and its C-terminal XDoc binds selectively to the ScaE cohesin. This surface-associated ORF protein and ScaB could thus account for the elusive cellulose-binding
function in this bacterium. The genome was used to develop a microarray (~6,000 sequences). Expression profiling with mRNA from bacteria grown with either cellobiose or as carbon source was performed. Of the up-regulated contigs, those containing dockerin sequences were further analyzed. The EndB type dockerins seem to be the most common in the up-regulated contigs. The multi-domain gene families of the dockerin containing contigs with the highest up-regulation were GH_11/CBM_4_9/GH_10/GH_11/polysaccharide deacetylase, CE_3/CBM 22/CesA, Ricin B-like lectin CBM/CBM 6/lipase GDSL, alpha-glucosidase, GH 30/CBM 22/CE_3/Ricin-like CBM/PL_11, and ScaC/ScaA/ScaB. These gene families range from 23-fold to 3-fold up-regulation respectively. A gene similar to a bifunctional xylanase found in R. flavefaciens 17 was the most highly up-regulated gene (10 to 23-fold). The putative scaffoldin proteins, ScaA, ScaB, and ScaC were up-regulated approximately 4-fold. The ScaA dockerin was the most highly
up-regulated. Multi-domain fiber degrading proteins appear to be highly up-regulated and it seems that the presence of a dockerin sequence is highly correlated with up-regulation.
Impacts
The Ruminococci are the predominant cellulolytic Gram-positive cocci inhabiting the rumen community. They are responsible for releasing simpler substrates from cellulolytic materials that other Eubacterial and Archaeal species can utilize. Applying genomic technologies to understanding this rate limiting step offers a new method to underscore not only differences between organisms in the rumen environment but also the sequence imprint that occurs from living in such a unique environment. The fact that R. flavefaciens strain FD-1 has a cellulosome complex which is orthologous to that of strain 17 suggests that the R. flavefaciens cellulosome system is the most complex, diverse and heterogeneous known in cellulolytic bacteria. Microarray analysis has enabled us to identify a complement of genes responsible for cellulose attachment and degradation and how expression of these genes changes cellulosome composition. This database of expressed sequences will allow us to
determine genes that are differentially expressed in response to plant cell wall components. The expressed genes will be the basis for studying the biochemistry of cohesin-dockerin interactions and cellulosome orthologues and allow us to unravel the complex molecular events in plant cell wall hydrolysis by R. flavefaciens. In the future, a combined genomic and proteomic approach is dictated to understand the regulation and assembly of this remarkable cadre of cellulosome components. Multi-domain fiber degrading proteins with a dockerin sequence are prime candidates for future systematic studies.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The current Ruminococcus flavefaciens FD-1 genome sequencing project at the University of Illinois at Urbana-Champaign is currently at 3.36x-coverage. We are manually curating the Phred/Phrap-mediated assembly in order to reduce the number of contigs (1,292) made up of 4,047,136 bases. There are several sequences with relevance to cellulose degradation, which are indicative of a Ruminococcus-like cellulosome. Homologues of the scaffoldin proteins ScaA (only 2 cohesins) and ScaB (9 cohesins and with 2 unusually short linkers, just a PT) and ScaC (85% including dockerin and cohesin missing the N-terminal part) have been found which have similar motifs and architecture to those described from R. flavefaciens 17. Additionally, further searches utilizing other cellulase components uncovered two putative ORFs with repeated X domains. Analysis of these domains suggests that they represent a new class of X domains from glyosyl hydrolases. These approaches will facilitate the
identification of relevant ORFs for further study using microarrays and proteomic-assisted technologies.
Impacts
The Ruminococci are the predominant cellulolytic Gram-positive cocci inhabiting the rumen community. They are responsible for releasing simpler substrates from cellulolytic materials that other Eubacterial and Archaeal species can utilize. Applying genomic technologies to understanding this rate limiting step offers a new method to underscore not only differences between organisms in the rumen environment but also the sequence imprint that occurs from living in such a unique environment.
Publications
- Antonopoulos, D.A., Bayer, E.A. and White, B.A. 2004. Identification of cellulase components from a random-phase genome sequence of Ruminococcus flavefaciens FD-1. In: Biotechnology of Lignocellulose Degradation and Biomass Utilization, (eds. K. Shimada, S. Hoshino, K. Ohmiya, K. Sakka, Y. Kobayashi and S. Karita) Uni Publishers Co., LTD., Tokyo, Japan. p. 309-316.
- Antonopoulos, D.A., Nelson, K.E., Morrison, M. and White, B.A. 2004. Strain specific genomic regions of Ruminococcus flavefaciens FD-1 as revealed by combinatorial random-phase genome sequencing and suppressive subtractive hybridization. Environ. Microbiol. 6:335-346.
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Progress 01/01/03 to 12/31/03
Outputs
The current Ruminococcus flavefaciens FD-1 genome sequencing project at the University of Illinois at Urbana-Champaign is currently at 2x-coverage. We are manually curating the Phred/Phrap-mediated assembly in order to reduce the number of contigs (1,500) made up of 14,437 reads. Automated BlastX searches of the non-redundant amino acid GenBank database using the assembled contigs as queries yields 151 sequences with similarity to fiber-degrading encoding sequences and/or sequences that may be important in substrate adhesion. There are also several sequences with relevance to cellulose degradation, which are indicative of a Ruminococcus like cellulosome. Homologues of the scaffoldin proteins ScaA and ScaB have been found which have similar motifs and architecture to those described from R. flavefaciens 17. Additionally, further searches utilizing other cellulase components uncovered two putative ORFs with repeated X domains. Analysis of these domains suggests that
they represent a new class of X domains from glyosyl hydrolases. These approaches will facilitate the identification of relevant ORFs for further study using microarrays and proteomic-assisted technologies.
Impacts
The Ruminococci are the predominant cellulolytic Gram-positive cocci inhabiting the rumen community. They are responsible for releasing simpler substrates from cellulolytic materials that other Eubacterial and Archaeal species can utilize. Applying genomic technologies to understanding this rate limiting step offers a new method to underscore not only differences between organisms in the rumen environment but also the sequence imprint that occurs from living in such a unique environment.
Publications
- Antonopoulos, D.A., Nelson, K.E., Morrisom, M. and White, B.A. 2003. Strain specific genomic regions of Ruminococcus flavefaciens FD-1 as revealed by combinatorial random-phase genome sequencing and suppressive subtractive hybridization. Environ. Microbiol. (In Press).
- Antonopoulos, D.A., Bayer, E.A. and White, B.A. 2004. Identification of cellulase components from a random-phase genome sequence of Ruminococcus flavefaciens FD-1. In: Biotechnology of Lignocellulose Degradation and Biomass Utilization, (eds. K. Shimada, S. Hoshino, K. Ohmiya, K. Sakka, Y. Kobayashi and S. Karita) Uni Publishers Co., LTD., Tokyo, Japan (In Press).
- Antonopoulos, D.A., Russell, W.M. and White, B.A. 2003. Phylogenetic reconstruction of Gram positive organisms based on comparative sequence analysis of molecular chaperones from the ruminal microorganism Ruminococcus flavefaciens FD-1 FEMS Microbiol. Lett. 227:1-7.
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Progress 01/01/02 to 12/31/02
Outputs
We are currently sequencing the genome of the cellulolytic bacterium Ruminococcus flavefaciens FD-1. A shotgun library was constructed from nebulized genomic DNA that was size-fractionated (1.5-3 kb), polished and cloned into pCR"4Blunt-TOPO". The cumulative number of bases sequenced so far with a Phred quality of 20 or higher is 4,117,421 from 10,272 reads. These have been assembled into 4,302 contigs which yields just under 1x coverage since the estimated size of this genome is 4.4 Mb based on PFGE of R. flavefaciens 17. Library quality was assessed periodically by checking the redundancy of sequences in the early phase of sequencing and efforts are now directed towards the next round of sequence coverage.
Impacts
Genomic information will accelerate the data gathering steps needed to address the important questions related to the regulation of cellulose hydrolysis. These approaches will revolutionize our understanding of the biochemistry, physiology and genetics of R. flavefaciens FD-1. These experiments will also allow us to unravel the complex molecular events in plant cell wall hydrolysis and apply this technology in mixed rumen cultures, artificial rumen systems and ultimately the animal itself.
Publications
- No publications reported this period
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