| Analysis of the genome of the cellulolytic microorganism Ruminococcus flavefaciens FD-1 was conducted via two parallel techniques---low coverage draft sequencing and suppressive subtractive hybridization (SSH)---in order to understand its ability to degrade more crystalline forms of cellulose relative to other Ruminococci. The genome of R. flavefaciens FD-1 was sequenced to 2x-coverage relative to the size of the R. albus 8 genome (3.86 Mb). Approximately 71.6% of the ORFs encoding several families of amino acid biosynthesis were identified and corroborate the sequence coverage (3.05 Mb of unique sequence recovered). Preliminary analysis has revealed homologs of scaffoldin proteins (ScaA and ScaB) that correspond to similar motifs described in R. flavefaciens 17. Additionally, further searches utilizing other cellulase components uncovered two ORFs with repeated fibronectin binding-like domains that may represent novel molecules involved in adhesion to fiber. These comparisons were corroborated and further enhanced with searches performed by TIGR's Annotation Engine, which revealed at least 189 full and partial ORFs that may encode proteins involved in fiber degradation. Neighbor-joining trees were constructed representing the evolutionary relationships between glycosyl hydrolase (GH) domains that were detected in these ORFs. Evidence from these analyses support "module swapping" of GH family 5 domains, and possible lateral gene transfer of at least one GH family 9 domain. The genome of a closely related strain, R. flavefaciens JM1, was used to "subtract" out regions common to the strains. Cloned products were sequenced (n = 384) and categorized as follows: no relation to deposited sequences in GenBank (42% of the subtracted library); 7% are loosely associated with fiber-degradation; and 6% with insertion elements, transposons, and phage-like ORFs. Although the strains differ in their respective 16S rRNA sequences by less than 2%, we estimate that 9.5% of the genome of R. flavefaciens FD-1 is missing from the JM1 strain. One sequence putatively identified as a bifunctional xylanase was utilized as a probe in Southern blot experiments to investigate the frequency of specific glycosyl hydrolase domains within the R. flavefaciens FD-1 genome. Multiple fragments were detected that indicate these domains are rearranged within the genome to provide novel permutations of glycosyl hydrolases.*.;*This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: Internet Browser; Microsoft Office. |
