Molecular Ecology and Evolution of Fibrobacter and Its Cellulases
Lin, Chuzhao
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https://hdl.handle.net/2142/72558
Description
Title
Molecular Ecology and Evolution of Fibrobacter and Its Cellulases
Author(s)
Lin, Chuzhao
Issue Date
1993
Doctoral Committee Chair(s)
Stahl, David A.
Department of Study
Veterinary Medical Science
Discipline
Veterinary Medical Science
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Ecology
Biology, Microbiology
Abstract
Fibrobacter is a major genus of fiber digesting bacteria of the bovine rumen. It is the only Gram ($-$) anaerobic cellulolytic bacteria identified at present. Strains of Fibrobacter are indistinguishable from each other using traditional phenotypic criteria. Of 21 substrates examined, only cellulose and its derivatives, glucose and cellobiose, supported the growth of Fibrobacter strains. However, there was a difference in the absolute amounts of carboxymethylcellulase enzyme activity in different Fibrobacter strains. Despite their apparent phenotypic similarities, great genetic diversity was evident among strains of Fibrobacter using 165 rRNA comparative sequence analysis. Strains of Fibrobacter formed a distinct phylogenetic group. Two species, F. succinogenes and F. intestinalis have been identified at present. The genetic diversity was confirmed by additional 165 rRNA sequence analysis and by genomic DNA hybridization. Genus-, species- and subspecies-specific 16S rRNA targeted oligonucleotide probes were designed for Fibrobacter. Using these probes, greater Fibrobacter diversity than represented in the pure culture isolates was demonstrated in environmental studies. Two new Fibrobacter 165 rRNA sequences that represent two subspecies within species F. succinogenes were recovered by PCR cloning from the cecum of a pony. In searching for differentiating characteristics, fiber digesting properties of Fibrobacter were further investigated. A cellulase gene, cel-3, that codes for endoglucanase 3 was chosen for comparative studies. Hybridization studies revealed that most Fibrobacter possess this gene. Phylogenetic analysis of the gene sequences demonstrated that this gene was conserved. The evolution of this gene coincides with the evolution of Fibrobacter. Analysis of this gene sequence revealed that cel-3 had different promoter and terminator structures as compared to E. coli. The gene product of cel-3, endoglucanase 3, is most likely a lipoprotein. The cloned cel-3 gene from F. succinogenes A3c was expressed in E. coli and the gene product purified to homogeneity. The N-terminal amino acid sequence matched that predicted from the gene sequence. This purified truncated gene product was used to elicit polyclonal antibody production in ascites fluid of mice. Using this antibody, the expression of cel-3 in different Fibrobacter strains was examined. Differential expression of endoglucanase 3 was indicated for different Fibrobacter strains examined. This is the first demonstration of a clear physiological difference among Fibrobacter.
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