Molecular Analysis of Roseburia Cecicola, a Motile Anaerobe, as a Foundation for Study of Bacterial Motility and Chemotaxis in the Murine Gastrointestinal Ecosystem
Martin, Joel Hastings
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https://hdl.handle.net/2142/71192
Description
Title
Molecular Analysis of Roseburia Cecicola, a Motile Anaerobe, as a Foundation for Study of Bacterial Motility and Chemotaxis in the Murine Gastrointestinal Ecosystem
Author(s)
Martin, Joel Hastings
Issue Date
1988
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
The obligately anaerobic, gram-negative, motile bacterium Roseburia cecicola, is being used as a model organism in the study. Motility may be essential for this organism to colonize its specific habitat in the mouse cecum. I purified the main protein (flagellin) of the flagellar apparatus of the organism. The purified protein was then used in raising polyclonal and monoclonal antibodies. In addition, the gene encoding the protein was cloned and sequenced.
The antibodies were used in immunofluorescence microscopy to localize the habitat of R. cecicola in the mouse cecum. The organism appeared to colonize only on or in the mucous layer overlying the cecal epithelium. To clone the gene, I obtained an amino terminal amino acid sequence of the flagellin protein, made the corresponding oligonucleotides, and used them to probe chromosomal DNA. While purifying that DNA at high molecular weight, I discovered that it degraded in R. cecicola cells and cell extracts exposed to air. Apparently, either a specific nuclease is activated or free radicals are formed when oxygen is present. The cloned gene is to be used in mutational analysis. Therefore, I attempted to develop a genetic transfer system by conjugation and transformation. Transformation was achieved at a low frequency and is the most promising system for genetic transfer. In addition, I inferred the amino acid sequence from the nucleotide sequence and compared it with such sequences from other bacteria. R. cecicola flagellin is most similar to that of B. subtilis (approximately 45%), especially in the amino terminal and carboxy terminal regions, following a general pattern reported for flagellin.
The 16S ribosomal RNA of R. cecicola has also been sequenced. A specific sequence (signature) was found that is normally found only in gram-positive bacteria. This finding and the discovery that its flagellin is most similar in structure to those of a gram-positive suggests that the organism has features of a gram positive, although it is gram-negative in ultrastructure. This flagellin protein is the first to be analyzed from a strict anaerobe. It has already revealed new taxonomic information on R. cecicola and will undoubtedly be useful in future motility studies. (Abstract shortened with permission of author.)
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