Genetic Analysis of Clostridium Acetobutylicum Using Two-Dimensional PFGE to Construct a Macrorestriction Map of ATCC 824 and Restriction Enzyme Profiles for Strain Analysis
Broussard, Suzanne Rene
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https://hdl.handle.net/2142/72226
Description
Title
Genetic Analysis of Clostridium Acetobutylicum Using Two-Dimensional PFGE to Construct a Macrorestriction Map of ATCC 824 and Restriction Enzyme Profiles for Strain Analysis
Author(s)
Broussard, Suzanne Rene
Issue Date
1994
Doctoral Committee Chair(s)
Blaschek, Hans,
Department of Study
Food Science
Discipline
Food Science
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Abstract
C. acetobutylicum and C. butyricum strains were analyzed using rare-cutting restriction enzymes by one-dimensional and two-dimensional PFGE. C. acetobutylicum strains were placed into three groups based on their restriction enzyme profiles following digestion with SalI, SmaI, and MluI. C. acetobutylicum strains ATCC 824, NCIB 8052, and NRRL B-591 were grouped together. A second group was composed of C. acetobutylicum strains ATCC 3179 and NRRL 4259. C. acetobutylicum P262 and C. butyricum NRRL B-593 were placed in individual groups. The four Clostridia groups displayed very low restriction enzyme profile similarities. The C. acetobutylicum ATCC 824 chromosome was further characterized with a macrorestriction map consisting of 92 restriction sites and seventeen genes. The macrorestriction map was constructed using combinations of SalI, SmaI, and MluI to digest the chromosomal DNA followed by two-dimensional PFGE.
Transformation of plasmid DNA into C. acetobutylicum ATCC 824 by electroporation was optimized by growing a culture to abs (600 nm) of 1.0, concentrated 400 fold and suspended in 10% PEG using a 0.1 cm cuvette. Successful electroporation requires the use of a high DNA concentration and a receptive host strain.
The phenomena of degeneration of C. acetobutylicum does not appear to be caused by chromosomal rearrangements or deletions. However, histone-like proteins appear to be more prominent in a degenerate strain of C. acetobutylicum ATCC 824 and may play a role in degeneration.
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