University of Illinois Urbana-Champaign

Genetic and Biochemical Characterization of the Escherichia Coli Cyclopropane Fatty Acid Synthase

Grogan, Dennis Wayne

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Description

Title
Genetic and Biochemical Characterization of the Escherichia Coli Cyclopropane Fatty Acid Synthase
Author(s)
Grogan, Dennis Wayne
Issue Date
1985
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 synthesis of cyclopropane fatty acids (CFA) is an interesting example of protein-lipid interaction which occurs in many bacterial species. Genetic and biochemical studies of this process have been hampered by the lack of any known physiological alteration associated with inability to form CFA and the lability of the corresponding enzyme, CFA synthase.
  • The putative structural gene for the Escherichia coli CFA synthase (cfa) was isolated as a chimeric plasmid and inserted into a bacteriophage lambda vector. This facilitated physical mapping and the construction of several useful cfa phage and plasmid derivatives.
  • Placing a selectable marker near the phenotypically silent cfa locus enabled this and several neighboring non-selectable markers to be mapped and ordered by conventional genetic techniques, placing cfa at 36.5 min on the E. coli linkage map. Selection for the loss of a cfa-linked Tn10 insertion yielded several classes of mutants harboring lesions in cfa or neighboring genes. Molecular analysis of the novel cfa mutants distinguished two null alleles apparently formed by inversions of the cfa-Tn10 chromosomal segment.
  • Although variable, levels of CFA and CFA synthase in different strains increased dramatically with gene amplification via cfa plasmid or phage. The temporal regulation of CFA synthesis was severely altered by elevated gene dosage, yet neither gross overproduction nor complete lack of CFA synthase showed any physiological impact.
  • Labeling of clone-specific proteins identified the cfa gene product as a 40,000 MW polypeptide. The kinetics of inactivation by a homologous series of N-alkyl maleimides indicated that the essential sulfhydryl group of CFA synthase lies in or near a hydrophobic region of the active site. Elevation of enzyme levels induced by various cfa plasmids in vivo increased the purity afforded by previously developed protocols. Analysis of highly purified enzyme confirmed the subunit molecular weight and indicated that the enzyme is much less abundant in crude extracts than previously thought.
Graduation Semester
1985
Type of Resource
text
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http://hdl.handle.net/2142/71170

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Graduate Dissertations and Theses at Illinois PRIMARY
Graduate Theses and Dissertations at Illinois
Dissertations and Theses - Microbiology