Escherichia coli transcription factor that both activates fatty acid synthesis and represses fatty acid degradation
Henry, Michael Francis
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Permalink
https://hdl.handle.net/2142/19536
Description
Title
Escherichia coli transcription factor that both activates fatty acid synthesis and represses fatty acid degradation
Author(s)
Henry, Michael Francis
Issue Date
1992
Doctoral Committee Chair(s)
Cronan, John E.
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, Molecular
Biology, Microbiology
Language
eng
Abstract
I have examined fadR regulation of fabA expression using a variety of biochemical and genetic techniques. First, I showed that introduction of fadR null alleles into strains carrying any of several different fabA temperature-sensitive alleles lowered the permissive growth temperature of the strain. The increased temperature sensitivity was shown to result from a decrease in the amount of unsaturated fatty acids (UFAs) present in the membranes of these strains. Dehydrase (FabA) activity was then assayed in isogenic $fad\sp+$ and fadR null strains and found to be decreased 10 to 12-fold in the fadR null strains. These results, in combination with results obtained by fabA-reporter-gene fusions (lacZ and cat) and direct Northern analysis, indicated transcriptional activation. Northern analysis also showed that the fabA gene was transcribed from two promoters located just upstream from the coding sequence; one of which was activated in a $fadR\sp+$ strain.
FadR DNA-binding to upstream elements of fabA was shown by mobility shift assays using both crude cell extracts, and radiochemically pure FadR protein synthesized in vitro. Additional DNA-binding assays defined the DNA-binding region to a 17-bp consensus sequence. Similar sequences were identified upstream of the fadL and the fadAB genes; both transcriptionally repressed by FadR. Finally, a combination of in vivo and in vitro assays were used to demonstrate that FadR DNA-binding was inhibited in the presence of long-chain acyl-CoAs.
This study was facilitated by developing a technique which uses the cointegrate intermediate of transposon Tn1000 as a means to lower the copy number of ColE1-type plasmids. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid was inhibited and replication proceeded only from the single-copy F factor replication origin. (Abstract shortened with permission of author.)
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