Biochemical and molecular genetic studies of uncoupler-resistant mutants of Bacillus subtilis.
Item
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Title
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Biochemical and molecular genetic studies of uncoupler-resistant mutants of Bacillus subtilis.
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Identifier
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AAI9207067
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identifier
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9207067
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Creator
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Dunkley, Eugene Alexander, Jr.
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Contributor
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Adviser: Terry Ann Krulwich
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Date
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1991
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Microbiology | Biology, Molecular
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Abstract
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Characterization of the protonophore-resistant mutants and revertants by others indicated that protonophore resistance correlated with a decreased ratio of unsaturated fatty acids to saturated fatty acids in the membrane phospholipids of the mutants; restoration of the level of unsaturated fatty acids using exogenous palmitoleic acid abolished the resistance. The study first established that exogenous C{dollar}\sb{lcub}16:1{rcub}{dollar} abolished protonophore resistance in duramycin-resistant strains of protonophore-resistant mutants of B. subtilis and B. megaterium. These strains have markedly deficient levels of phosphatidylethanolamine and cardiolipin, which are the major sites of incorporation of unsaturated fatty acids in B. subtilis. Assays of the fatty acyl-CoA desaturase of wild-type B. subtilis indicated that the activity resembled the fatty acyl-CoA desaturase of eukaryotic organisms in that it required O{dollar}\sb2{dollar} and an electron donor, was cyanide-sensitive and carbon monoxide-insensitive. This O{dollar}\sb2{dollar}-NADH-dependent formation of unsaturated fatty acids was deficient in the protonophore-resistant mutants. Temperature-sensitive protonophore-resistant revertants exhibited fatty acid desaturation that was active at temperatures which were non-permissive for protonophore-resistant growth, and inactive at temperatures which were permissive for protonophore-resistant growth.;Five libraries of insertional mutations introduced into B. subtilis by Tn917-mediated transposition were screened for CCCP resistance to determine whether the site(s) of insertion unrelated to the desaturase could lead to protonophore resistance, and to identify the genes encoding or positively regulating components of the fatty acid desaturase. Twenty-five protonophore-resistant strains were isolated. Nineteen of these isolates were phenotypically reversible by the addition of palmitoleic acid to the growth medium, while the remainder were not reversible. Six of the "C{dollar}\sb{lcub}16:1{rcub}{dollar}-reversible" and three of the "non-reversible" strains were assayed for fatty acyl-CoA desaturase activity. All of the strains assayed were deficient in desaturase activity in comparison to the wild-type. However, two of the "C{dollar}\sb{lcub}16:1{rcub}{dollar}-reversible" strains had noticeable desaturase activity, indicating a degree of heterogeneity among that category of mutants.;Analysis of the flanking region of transposon insertion was conducted on one of the "C{dollar}\sb{lcub}16:1{rcub}{dollar}-reversible" strains which was deficient in desaturase activity. No sequence similarity to the fatty acid desaturase or its components has yet been found, but the insertion site has been localized to a position 4.2 kb from the spo0F gene of B. subtilis. (Abstract shortened with permission of author.).
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
