Sodium ion extrusion systems of Bacillus subtilis.
Item
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Title
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Sodium ion extrusion systems of Bacillus subtilis.
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Identifier
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AAI9630448
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identifier
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9630448
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Creator
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Cheng, Jianbo.
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Contributor
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Adviser: Terry Ann Krulwich
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Date
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1996
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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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Biology, Microbiology | Chemistry, Biochemistry
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Abstract
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Na{dollar}\sp+{dollar} extrusion systems of Bacillus subtilis were studied by screening Tn917-mediated transpositional libraries. Mutant B. subtilis strains were isolated on the basis of growth inhibition by Na{dollar}\sp+{dollar} at elevated pH. Two mutant strains, JC901 and JC111 were characterized in this study. Both strains exhibited a Na{dollar}\sp+{dollar}-sensitive phenotype at elevated pH, and were deficient in energy-dependent Na{dollar}\sp+{dollar} extrusion. Efflux of {dollar}\rm\sp{lcub}22{rcub}Na\sp+{dollar} from whole cells was significantly reduced in the mutants upon re-energization. The capacity of the JC901 strain for pH homeostasis at pH 8.5 was unaffected relative to the wild type strain, while that of the JC111 strain was impaired. The site of transposition of JC901 is near the 3{dollar}\sp\prime{dollar}-terminal end of a gene predicted to encode a membrane protein with six transmembrane spanning regions, designated natB. Together with an upstream gene natA, natB could function as an ABC type transport system. This system would belong to a subset of ABC type superfamily which are composed of only two types of polypeptides. A DNA fragment containing natAB cloned into an Escherichia coli vector enhanced the Na{dollar}\sp+{dollar}-resistance of an Na{dollar}\sp+/H\sp+{dollar}-deficient strain of E. coli. The insertional site of the transposon in JC111 was the promoter region of a gene which was previously identified by others as the tetA(L) locus. The gene product is a tetracycline efflux protein, which leads to pronounced tetracycline resistance when the gene is present in multicopy, but the function of the single copy of tetA(L) that is normally present has been unclear. Studies of a tetA(L) gene deletion strain indicate that, in addition to resistance of a low concentration of tetracycline, the tetA(L) gene product in B. subtilis plays a significant role in Na{dollar}\sp+{dollar}-resistance, and a major role in Na{dollar}\sp+{dollar}- and K{dollar}\sp+{dollar}-dependent pH homeostasis at high pH. The cloned tetA(L) gene enhanced the Na{dollar}\sp+{dollar}-resistance of an Na{dollar}\rm\sp+/H\sp+{dollar}-deficient strain of E. coli. To further characterize the tetA(L) gene product, a his-tagged tetA(L) gene was overexpressed in E. coli, and the gene product was purified and reconstituted in proteoliposomes. The purified preparation of TetA(L) cross-reacted with antibody raised against synthetic peptide corresponding to the N-terminus of TetA(L). The protein reconstitutes {dollar}\Delta{dollar}pH-dependent transport of tetracycline, in the presence of cobalt, and of Na{dollar}\sp+{dollar} in proteoliposomes. This strongly supports the conclusion that TetA(L) is a multifunctional antiporter and represents the first successful reconstitution of a Tet protein alone in proteoliposomes.
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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.
