In vivo and in vitro studies on interactions between the components of the hemolysin (HlyA) secretion machinery of Escherichia coli |
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Authors: | S Schl?r A Schmidt E Maier R Benz W Goebel and I Gentschev |
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Institution: | (1) Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften, Am Hubland, D-97074 Würzburg, Germany Fax: +49-931-888-4402 e-mail: gentsch@biozentrum.uni-wuerzburg.de, DE;(2) Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut für Biowissenschaften, Am Hubland, D-97074 Würzburg, Germany, DE |
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Abstract: | The glycopeptide antibiotic vancomycin blocks cell wall synthesis in Escherichia coli only when it can reach its target site in the periplasm. In vivo, sensitivity to vancomycin is enhanced in the presence of
the hemolysin (hly) determinant of E. coli or its translocator portion hlyBD. Two different mutations in hlyD alter the cell's susceptibility to vancomycin: mutations in the tolC-homologous region of hlyD increase vancomycin resistance, whereas mutations at the 3′-terminus of hlyD lead to hypersensitivity to vancomycin and to the accumulation of large periplasmic and cytoplasmic pools of this antibiotic
in E. coli. These effects are only observed in the presence of functional HlyB and TolC, the two other components of the hemolysin secretion
machinery. A defect in TolC causes hyperresistance to vancomycin, even when present together with a mutant HlyD protein which
in the presence of TolC renders E. coli hypersensitive to vancomycin. Lipid bilayer experiments in vitro revealed specific interactions between TolC and vancomycin
or HlyD protein. Second-site suppressor mutations in hlyD and hlyB were obtained, which abolish the hypersensitive phenotype caused by the 3′-terminal mutations in hlyD. Our results are compatible with the idea that (a) TolC, together with the TolC-homologous part of HlyD, forms a pore in
the outer membrane through which hemolysin is released and vancomycin taken up; and (b) the C-terminal sequence of HlyD interacts
with periplasmic loop(s) of HlyB to form a closed channel spanning the periplasm.
Received: 7 April 1997 / Accepted: 28 May 1997 |
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Keywords: | Hemolysin secretion HlyD mutants HlyD/HlyB suppressor mutants TolC Vancomycin |
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