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Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis

Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus... Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis ▿ § Anna-Barbara Hachmann 1 , † , Elif Sevim 1 , ‡ , Ahmed Gaballa 1 , David L. Popham 2 , Haike Antelmann 3 and John D. Helmann 1 , * 1 Department of Microbiology, Cornell University, Ithaca, New York 14853-8101 2 Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061 3 Institute for Microbiology, Ernst Moritz Arndt University of Greifswald, D-17487 Greifswald, Germany ABSTRACT Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca 2+ -dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap R 1, from the model bacterium Bacillus subtilis 168. Dap R 1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. Dap R 1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of Dap R 1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator gene mreB , the stringent response gene relA , and the phosphatidylglycerol synthase gene pgsA . Genetic reconstruction studies indicated that the pgsA ( A64V ) allele is primarily responsible for DAP resistance. Allelic replacement with wild-type pgsA restored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca 2+ -DAP complex. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antimicrobial Agents and Chemotherapy American Society For Microbiology

Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis

Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis

Antimicrobial Agents and Chemotherapy , Volume 55 (9): 4326 – Sep 1, 2011

Abstract

Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis ▿ § Anna-Barbara Hachmann 1 , † , Elif Sevim 1 , ‡ , Ahmed Gaballa 1 , David L. Popham 2 , Haike Antelmann 3 and John D. Helmann 1 , * 1 Department of Microbiology, Cornell University, Ithaca, New York 14853-8101 2 Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061 3 Institute for Microbiology, Ernst Moritz Arndt University of Greifswald, D-17487 Greifswald, Germany ABSTRACT Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca 2+ -dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap R 1, from the model bacterium Bacillus subtilis 168. Dap R 1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. Dap R 1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of Dap R 1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator gene mreB , the stringent response gene relA , and the phosphatidylglycerol synthase gene pgsA . Genetic reconstruction studies indicated that the pgsA ( A64V ) allele is primarily responsible for DAP resistance. Allelic replacement with wild-type pgsA restored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca 2+ -DAP complex.

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References (61)

Publisher
American Society For Microbiology
Copyright
Copyright © 2011 by the American society for Microbiology.
ISSN
0066-4804
eISSN
1098-6596
DOI
10.1128/AAC.01819-10
pmid
21709092
Publisher site
See Article on Publisher Site

Abstract

Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis ▿ § Anna-Barbara Hachmann 1 , † , Elif Sevim 1 , ‡ , Ahmed Gaballa 1 , David L. Popham 2 , Haike Antelmann 3 and John D. Helmann 1 , * 1 Department of Microbiology, Cornell University, Ithaca, New York 14853-8101 2 Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061 3 Institute for Microbiology, Ernst Moritz Arndt University of Greifswald, D-17487 Greifswald, Germany ABSTRACT Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca 2+ -dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap R 1, from the model bacterium Bacillus subtilis 168. Dap R 1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. Dap R 1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of Dap R 1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator gene mreB , the stringent response gene relA , and the phosphatidylglycerol synthase gene pgsA . Genetic reconstruction studies indicated that the pgsA ( A64V ) allele is primarily responsible for DAP resistance. Allelic replacement with wild-type pgsA restored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca 2+ -DAP complex.

Journal

Antimicrobial Agents and ChemotherapyAmerican Society For Microbiology

Published: Sep 1, 2011

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