Studies on the molecular organisation and permeability of the outer membrane of Escherichia coli K-12
Studies on the molecular organisation and permeability of the outer membrane of Escherichia coli K-12
The outer membrane of Gram-negative bacteria such as Escherichia coli functions as an important and highly selective permeability barrier. This thesis describes an investigation of the molecular basis for the permeability barrier effect using defined permeability mutants of the TraT protein, a major outer membrane lipoprotein specified by F-like plasmids. The precise nature of the defect in mutant TraT proteins causing increased membrane permeability was studied by site-directed mutagenesis of codon 126 (Leu) of the protein specified by plasmid R6-5. Substitution of Pro, His, Arg, Glu, Asp or Gln residues increased the penetration of hydrophobic antibiotics whereas substitution of Val, Ile, Ser or Thr had no significant effect. Thus, while the introduction of a charged residue at this position invariably caused an increase in outer membrane permeability, certain uncharged amino acid residues had similar effects. All of the mutant proteins retained the ability to assemble into TraT oligomers and to function in surface exclusion suggesting that the mutated region is not involved in the strong subunit: subunit interactions necessary for oligomer formation or for surface exclusion. 258 suppressor derivatives of the TraT pemeability mutants specified by pDOC40 and pDOC44 were isolated by selection for restoration of vancomycin resistance. The suppressor strains were grouped into four classes according to the patterns of resistance to hydrophobic antibiotics and to alterations in their cell envelope components. Restoration of resistance to hydrophobic antibiotics in addition to vancomycin was associated with the appearance of new cell envelope proteins of Mrs 20,000 and 28,000 and with more complex cell envelope protein alterations affecting the OmpA and porin proteins. By contrast, acquisition of resistance to vancomycin alone correlated with alteration of LPS to the Rc chemotype. Representative suppressor loci, provisionally designated sip for suppression of increased permeability, were cloned and mapped. The results obtained indicate that the sipB locus is located in the 11 min region whereas sipC and sipD both map to 82 min. The sipA locus reduced the copy number of plasmids carrying mutant traT genes thereby decreasing the amounts of the corresponding TraT proteins in the outer membrane. Evidence is presented that the sipA locus defines a new gene involved in plasmid copy number control.
University of Southampton
1990
Qi, Shu-Yun
(1990)
Studies on the molecular organisation and permeability of the outer membrane of Escherichia coli K-12.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The outer membrane of Gram-negative bacteria such as Escherichia coli functions as an important and highly selective permeability barrier. This thesis describes an investigation of the molecular basis for the permeability barrier effect using defined permeability mutants of the TraT protein, a major outer membrane lipoprotein specified by F-like plasmids. The precise nature of the defect in mutant TraT proteins causing increased membrane permeability was studied by site-directed mutagenesis of codon 126 (Leu) of the protein specified by plasmid R6-5. Substitution of Pro, His, Arg, Glu, Asp or Gln residues increased the penetration of hydrophobic antibiotics whereas substitution of Val, Ile, Ser or Thr had no significant effect. Thus, while the introduction of a charged residue at this position invariably caused an increase in outer membrane permeability, certain uncharged amino acid residues had similar effects. All of the mutant proteins retained the ability to assemble into TraT oligomers and to function in surface exclusion suggesting that the mutated region is not involved in the strong subunit: subunit interactions necessary for oligomer formation or for surface exclusion. 258 suppressor derivatives of the TraT pemeability mutants specified by pDOC40 and pDOC44 were isolated by selection for restoration of vancomycin resistance. The suppressor strains were grouped into four classes according to the patterns of resistance to hydrophobic antibiotics and to alterations in their cell envelope components. Restoration of resistance to hydrophobic antibiotics in addition to vancomycin was associated with the appearance of new cell envelope proteins of Mrs 20,000 and 28,000 and with more complex cell envelope protein alterations affecting the OmpA and porin proteins. By contrast, acquisition of resistance to vancomycin alone correlated with alteration of LPS to the Rc chemotype. Representative suppressor loci, provisionally designated sip for suppression of increased permeability, were cloned and mapped. The results obtained indicate that the sipB locus is located in the 11 min region whereas sipC and sipD both map to 82 min. The sipA locus reduced the copy number of plasmids carrying mutant traT genes thereby decreasing the amounts of the corresponding TraT proteins in the outer membrane. Evidence is presented that the sipA locus defines a new gene involved in plasmid copy number control.
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Published date: 1990
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Local EPrints ID: 462055
URI: http://eprints.soton.ac.uk/id/eprint/462055
PURE UUID: ca772128-f2a5-40ff-ab2f-008eaf084ff9
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Date deposited: 04 Jul 2022 19:00
Last modified: 04 Jul 2022 19:00
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Author:
Shu-Yun Qi
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