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Escherichia coli TolA tolerates multiple amino-acid substitutions as revealed by screening randomized variants for membrane integrity and phage receptor function

Karlsson, Fredrik LU ; Malmborg Hager, Ann-Christin LU and Borrebaeck, Carl LU (2006) In FEMS Microbiology Letters 259(1). p.81-88
Abstract
Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at... (More)
Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at positions 415-420. These libraries were introduced into the tolA strain K17DE3tolA/F+ and several variants, containing complementing, multiple amino-acid substitutions, were identified. However, most randomized variants did not complement the tolA strain K17DE3tolA/F+. The TolA variants that restored sensitivity to phage infection displayed a considerable sequence variation, while the few variants that restored tolerance to detergent were from the same library. A comparison of the generated residue variation and natural variation, suggests that structural dependence overrides contact residue dependence. Thus, library screening can be efficient in identifying TolA variants with different functionally associated characteristics. (Less)
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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
functional screening, randomized TolA, Escherichia coli
in
FEMS Microbiology Letters
volume
259
issue
1
pages
81 - 88
publisher
Oxford University Press
external identifiers
  • wos:000237395200013
  • pmid:16684106
  • scopus:33646775316
  • pmid:16684106
ISSN
1574-6968
DOI
10.1111/j.1574-6968.2006.00256.x
language
English
LU publication?
yes
id
46900603-ebe8-47aa-bcc8-12f036f6b84f (old id 410100)
date added to LUP
2016-04-01 15:37:10
date last changed
2021-02-17 07:40:07
@article{46900603-ebe8-47aa-bcc8-12f036f6b84f,
  abstract     = {Escherichia coli TolA is a cytoplasmic membrane protein required for outer membrane integrity and the translocation of F-specific filamentous (Ff) bacteriophage DNA. Both phage infection and membrane integrity depend on several TolA interactions, e.g. those of the TolA C-terminal domain (TolAIII). Membrane integrity involves interaction with two host proteins and phage translocation requires direct interaction with the N-terminal domain (N1) of Ff phage protein g3p. Although cocrystallization of TolAIII and N1g3p has identified several contact points, it is still uncertain which residues are selectively involved in the different TolA functions. Thus, four different limited substitution libraries of TolA were created, targeting contacts at positions 415-420. These libraries were introduced into the tolA strain K17DE3tolA/F+ and several variants, containing complementing, multiple amino-acid substitutions, were identified. However, most randomized variants did not complement the tolA strain K17DE3tolA/F+. The TolA variants that restored sensitivity to phage infection displayed a considerable sequence variation, while the few variants that restored tolerance to detergent were from the same library. A comparison of the generated residue variation and natural variation, suggests that structural dependence overrides contact residue dependence. Thus, library screening can be efficient in identifying TolA variants with different functionally associated characteristics.},
  author       = {Karlsson, Fredrik and Malmborg Hager, Ann-Christin and Borrebaeck, Carl},
  issn         = {1574-6968},
  language     = {eng},
  number       = {1},
  pages        = {81--88},
  publisher    = {Oxford University Press},
  series       = {FEMS Microbiology Letters},
  title        = {Escherichia coli TolA tolerates multiple amino-acid substitutions as revealed by screening randomized variants for membrane integrity and phage receptor function},
  url          = {http://dx.doi.org/10.1111/j.1574-6968.2006.00256.x},
  doi          = {10.1111/j.1574-6968.2006.00256.x},
  volume       = {259},
  year         = {2006},
}