Use of transposon TnphoA to identify genes for cell envelope proteins of Escherichia coli required for long-chain fatty acid transport: The periplasmic protein Tsp potentiates long-chain fatty acid transport

A. Azizan, P. N. Black

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

TnphoA was used to mutagenize the chromosome in an effort to identify membrane-bound and exported components of the long-chain fatty acid transport system of Escherichia coli. This strategy identified three classes of fusions that were unable to grow or grew at reduced rates on minimal agar plates containing the long-chain fatty acid oleate (C(18:1)), (i) fadL-phoA, (ii) tolC-phoA, and (iii) tsp-phoA. fadL-phoA and tolC-phoA fusions were unable to grow on oleate as the sole carbon and energy source, while the tsp-phoA fusion had a markedly reduced growth rate. As expected, fadL-phoA fusions were unable to grow on oleate plates because the outer membrane-bound fatty acid transport protein FadL was defective. The identification of multiple fadL-phoA fusions demonstrated that this strategy of mutagenesis specifically targeted membrane-bound and exported components required for growth on long- chain fatty acids. tolC-phoA fusions were sensitive to fatty acids (particularly medium chain) and thus unable to grow, whereas the reduced growth rate of tsp-phoA fusions on oleate was apparently due to changes in the energized state of the outer membrane or inner membrane. tsp-phoA fusions transported the long-chain fatty acid oleate at only 50% of wild-type levels when cells were energized with 1 mM DL-lactate. Under conditions in which transport was measured in the absence of lactate, tsp-phoA fusion strains and wild-type strains had the same levels of oleate transport. The tsp+ clone pAZA500 was able to restore wild-type transport activity to the tsp-phoA strain under lactate-energized conditions. These results indicate that the periplasmic protein Tsp potentiates long-chain fatty acid transport.

Original languageEnglish
Pages (from-to)6653-6662
Number of pages10
JournalJournal of Bacteriology
Volume176
Issue number21
Publication statusPublished - 1994
Externally publishedYes

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Periplasmic Proteins
Escherichia coli Proteins
Oleic Acid
Fatty Acids
Membranes
Genes
Lactic Acid
Fatty Acid Transport Proteins
Growth
Mutagenesis
Agar
Carbon
Clone Cells
Chromosomes
Escherichia coli

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

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title = "Use of transposon TnphoA to identify genes for cell envelope proteins of Escherichia coli required for long-chain fatty acid transport: The periplasmic protein Tsp potentiates long-chain fatty acid transport",
abstract = "TnphoA was used to mutagenize the chromosome in an effort to identify membrane-bound and exported components of the long-chain fatty acid transport system of Escherichia coli. This strategy identified three classes of fusions that were unable to grow or grew at reduced rates on minimal agar plates containing the long-chain fatty acid oleate (C(18:1)), (i) fadL-phoA, (ii) tolC-phoA, and (iii) tsp-phoA. fadL-phoA and tolC-phoA fusions were unable to grow on oleate as the sole carbon and energy source, while the tsp-phoA fusion had a markedly reduced growth rate. As expected, fadL-phoA fusions were unable to grow on oleate plates because the outer membrane-bound fatty acid transport protein FadL was defective. The identification of multiple fadL-phoA fusions demonstrated that this strategy of mutagenesis specifically targeted membrane-bound and exported components required for growth on long- chain fatty acids. tolC-phoA fusions were sensitive to fatty acids (particularly medium chain) and thus unable to grow, whereas the reduced growth rate of tsp-phoA fusions on oleate was apparently due to changes in the energized state of the outer membrane or inner membrane. tsp-phoA fusions transported the long-chain fatty acid oleate at only 50{\%} of wild-type levels when cells were energized with 1 mM DL-lactate. Under conditions in which transport was measured in the absence of lactate, tsp-phoA fusion strains and wild-type strains had the same levels of oleate transport. The tsp+ clone pAZA500 was able to restore wild-type transport activity to the tsp-phoA strain under lactate-energized conditions. These results indicate that the periplasmic protein Tsp potentiates long-chain fatty acid transport.",
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AU - Black, P. N.

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