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.
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 (C18:1), (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.
Azizan
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Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163.
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D000469:Alkaline Phosphatase / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D050612:Fatty Acid Transport Proteins; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; C057119:FadR protein, Bacteria; D050612:Fatty Acid Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009829:Oleic Acids; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C065691:fadL protein, E coli; C073126:tolC protein, E coli; D019301:Oleic Acid; D000469:Alkaline Phosphatase