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%o 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.Biological membrane transport systems are highly selective, allowing specific passage of the exogenous nutrients required for cell growth and maintenance. The cell envelope of the gram-negative bacterium Escherichia coli represents a formidable barrier for the uptake of these compounds; it is composed of two functionally distinct membranes that are separated by the periplasmic space. The outer membrane is asymmetric, containing an outer layer of lipopolysaccharide and an inner layer of phospholipid. The inner leaflet of the outer membrane is associated with a layer of peptidoglycan. The outer membrane layer contains specific proteins which facilitate nutrient transport and are classified into three groups on the basis of their functional properties: (i) nonspecific or general porins, (ii) specific channels, and (iii) high-affinity, energy-dependent transport systems (33, 34). The inner membrane is more typical of a phospholipid membrane and contains, in addition to proteins that facilitate nutrient transport, the enzymes for phospholipid biosynthesis and oxidative phosphorylation (9). The periplasmic space separates the two membrane layers and contains a number of proteins that have a catabolic function and are involved in nutrient uptake or required for detoxification. Because of the permeability properties of the bacterial cell env...
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