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 (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...

Transmembrane Movement of Exogenous Long-Chain Fatty Acids: Proteins, Enzymes, and Vectorial Esterification

“…Studies of the purified FACS FadD have borne this out: this enzyme can activate fatty acids with chain lengths varying from C 6:0 to C 20:4 , with the highest specificity for C 14:0 to C 18:0 fatty acid substrates (82,83). The periplasmic protease Tsp is also required for optimal levels of transport, although its precise role in this process remains undefined (10). There is also evidence supporting the existence of a specific fatty acid/H ϩ cotransporter in the inner membrane; the structural gene encoding this protein has not been identified (81,84).…”

Bacterial Long Chain Fatty Acid Transport: Gateway to a Fatty Acid-responsive Signaling System

“…Strains defective in the tsp gene are able to transport exogenous LCFAs but do not respond to an added exogenous energy source (D-lactate or glucose). One particular note of interest is that Tsp shares sequence homologies to the retinoid binding proteins, suggesting it may be involved in fatty acid binding although this has not been defined (33). Osmotic shock not only disrupts the protein constituents of the periplasmic space but likewise the chemical makeup, including pH.…”

Determination of the native form of FadD, the Escherichia coli fatty acyl-CoA synthetase, and characterization of limited proteolysis by outer membrane protease OmpT

“…Tsp protease had been implicated in E. coli LCFA uptake, but its precise role is unclear [15]. To determine if the Tsp protease was Purification and characterization of native and cleaved forms of FadD…”

“…FadL is thought to facilitate the permeation of medium-chain fatty acids and LCFAs across the outer membrane [13], which are then sequestered inside the cell by vectoral thio-esterification catalysed by FadD [14]. The periplasmic protease Tsp has also been implicated in LCFA uptake, but its precise role remains unclear [15].…”