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The fatty acid transport protein FadL from Escherichia coli is predicted to be rich in beta-structure and span the outer membrane multiple times to form a long-chain fatty acid specific channel. Proteolysis of FadL within whole cells, total membranes, and isolated outer membranes identified two trypsin-sensitive sites, both predicted to be in externally exposed loops of FadL. Amino acid sequence analysis of the proteolytic fragments determined that the first followed R93 and yielded a peptide beginning with 94S-L-K-A-D-N-I-A-P-T-A104 while the second followed R384 and yielded a peptide beginning with 385S-I-S-I-P-D-Q-D-R-F-W395. Proteolysis using trypsin eliminated the bacteriophage T2 binding activity associated with FadL, suggesting the T2 binding domain within FadL requires elements within one of these extracellular loops. A peptide corresponding to the amino-terminal region of FadL (FadL28-160) was purified and shown to inactivate bacteriophage T2 in a concentration-dependent manner, supporting the hypothesis that the amino-proximal extracellular loop of the protein confers T2 binding activity. Using an artificial neural network (NN) topology prediction method in combination with Gibbs motif sampling, a predicted topology of FadL within the outer membrane was developed. According to this model, FadL spans the outer membrane 20 times as antiparallel beta-strands. The 20 antiparallel beta-strands are presumed to form a beta-barrel specific for long-chain fatty acids. On the basis of our previous studies evaluating the function of FadL using site-specific mutagenesis of the fadL gene, proteolysis of FadL within outer membranes, and studies using the FadL28-160 peptide, the predicted extracellular regions between beta-strands 1 and 2 and beta-strands 3 and 4 are expected to contribute to a domain of the protein required for long-chain fatty acid and bacteriophage T2 binding. The first trypsin-sensitive site (R93) lies between predicted beta-strands 3 and 4 while the second (R384) is between beta-strands 17 and 18. The trypsin-resistant region of FadL is predicted to contain 13 antiparallel beta-strands and contribute to the long-chain fatty acid specific channel.

The gram negative bacterium Escherichia coli has evolved a highly specific system for the transport of exogenous long-chain fatty acids (C12-C18) across the cell envelope that requires the outer membrane protein FadL and the inner membrane associated fatty acyl CoA synthetase. The transport of oleate (C18:1) across the cell envelop responds to metabolic energy. In order to define the source of metabolic energy which drives this process, oleate transport was measured in wild-type and ATP synthase-defective (Deltaatp) strains which were (i) subjected to osmotic shock and (ii) starved and energized with glucose or d-lactate in the presence of different metabolic inhibitors. Osmotic shock did not eliminate transport but rather reduced the rate to 33-55% of wild-type levels. These results suggested a periplasmic protein may participate in this process or that osmotic shock disrupts the energized state of the cell which in turn reduces the rate of oleate transport. Transport systems which are osmotically sensitive also require ATP. The process of long-chain fatty acid transport requires ATP generated either by substrate-level or oxidative phosphorylation. Following starvation, the basal rate of transport for wild-type cells was 340.4 pmol/min/mg protein compared to 172.0 pmol/min/mg protein for the Deltaatp cells. When cells are energized with glucose, the rates of transport were increased and comparable (1242.6 and 1293.8 pmol/min/mg protein, respectively). This was in contrast to cells energized with d-lactate in which only the wild-type cells were responsive. The role of ATP is likely due to the ATP requirement of fatty acyl CoA synthetase for catalytic activity. The process of oleate transport is also influenced by the energized state of the inner membrane. In the presence of carbonyl cyanide-m-chlorophenylhydrazone oleate transport is depressed to 30-50% of wild-type levels in wild-type and Deltaatp strains under starvation conditions. These results are mirrored in cells energized with glucose and d-lactate, indicating that an energized membrane is required for optimal levels of oleate transport. These data support the hypothesis that the fatty acid transport system of E. coli responds to both intracellular pools of ATP and an energized membrane for maximal proficiency., (Copyright 1999 Academic Press.)

The binding of exogenous fatty acids to the outer-membrane protein FadL of Escherichia coli is specific for long-chain fatty acids (C14-C18). Oleoyl alcohol [(Z)-9-octadecen-1-ol] and methyl oleate were unable to displace FadL-specific binding of [3H]oleate (C18:1), suggesting that the carboxylate of the long-chain fatty acid was required for binding. Therefore the binding of exogenous fatty acids to FadL is governed, in part, by the carboxy group of the long-chain fatty acid. Treatment of whole cells with 1 mM diethyl pyrocarbonate (DEPC) depressed binding by 43-73% over the range of oleate concentrations used (10-500 nM). On the basis of these results and the notion that histidine residues often play a role involving proton transfer and charge-pairing, the five histidine residues within FadL (His110, His226, His327, His345 and His418) were replaced by alanine using site-directed mutagenesis. Altered FadL proteins were correctly localized in the outer membrane at wild-type levels and retained the heat-modifiable property characteristic of the wild-type protein. Initial screening of these fadL mutants revealed that the replacement of His110 by Ala resulted in a decreased growth rate on minimal oleate/agar plates. The rates of long-chain fatty acid transport for delta fadL strains harbouring each mutation on a plasmid, with the exception of fadLH110A, were the same, or nearly the same, as those for the wild-type. fadLH110A was also defective in binding, arguing that the functional effect of this mutation was at the level of long-chain-fatty-acid binding.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The outer membrane protein FadL (product of the fadL gene) of Escherichia coli is required for the specific binding and transport of exogenous long-chain fatty acids prior to metabolic utilization. The carboxyl end of FadL has been proposed to play a crucial role by facilitating the transport of long-chain fatty acids. In an attempt to define specific amino acid residues within carboxyl region of FadL essential for activity, a series of deletion and point mutations within the 3' end of the fadL+ gene have been constructed and characterized. These fadL mutants were classified into three categories based on functional properties attributable to the altered FadL proteins: (i) those that had essentially wild-type levels of long-chain fatty acid binding and transport, (ii) those that had wild-type levels of long-chain fatty acid binding but were defective in transport, and (iii) those that were defective for both long-chain fatty acid binding and transport. These findings demonstrate that amino acid residues Phe448, Pro428, Val410, and Ser397 are required for optimal levels of long-chain fatty acid transport and that amino acid residues Pro428 and Val410 are essential for long-chain fatty acid binding.

The fadL gene of Escherichia coli encodes an outer membrane protein (FadL) that plays a central role in the uptake of exogenous long-chain fatty acids. The nucleotide sequence of the fadL gene revealed a single open reading frame of 1,344 bp encoding a protein with 448 amino acid residues and a molecular weight of 48,831. The transcriptional start, analyzed by primer extension, was shown to be 95 bp upstream from the translational start. Apparent -10 and -35 regions were found at -12 and -37 bp upstream from the transcriptional start. Three regions with hyphenated dyad symmetry (two between the transcriptional start and the translational start and one upstream from the -10 and -35 regions) were identified that may play a role in the expression of fadL. The protein product of the fadL gene contained a signal sequence and signal peptidase I cleavage site similar to that defined for other E. coli outer membrane proteins. The N-terminal sequence of mature FadL protein was determined by automated amino acid sequencing of protein purified from the outer membrane of a strain harboring fadL under the control of a T7 RNA polymerase-responsive promoter. This amino acid sequence, Ala-Gly-Phe-Gln-Leu-Asn-Glu-Phe-Ser-Ser, verified the signal peptidase I cleavage site on pre-FadL and confirmed the N-terminal amino acid sequence of FadL predicted from the DNA sequence. Mature FadL contained 421 amino acid residues, giving a molecular weight of 45,969. The amino acid composition of FadL deduced from the DNA sequence suggested that this protein contained an abundance of hydrophobic amino acid residues and lacked cysteinyl residues. The hydrophobic amino acids within FadL were predicted to contribute to at least five regions of the protein with an overall hydrophobic character. The amino acid sequence of FadL was used to search GenBank for other proteins with amino acid sequence homology. These data demonstrated that FadL and the heat-modifiable outer membrane protein P1 of Haemophilus influenzae type b were 60.5% conserved and 42.0% identical over 438 amino acid residues.

The product of the fadL gene (FadL) is a central component of the long-chain fatty acid transport system of Escherichia coli. When fatty acid activation is blocked by a mutation in the structural gene for acyl CoA synthetase (fadD) transport is inhibited allowing a FadL-specific fatty acid binding activity to be measured. This binding activity was 4- to 6-fold greater in the fadL+ fadD strain LS6928 when compared to the delta fadLfadD strain LS6929. With long-chain fatty acids, this binding activity was saturable and it was estimated that there were approx. 35,000 FadL-specific oleic acid binding sites per cell in the fadL+ strain LS6928. The FadL-specific fatty acid binding affinity was highest for oleic acid (18:1) and palmitic acid (16:0) giving apparent KD values of 2.3.10(-7) M and 8.8.10(-7) M, respectively. FadL-specific binding affinity of myristic acid (14:0) was nearly an order of magnitude less and no FadL-specific binding of decanoic acid (10:0) could be measured. Two lines of evidence suggest that FadL-fatty acid binding occurs by a hydrophobic interaction: (1) There was a preference for the long-chain substrates oleic acid and palmitic acid; and (2) oleic acid binding activity was not significantly changed over the pH range 5.0 to 8.0. The FadL-specific binding of oleic acid in the fadL+ strain LS6928 could be blocked by preincubation with antisera raised against purified FadL providing a clear correlation between the activity and identity of FadL. The binding activity associated with FadL was measured in vesicles of the outer membrane following passage over the hydrophobic resin Lipidex 1000. The KD of oleic acid binding attributable to FadL in outer membranes vesicles (6.0.10(-7) M) was in close agreement with that determined in whole cells. Overall, these studies demonstrated that FadL binds long-chain fatty acids with a relatively high affinity prior to their transport across the outer membrane.

The fadL+ gene of Escherichia coli encodes an outer membrane protein (FadL) essential for the uptake of long-chain fatty acids (C12 to C18). The present study shows that in addition to being required for uptake of and growth on the long-chain fatty acid oleate (C18:1), FadL acts as a receptor of bacteriophage T2. Bacteriophage T2-resistant (T2r) strains lacked FadL and were unable to take up and grow on long-chain fatty acids. Upon transformation with the fadL+ clone pN103, T2r strains became sensitive to bacteriophage T2 (T2s), became able to take up long-chain fatty acids at wild-type levels, and contained FadL in the outer membrane.