Functional characterization of ExFadLO, an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa.
Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance.
Unitat de Microbiologia, Facultat de Farmàcia, Universitat de Barcelona, Joan XXIII s/n, Barcelona E-08028, Spain.
Eriel
E
Martínez
Mónica
M
Estupiñán
F I Javier
FI
Pastor
Montserrat
M
Busquets
Pilar
P
Díaz
Angeles
A
Manresa
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D050612:Fatty Acid Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005816:Genetic Complementation Test; D006880:Hydroxy Acids / Q000378:metabolism*; D009154:Mutation; D009829:Oleic Acids / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D010802:Phylogeny; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*
C107324:10-hydroxy-8-octadecenoic acid; C432089:7,10,12-trihydroxy-8-octadecenoic acid; D001425:Bacterial Outer Membrane Proteins; D050612:Fatty Acid Transport Proteins; D006880:Hydroxy Acids; D009829:Oleic Acids; D010100:Oxygen