A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa

A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still show ed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene , estA, was identified, cloned, and sequenced, revealing an open reading fr ame of 1,941 bp, The product of estA is a 69.5-kDa protein, which is probab ly processed by removal of an N-terminal signal peptide to yield a 67-kDa m ature protein. A molecular mass of 66 kDa was determined for S-35-labeled E stA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autora diography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed h igh similarity to an open reading frame of unknown function located in the trpE-trpG region of P. putida and to a gene encoding an outer membrane este rase of Salmonella typhimurium. Amino acid sequence alignments led us to pr edict that this esterase is an autotransporter protein which possesses a ca rboxy-terminal beta-barrel domain, allowing the secretion of the amino-term inal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and Escherichia coli and subsequent cell fractionation rev ealed that the enzyme was associated with the cellular membranes. Trypsin t reatment of whole cells released a significant amount of esterase, indicati ng that the enzyme was located in the outer membrane with the catalytic dom ain exposed to the surface, To our knowledge, this esterase is unique in th at it exemplifies in P. aeruginosa (i) the first enzyme identified in the o uter membrane and (ii) the first example of a type TV secretion mechanism.