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Journal of Bacteriology, November 1999, p. 6977-6986, Vol. 181, No. 22
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Novel Lipolytic Enzyme Located in the Outer
Membrane of Pseudomonas aeruginosa
Susanne
Wilhelm,1
Jan
Tommassen,2 and
Karl-Erich
Jaeger1,*
Lehrstuhl Biologie der Mikroorganismen, Ruhr
Universität, D-44780 Bochum, Germany,1 and
Department of Molecular Microbiology and Institute of
Biomembranes, Utrecht University, NL-3584 CH Utrecht, The
Netherlands2
Received 13 July 1999/Accepted 8 September 1999
A lipase-negative deletion mutant of Pseudomonas
aeruginosa PAO1 still showed 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 frame of 1,941 bp. The product of
estA is a 69.5-kDa protein, which is probably processed by
removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for
35S-labeled EstA by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and autoradiography. 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 high 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 esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a
carboxy-terminal
-barrel domain, allowing the secretion of the
amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and
Escherichia coli and subsequent cell fractionation revealed
that the enzyme was associated with the cellular membranes. Trypsin
treatment of whole cells released a significant amount of esterase,
indicating that the enzyme was located in the outer membrane with the
catalytic domain exposed to the surface. To our knowledge, this
esterase is unique in that it exemplifies in P. aeruginosa
(i) the first enzyme identified in the outer membrane and (ii) the
first example of a type IV secretion mechanism.
*
Corresponding author. Mailing address: Ruhr
Universität Bochum, Lehrstuhl Biologie der Mikroorganismen,
Universitätsstrasse 150, D-44780 Bochum, Germany. Phone: 49 234 322 3101. Fax: 49 234 321 4425. E-mail:
karl-erich.jaeger{at}ruhr-uni-bochum.de.
Journal of Bacteriology, November 1999, p. 6977-6986, Vol. 181, No. 22
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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