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

Abstract

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 (35)S-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 beta-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.

FIG. 1

Lipolytic activity of P. aeruginosa strains, wild-type PAO1, lipase-negative mutant PABS1, and PABS1pLAFR3-21.P containing a cosmid with a 22.1-kb insert of chromosomal DNA from P. aeruginosa PAO1. (A) Halo formation of bacterial colonies on esterase indicator plates after incubation for 3 days at 30°C. (B) Lipolytic activities of culture supernatants in liquid assays, assayed with p-nitrophenylpalmitate (C₁₆) for lipase activity and p-nitrophenylcaproate (C₆) for esterase activity. Relative enzyme activities were determined as the ratio of OD₄₁₀ (enzyme activity) to OD₅₈₀ (cell density) per milliliter of culture supernatant.

FIG. 2

Autoradiography of l-[³⁵S]methionine–l-[³⁵S]cysteine-labeled proteins from cell lysates of E. coli BL21(DE3)(pLysS) containing pSKX− separated by SDS-PAGE. Samples were labeled prior to induction of T7 RNA polymerase expression (t = 0; lane A), 30 min after induction but prior to inhibition of E. coli RNA polymerase with rifampin (t = 30; lane B), and 30 min (t = 60; lane C) and 60 min (t = 90; lane D) after addition of rifampin. Lane M contains prestained molecular mass markers (Bio-Rad).

FIG. 3

Nucleotide sequence and derived amino acid sequence of the esterase gene estA. The putative Shine-Dalgarno sequence is underlined, the consensus sequence for the RpoN (ς⁵⁴)-dependent promoter is marked by dashed lines, and the putative signal sequence is indicated by an arrow.

FIG. 4

Sequence comparison between P. aeruginosa esterase (EstA) and members of a novel family of lipolytic enzymes (55). Identical amino acids are shaded in grey; numbers in parentheses refer to the number of amino acid residues between the conserved blocks. The putative catalytic triad residues (∗) are printed in bold, and the G-D-S-L-S consensus motif is underlined.

FIG. 5

Multiple alignment of C-terminal domains. EstA, outer membrane proteins, and secreted proteins belonging to the autotransporter family (31) were compared. Boxes indicate putative amphiphathic β-barrels, and the two fully conserved amino acids from the ATF are marked with an asterisk.

FIG. 6

Distribution of esterase (A) and glucose-6-phosphate dehydrogenase (B) activity in cellular compartments of P. aeruginosa PABS1 and E. coli JM109 containing estA on pBBX+. The percentages of total enzyme activities present in culture supernatants (SN), cytoplasmic-periplasmic fractions (CP/PP), and membranes (M) are given.

FIG. 7

One possible model of the C-terminal domain of P. aeruginosa esterase (EstA) located in the outer membrane. The C-terminal domain (∼G 374 to F 622) is predicted to consist of 11 amphipathic β-strands with 10 to 12 amino acid residues per strand, sufficient to traverse the hydrophobic core of the membrane. The two amino acids fully conserved in proteins belonging to the autotransporter family (31) are marked with asterisks; amino acids printed in bold indicate the hydrophobic side of the β-strands; and amino acids shaded in grey represent two girdles of aromatic residues, which seem to be present in outer membrane proteins of known structure.

FIG. 8

Effect of trypsin treatment on esterase activities of whole cells of P. aeruginosa and E. coli containing estA on pBBX+.