Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1

Abstract

Pseudomonas fluorescens, a gram-negative psychrotrophic bacterium, secretes a thermostable lipase into the extracellular medium. In our previous study, the lipase of P. fluorescens SIK W1 was cloned and expressed in Escherichia coli, but it accumulated as inactive inclusion bodies. Amino acid sequence analysis of the lipase revealed a potential C-terminal targeting sequence recognized by the ATP-binding cassette (ABC) transporter. The genetic loci around the lipase gene were searched, and a secretory gene was identified. Nucleotide sequencing of an 8.5-kb DNA fragment revealed three components of the ABC transporter, tliD, tliE, and tliF, upstream of the lipase gene, tliA. In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. tliDEF showed high similarity to ABC transporters of Pseudomonas aeruginosa alkaline protease, Erwinia chrysanthemi protease, Serratia marcescens lipase, and Pseudomonas fluorescens CY091 protease. tliDEF and the lipase structural gene in a single operon were sufficient for E. coli cells to secrete the lipase. In addition, E. coli harboring the lipase gene secreted the lipase by complementation of tliDEF in a different plasmid. The ABC transporter of P. fluorescens was optimally functional at 20 and 25 degrees C, while the ABC transporter, aprD, aprE, and aprF, of P. aeruginosa secreted the lipase irrespective of temperature between 20 and 37 degrees C. These results demonstrated that the lipase is secreted by the P. fluorescens SIK W1 ABC transporter, which is organized as an operon with tliA, and that its secretory function is temperature dependent.

FIG. 1

Restriction endonuclease map of the various plasmids used. The relative positions of genes in P. fluorescens SIK W1 and restriction enzyme sites are depicted at the top. Inserts of subcloned plasmids are represented by the heavy line with pertinent enzyme sites at both ends. The names of subcloned plasmids and vectors used are given above the heavy line. Restriction enzymes: B, BamHI; Bs, BsrBI; Cl, ClaI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; Sau, Sau3AI; X, XhoI.

FIG. 2

Multiple-sequence alignment of the C termini of lipases from P. fluorescens SIK W1 (SIK), P. fluorescens B52 (B52), and P. fluorescens LS107d2 (LS107). Identical residues in three different lipases are shaded in black, and identical residues in two different lipases are shaded in gray. Sequence alignment was performed with CLUSTALW (54), and gaps were introduced to maximize the homology. Position numbers for the sequences are given to the right. Solid boxes represent glycine-rich boxes; the dot-dash box represents a putative amphipathic α-helix; the dotted box represents the extreme C-terminal motif.

FIG. 3

Immunodetection of the lipase in cell extracts and culture supernatant of E. coli carrying various dual plasmids. E. coli XL1-Blue harboring dual plasmids was grown at different temperatures in LB medium without induction. The cells were harvested when they reached an optical density at 600 nm of 4 for the 20, 25, and 30°C cultures and an optical density at 600 nm of 2.5 for the 37°C culture. The cell extract and culture supernatant were prepared as described in Materials and Methods. Cell extract of 20 μl (culture equivalent) and culture supernatant of 240 μl (culture equivalent) were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed by immunodetection. Lanes: 1, E. coli (pHOPE + pSTV29); 2, E. coli (pHOPE + pAGS8); 3, E. coli (pHOPE + pABC-BsSTV); 4, E. coli (pHOPE + pABC-ACYC).