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

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 Escherich ia coli, but it accumulated as inactive inclusion bodies. Amino acid sequen ce analysis of the lipase revealed a potential C-terminal targeting sequenc e recognized by the ATP-binding cassette (ABC) transporter, The genetic loc i 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, till, In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. tliDEF showed high similarity to ABC transport ers of Pseudomonas aeruginosa alkaline protease, Erwinia chrysanthemi prote ase, 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 differen t plasmid, The ABC transporter of P. fluorescens was optimally functional a t 20 and 25 degrees C, while the ABC transporter, aprD, aprE, and aprF, of P, aeruginosa secreted the lipase irrespective of temperature between 20 an d 37 degrees C. These results demonstrated that the lipase is secreted by t he P. fluorescens SIK W1 ABC transporter, which is organized as an operon w ith tliA, and that its secretory function is temperature dependent.