Identification of the tliDEF ABC Transporter Specific for Lipase in Pseudomonas fluorescens SIK W1

Abstract

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°C, while the ABC transporter, aprD , aprE , and aprF , of P. aeruginosa secreted the lipase irrespective of temperature between 20 and 37°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.