This study identified and characterized lipase-deficient (lip) mutants of Pseudomonas aeruginosa and mapped and cloned the lipase structural gene. Using N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis on strain PA0 2302, researchers isolated two non-pleiotropic lip mutants (29-1 and 6-1) that lacked extracellular lipase activity but retained other exoproducts and cell-bound esterases. Genetic mapping through conjugation and bacteriophage transduction located the lip locus at approximately 57 minutes on the P. aeruginosa chromosome, closely linked to pyrF. The lipase gene was successfully cloned as a 3.1 kilobase fragment on shuttle vector pKT248 and fully complemented the lipase deficiency in both mutants. Outer membrane protein and lipopolysaccharide profiles were identical between mutants and parent strain, confirming the mutations affected the lipase structural gene rather than general secretion pathways. The cloned gene showed minimal expression in E. coli, consistent with poor recognition of Pseudomonas promoters in heterologous hosts.

Key findings

• Two non-pleiotropic lipase-deficient mutants (29-1 and 6-1) were isolated from P. aeruginosa, with mutations specifically affecting lipase production while preserving other exoproducts and cell-bound esterases
• The lipase structural gene (lip) was mapped to 57 minutes on the P. aeruginosa chromosome, closely linked to pyrF with 60% cotransduction frequency
• A 3.1 kilobase SalI DNA fragment containing the lipase gene was cloned and successfully complemented the lipase deficiency in both mutant strains
• Identical outer membrane protein and lipopolysaccharide profiles between wild-type and lip mutants confirmed the mutations affected the lipase structural gene rather than excretion or regulatory pathways