This study presents the molecular genetic characterization of the extracellular lipase gene (lipA) from Pseudomonas aeruginosa PAO1, a gram-negative bacterium. The researchers successfully cloned and sequenced the lipA gene, which consists of 936 bp encoding a 311-amino acid proenzyme with a 26-amino acid signal sequence. The mature lipase protein has a predicted molecular weight of approximately 30,134 Da and an isoelectric point of 5.6. Sequence analysis revealed a conserved lipase active center motif (IGHSHGG) and greater than 60% homology to lipases from other Pseudomonas species. The study also identified a neighboring open reading frame (ORF2, termed lipH) located 220 bp downstream of lipA, encoding a 283-amino acid cytoplasmic protein with approximately 40% homology to the LimA protein of P. cepacia. Southern blot analysis demonstrated that lipA sequences hybridized strongly with all tested P. aeruginosa strains but showed minimal homology with other Pseudomonas species, except P. alcaligenes. Complementation studies revealed that lipH is essential for active extracellular lipase expression in certain lipase-defective mutants, indicating its role in lipase maturation or secretion.

Key findings

• The lipA gene encodes a 311-amino acid proenzyme with a conserved lipase active site (IGHSHGG) and approximately 30 kDa mature protein size
• A neighboring gene lipH is required for expression of active extracellular lipase in some lipase-defective mutants, indicating involvement in lipase maturation or function
• The lipase gene is highly conserved among P. aeruginosa strains but shows limited homology to lipase genes in other Pseudomonas species
• The gene product shows greater than 60% amino acid homology to other Pseudomonas lipases, suggesting evolutionary conservation of this enzymatic function