CHARACTERIZATION OF LIPASE-DEFICIENT MUTANTS OF ACINETOBACTER-CALCOACETICUS BD413 - IDENTIFICATION OF A PERIPLASMIC LIPASE CHAPERONE ESSENTIAL FOR THE PRODUCTION OF EXTRACELLULAR LIPASE

Acinetobacter calcoaceticus BD413 produces an extracellular lipase, wh ich is encoded by the lipA gene, Five lipase-deficient mutants have be en generated via random insertion mutagenesis, Phenotypic characteriza tion of these mutants revealed the presence of as many as four lipolyt ic enzymes in A, calcoaceticus. Biochemical evidence classified four o f the mutants as export mutants, which presumably are defective in tra nslocation of the lipase across the outer membrane, The additional mut ant, designated AAC302, displays a LipA(-) pheno-type, and yet the mut ation in this strain was localized 0.84 kbp upstream of lipA. Sequence analysis of this region revealed an open reading frame, designated li pB, that is disrupted in AAC302, The protein encoded by this open read ing frame shows extensive similarity to a chaperone-like helper protei n of several pseudomonads, required for the production of extracellula r lipase, Via complementation of AAC302 with a functional extra-chromo somal copy of lipA, it could be determined that LipB is essential for lipase production, As shown by the use of a translational LipB PhoA fu sion construct, the C-terminal part of LipB of A. calcoaceticus BD413 is located outside the cytoplasm, Sequence analysis further strongly s uggests that A. calcoaceticus LipB is N terminally anchored in the cyt oplasmic membrane, Therefore, analogous to the situation in Pseudomona s species, the lipase helper protein presumably is active in the perip lasm, In contrast to the situation in Pseudomonas species, however, li pB in A. calcoaceticus is located upstream of the structural lipase ge ne, lipB and lipA form a bicistronic operon, and the two genes are cot ranscribed from an Escherichia coli sigma(70)-type promoter, The rever sed order of genes, in comparison with the situation in Pseudomonas sp ecies, suggests that LipA and LipB are produced in equimolar amounts, Therefore, the helper protein presumably does not only have a catalyti c function, e.g., in folding of the lipase, but is also likely to act as a lipase-specific chaperone. detailed model of the export route of the lipase of A, calcoaceticus BD413 is proposed.