title: Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase.
abstract: Acinetobacter calcoaceticus BD413 produces an extracellular lipase, which is encoded by the lipA gene. Five lipase-deficient mutants have been generated via random insertion mutagenesis. Phenotypic characterization of these mutants revealed the presence of as many as four lipolytic enzymes in A. calcoaceticus. Biochemical evidence classified four of the mutants as export mutants, which presumably are defective in translocation of the lipase across the outer membrane. The additional mutant, designated AAC302, displays a LipA- phenotype, and yet the mutation in this strain was localized 0.84 kbp upstream of lipA. Sequence analysis of this region revealed an open reading frame, designated lipB, that is disrupted in AAC302. The protein encoded by this open reading frame shows extensive similarity to a chaperone-like helper protein of several pseudomonads, required for the production of extracellular lipase. Via complementation of AAC302 with a functional extrachromosomal copy of lipA, it could be determined that LipB is essential for lipase production. As shown by the use of a translational LipB-PhoA fusion construct, the C-terminal part of LipB of A. calcoaceticus BD413 is located outside the cytoplasm. Sequence analysis further strongly suggests that A. calcoaceticus LipB is N terminally anchored in the cytoplasmic membrane. Therefore, analogous to the situation in Pseudomonas species, however, lipB in A. calcoaceticus is located upstream of the structural lipase gene. lipB and lipA form a bicistronic operon, and the two genes are cotranscribed from an Escherichia coli sigma 70-type promoter. The reversed order of genes, in comparison with the situation in Pseudomonas species, suggests that LipA and LipB are produced in equimolar amounts. Therefore, the helper protein presumably does not only have a catalytic function, e.g., in folding of the lipase, but is also likely to act as a lipase-specific chaperone. A detailed model of the export route of the lipase of A. calcoaceticus BD413 is proposed.
authors.affiliation: Department of Microbiology, E. C. Slater Institute, BioCentrum Amsterdam, The Netherlands.
authors.forename: R G
authors.initials: RG
authors.lastname: Kok
authors.forename: J J
authors.initials: JJ
authors.lastname: van Thor
authors.forename: I M
authors.initials: IM
authors.lastname: Nugteren-Roodzant
authors.forename: B
authors.initials: B
authors.lastname: Vosman
authors.forename: K J
authors.initials: KJ
authors.lastname: Hellingwerf
mesh_terms: D016954:Acinetobacter calcoaceticus / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002451:Cell Compartmentation; D005110:Extracellular Space / Q000201:enzymology; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009876:Operon; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D008049:Lipase