Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase

Abstract

At the onset of the stationary phase in rich media, the gram-negative soil bacterium Acinetobacter calcoaceticus BD413 produces several lipolytic enzymes (40), which have partly overlapping substrate specificities. BD413 estA, encoding one of the cell-bound esterases (40), and lipA, encoding the extracellular lipase (43), have been cloned and characterized. The regulation of their expression is currently being investigated. In addition, we also aim at defining posttranscriptional factors involved in regulation of enzyme production. The latter aspect is especially important for the extracellular lipase of A. calcoaceticus BD413, since several components involved in export of the protein, including concurrent protein processing events, may have significant control over lipase production.The extracellular lipase of A. calcoaceticus BD413 presumably is exported from the cell via a two-step translocation process (43): across the cytoplasmic membrane, this translocation will be mediated by the Sec system (59, 66), whereas translocation through the outer membrane proceeds via a translocation complex similar to the Xcp system in Pseudomonas spp. (69) and the Pul system in Klebsiella spp. (59). These assumptions are based upon the identification of an N-terminal export signal peptide, indicative of the involvement of a Seclike system, and upon the high degree of similarity of A. calcoaceticus LipA to Pseudomonas lipases that are secreted via a two-step mechanism. Moreover, we have demonstrated that a periplasmic processing enzyme, a protein disulfide oxidoreductase, is required for high-level production of lipase in A. calcoaceticus BD413 (43).In Pseudomonas spp., production of lipases that are similar to Acinetobacter LipA requires the presence of a lipase-specific helper protein, which is N terminally anchored in the cytoplasmic membrane, with its C-terminal domain in the periplasm. Activity of this type of protein is essential for the second lipase translocation step, across the outer membrane (14, 34). In the periplasm, such helper proteins have been shown to mediate the formation of secondary structure elements in the periplasmic form of the lipase, suggesting a chaperone-like function of the helper protein. Unlike the situation in pseudomonads, as yet no essential lipase helper protein has been identified in A. calcoaceticus. In Pseudomonas species, these proteins are invariably encoded downstream of the structural lipase gene, and this is not the case in A. calcoaceticus BD413 (43).Previously, we have reported on the identification of two

Physiological factors affecting production of extracellular lipase (LipA) in Acinetobacter calcoaceticus BD413: fatty acid repression of lipA expression and degradation of LipA

“…The extracellular lipase (LipA) has recently been purified and characterized, and the encoding lipA gene has been cloned (23). Secretion of LipA requires the activity of LipB, a specific LipA chaperone that is anchored into the cytoplasmic membrane and faces the periplasm (5,24). Both the lipase and the lipase chaperone of A. calcoaceticus have high degrees of sequence identity with their respective counterparts in pseudomonads, suggesting strong similarities in the lipase production mechanisms in these genera.…”

“…The gram-negative soil bacterium Acinetobacter calcoaceticus BD413 produces a single extracellular lipase (EC 3.1.1.3) along with several other (cell-bound) lipolytic enzymes (22)(23)(24). The extracellular lipase (LipA) has recently been purified and characterized, and the encoding lipA gene has been cloned (23).…”

“…Both the lipase and the lipase chaperone of A. calcoaceticus have high degrees of sequence identity with their respective counterparts in pseudomonads, suggesting strong similarities in the lipase production mechanisms in these genera. Indeed, identification of several similar components involved in the secretion of lipase has allowed us to characterize the mechanism of lipase production by A. calcoaceticus in considerable detail (23,24).…”

In Vitro Analysis of Roles of a Disulfide Bridge and a Calcium Binding Site in Activation of Pseudomonas sp. Strain KWI-56 Lipase

“…The formation of complex was proposed to weaken the activity of lipase, thus preventing membrane degradation. Furthermore, the proper folding of lipase in the periplasm of Pseudomonas cells appears to be essential for its Xcp-mediated translocation across the outer membrane (25).…”

Combining localized PCR mutagenesis and natural transformation in direct genetic analysis of a transcriptional regulator gene, pobR

“…All recombinant DNA techniques were performed as described previously (21,22) and according to Sambrook et al (32). Isolation of bacterial chromosomal DNA, to be used as template DNA in PCRs with Taq polymerase, was done with Instagene DNA Purification Matrix (BioRad) according to the protocol provided by the supplier.…”