Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases

Abstract

Acinetobacter calcoaceticus BD413, when grown in batch culture in nutrient broth, produces both extracellular lipase activity and cell-bound esterase activity during and after the transition between exponential growth and the stationary phase. From a library of A. calcoaceticus DNA in Escherichia cofi, plasmids were isolated that enabled E. coli to grow on media with tributyrin as the sole carbon source. Assays with model substrates classified the product of the cloned gene as an esterase. Via deletion analysis, the esterase gene was mapped on a 1.8 kbp chromosomal DNA fragment. This fragment was sequenced and found to contain one open reading frame, termed estA, which encodes a protein of 40.0 kDa. The amino acid sequence of this protein shows homology to a number of lipolytic enzymes, most notably to esterases. Deletion of estA only partially abolished cell-bound esterase activity in A. calcoaceticus, indicating that BD413 forms at least two esterases. Both esterases show the same temporal regulation of expression. /?-Galactosidase activity was measured in strains in which a promoterless lac2 gene was inserted into estA. Induction of lac2 expression in these strains also occurred at the end of exponential growth in batch cultures, indicating that production of the esterase is regulated at the genetic level.

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

“…Egg yolk plates (49,75), olive oil plates (containing an emulsion of olive oil and rhodamine B) (44), and tributyrin plates (containing an emulsion of tributyrin) were made as described previously (40). Tween plates contained 1% (vol/vol) Tween 80 and 0.5 mM CaCl 2 .…”

“…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.…”

“…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.…”

Purification of antibiotics produced byLentinus squarrosulus and preliminary characterization of a compound active againstRigidoporus lignosus

“…In this assay, strain AAC350 was used as DNA donor, and wild-type strain BD413 as recipient. AAC350 contains an aph3 gene (encoding kanamycin resistance) from pPJ1 [15] on a PvulI fragment, inserted in the EcoRV site of the estA gene of A. calcoaceticus BD413 [8]. Samples of AAC350 collected at different stages of growth were filtered over 0.2 txm cellulose-acetate filters, and the supernatants were stored overnight at 4~ The next day approximately 1 • 108 competent BD413 cells were resuspended in 0.5 ml filtrate and incubated for 60 min at 30~ in a shaking waterbath to allow transformation.…”

Detection of α/β‐hydrolase fold in the cell surface esterases of Acinetobacter species using an analysis of 3D profiles

“…Antibodies prepared against the over-expressed recombmant esterase m E. ('()it were used to locate the enzyme primarily in the membrane fractions of A lll'ofjii RAG-I [I]. A cell-bound esterase from A_ calcoacetlcus BD413 has also been expressed in E. coli [8]. Analysis of the conserved motifs in the primary sequence of the two esterases and aligments with related proteins using BLAST and FASTA program~.…”