GROWTH-PHASE-DEPENDENT EXPRESSION OF THE LIPOLYTIC SYSTEM OF ACINETOBACTER-CALCOACETICUS BD413 - CLONING OF A GENE ENCODING ONE OF THE ESTERASES

Acinetobacter calcoaceticus BD413, when grown in batch culture in nutr ient 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 D NA in Escherichia coli, 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 ester ase. Via deletion analysis, the esterase gene was mapped on a 1.8 kbp chromosomal DNA fragment. This fragment was sequenced and found to con tain one open reading frame, termed estA, which encodes a protein of 4 0.0 kDa. The amino acid sequence of this protein shows homology to a n umber of lipolytic enzymes, most notably to esterases. Deletion of est A only partially abolished cell-bound esterase activity in A. calcoace ticus, indicating that BD413 forms at least two esterases. Both estera ses show the same temporal regulation of expression. Beta-Galactosidas e activity was measured in strains in which a promoterless lacZ gene w as inserted into estA. Induction of lacZ expression in these strains a lso occurred at the end of exponential growth in batch cultures, indic ating that production of the esterase is regulated at the genetic leve l.