THE LIPASE GENE OF BACILLUS SUBTILIS 168

Abstract

Among Gram-positive bacteria, strain 168 of Bacillus subtilis constitutes a tool of choice for basic genetic and molecular studies. We madethe observation that this strain exhibits an extracellular lipolytic activity. We have undertaken to characterize the corresponding gene(s). Shotgun cloning of Bacillus subtilis 168 DNAin E. coli yielded two types of lipasepositive clones designated lipA and lipB. However, the lipB enzyme wasrather an esterase, on the basis of the preferential cleavage of esters of short chain fatty acids and of the absence of fluorescent reaction on triolein/rhodamin G medium. By multiple Tn5 transposon inactivations, gene lipA was estimated to be about 700 basepairs long. Both genes were inactivated in B. subtilis by reciprocal recombination with the homologous gene disrupted in vitro by a DNA segmentcontaining an antibiotic resistance (lipA::Km; lipB::Cm). The resulting strain expressed very little - if any - residual extracellular lipase-esterase activity. Mapping experiments indicated that lipA is a new locus at about 22° whereaslipB, at about 306° could correspondto an esterase gene (estB) previously described. Sequencing of genelipA is in progress. Besides, we are seeking conditions to | optimize the yield of extracellular lipase (culture conditions, genetic background, transcription signals) as a prerequisite for its large scale purification.