Cloning and Gene Replacement Mutagenesis of a Pseudomonas atlantica Agarase Gene

Abstract

An agarase gene ( agrA ) was isolated by cloning genomic DNA prepared from Pseudomonas atlantica . The agarase activity in recombinant Escherichia coli was found in cell-free culture supernatants and could pass through a 0.45-μm-pore-size membrane separating cells from agar, suggesting that the gene product was exported in E. coli . The enzyme was specific for agar and agarose and did not digest alginate or carrageenan. Mutations generated by transposon mini-Mu d1( lacZ Km r ) were used to define the agrA coding region, as well as the direction of transcription of the gene. A procedure was developed to produce a P. atlantica agrA mutant. This required construction of an agrA::kan insertion mutation in vitro and subsequent introduction of the defect into the chromosome of P. atlantica by recombinational exchange. Transformation of P. atlantica with plasmids containing agrA::kan utilized a Tris-polyethylene glycol 6000-CaCl 2 treatment for making competent cells. Replacement of wild-type agrA with agrA::kan resulted in loss of agarase activity. Uses of the agrA gene probe and an Agr − mutant for environmental studies are discussed.