DIRECT SCREENING OF RECOMBINANTS IN GRAM-POSITIVE BACTERIA USING THE SECRETED STAPHYLOCOCCAL NUCLEASE AS A REPORTER

A system for direct screening of recombinant clones in Lactococcus lac tis, based on secretion of the staphylococcal nuclease (SNase) in the organism, was developed. The nuc gene (encoding SNase) was cloned on b oth rolling-circle and theta-replicating plasmids. L. lactis strains c ontaining these nuc(+) plasmids secrete SNase and are readily detectab le by a simple plate test. A multicloning site (MCS) was introduced ju st after the cleavage site between leader peptide and the mature SNase , without affecting nuclease activity. Cloning foreign DNA fragments i nto any site of the MCS interrupts nuc and thus results in nuc mutant clones which are easily distinguished from nuc(+) clones on plates. Th e utility of this system for L. lactis was demonstrated by cloning an antibiotic resistance marker and Escherichia coli chromosomal DNA frag ments into the MCS of the nucMCS cassette. Both cloning vectors contai ning the nucMCS cassette were also introduced into Streptococcus saliv arius subsp. thermophilus, in which direct screening of nuc mutant rec ombinant clones was also achieved. The potential uses of nuc as a secr etion reporter system are discussed.