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 lactis, based on secretion of the staphylococcal nuclease (SNase) in the organism, was developed. The nuc gene (encoding SNase) was cloned on both rolling-circle and theta-replicating plasmids. L. lactis strains containing these nuc+ plasmids secrete SNase and are readily detectable by a simple plate test. A multicloning site (MCS) was introduced just after the cleavage site between leader peptide and the mature SNase, without affecting nuclease activity. Cloning foreign DNA fragments into any site of the MCS interrupts nuc and thus results in nuc mutant clones which are easily distinguished fron nuc+ clones on plates. The utility of this system for L. lactis was demonstrated by cloning an antibiotic resistance marker and Escherichia coli chromosomal DNA fragments into the MCS of the nucMCS cassette. Both cloning vectors containing the nucMCS cassette were also introduced into Streptococcus salivarius subsp. thermophilus, in which direct screening of nuc mutant recombinant clones was also achieved. The potential uses of nuc as a secretion reporter system are discussed.
Le Loir
Y
Y
Laboratorie de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France.
Gruss
A
A
Ehrlich
S D
SD
Langella
P
P
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004274:DNA, Recombinant; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000235:genetics*; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D021382:Protein Sorting Signals / Q000235:genetics
D017931:DNA Primers; D004274:DNA, Recombinant; D021382:Protein Sorting Signals; D008836:Micrococcal Nuclease