The development of Tn Nuc and its use for the isolation of novel secretion signals in Lactococcus lactis

Abstract

We have previously used Tn 917 for the identification and characterization of regulated promoters from Lactococcus lactis [Israelsen et al., Appl. Environ. Microbiol. 61 (1995) 2540–2547]. We describe here the construction of a new Tn 917-transposon derivative, termed Tn Nuc, which includes the Staphylococcus aureus nuclease gene ( nuc) as a reporter for secretion. Transposition of Tn Nuc into the L. lactis chromosome allows the generation of fusions in-frame with the nuc gene. Tn Nuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e. clones with Lac + phenotype result from transposition of Tn Nuc into a functional gene on the L. lactis chromosome. The presence of a functional signal sequence at the upstream flanking region of the left repeat of the transposed element results in the detection of nuclease activity using a sensitive plate assay. Tn Nuc was used for the identification of novel secretion signals from L. lactis. The sequences identified included known and unknown lactococcal-secreted proteins containing either a signal peptidase-I or -II recognition sequence. In one case, the gene identified codes for a transmembrane protein. The sequences identified were used to study functionality when located in a plasmid under the control of the pH and growth phase-dependent promoter P170 [Madsen et al., Mol. Microbiol. 32 (1999) 75–87]. In all cases, concurrent secretion of nuclease was observed during induction of P170 in a fermentor.