title: The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis.
abstract: We have previously used Tn917 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 Tn917-transposon derivative, termed TnNuc, which includes the Staphylococcus aureus nuclease gene (nuc) as a reporter for secretion. Transposition of TnNuc into the L. lactis chromosome allows the generation of fusions in-frame with the nuc gene. TnNuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e. clones with Lac+ phenotype result from transposition of TnNuc 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. TnNuc 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.
authors.affiliation: Department of Lactic Acid Bacteria, Biotechnological Institute, Hørsholm, Denmark.
authors.forename: P
authors.initials: P
authors.lastname: Ravn
authors.forename: J
authors.initials: J
authors.lastname: Arnau
authors.forename: S M
authors.initials: SM
authors.lastname: Madsen
authors.forename: A
authors.initials: A
authors.lastname: Vrang
authors.forename: H
authors.initials: H
authors.lastname: Israelsen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D021382:Protein Sorting Signals; D008836:Micrococcal Nuclease