Open AccessNew Thermosensitive Delivery Vector and Its Use To Enable Nisin-Controlled Gene Expression in Lactobacillus gasseri
Author(s) -
Thomas R. Neu,
Bernhard Henrich
Publication year - 2003
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.69.3.1377-1382.2003
Subject(s) - replicon , lactobacillus gasseri , nisin , biology , plasmid , lactococcus lactis , reporter gene , lactobacillus , gene , gene expression , expression vector , marker gene , transduction (biophysics) , bacteria , genetics , microbiology and biotechnology , biochemistry , recombinant dna , lactic acid
Derivatives of a cryptic plasmid from Lactobacillus curvatus showed temperature-sensitive replication in thermophilic lactobacilli. The thermosensitive replicon was used to construct the new delivery vector pTN1, which allows site-specific replacement of chromosomal DNA sequences. pTN1 carries an erythromycin resistance marker suitable for selection of single-copy integrants and replicates readily at 35 degrees C, whereas replication is efficiently shut down at 42 degrees C. To demonstrate the functionality of pTN1, the signal transduction genes (nisRK) of the nisin-controlled expression system were integrated downstream of the pepN gene into the chromosome of Lactobacillus gasseri. In the resulting strain, UKLbg1, expression of nisRK was likely driven by cotranscription with pepN and enabled nisin-dependent induction of a fusion of a reporter gene (pepI) to the nisA promoter. The induction rates were correlated with the amount of nisin used, and maximum pepI expression was achieved with nisin concentrations (above 25 ng/ml) at which growth of the bacteria was already inhibited.