ABSTRACT 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°C, whereas replication is efficiently shut down at 42°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.