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Applied and Environmental Microbiology, March 2003, p. 1377-1382, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1377-1382.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

New Thermosensitive Delivery Vector and Its Use To Enable Nisin-Controlled Gene Expression in Lactobacillus gasseri

T. Neu and B. Henrich*

Abteilung Mikrobiologie, Fachbereich Biologie, Universität Kaiserslautern, D-67653 Kaiserslautern, Germany

Received 30 August 2002/ Accepted 10 December 2002

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.

* Corresponding author. Mailing address: Abteilung Mikrobiologie, Fachbereich Biologie, Universität Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany. Phone: 49 631 205 2347. Fax: 49 631 205 3799. E-mail: henrich{at}rhrk.uni-kl.de.

Applied and Environmental Microbiology, March 2003, p. 1377-1382, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1377-1382.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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