Heterologous expression and activity studying of a nisin resistance gene in <i>Lactobacillus delbrueckii</i> subsp. bulgaricus

Abstract

Background: Lactic acid bacteria are generally regarded as safe microorganisms and widely used in industry and medicine. We are trying to add additional properties to them by gene engineering. However, the genetically modified bacteria are not acceptable to use in food and medicine due to the presence of antibiotic resistance genes in plasmids. Thus, it is necessary to develop food-grade selection markers. Methods: To evaluate the activity of nisin resistance gene (nsr) cloned from Lactococcus lactis as a food-grade selective marker in transformant bacterial strains, we isolated L.lactis nisin resistant strains from fresh milk, and cloned the nsr gene to Lactobacillus delbrueckii subsp. bulgaricus, then studied the activity of nsr in positive transformant bacterial strains. Results: Six nisin resistant strains were isolated from fresh milk on M17 plates supplemented with nisin at a final concentration of 400 μg/ml. All of these isolates were identified as L.lactis by morphological, physiological, biochemical and 16S rDNA sequence analysis. A pair of primers was designed on the basis of the DNA sequences of reported nsr. PCR amplification was carried out with chromosome and plasmid DNA as templates from these six strains respectively, and an expected PCR product was obtained from one chromosome. After the amplicon was confirmed as nsr gene, it was cloned into E.coli-Lactobacillus shuttle vector pMG36e, resulting in pMG36ensr. The construct was obtained when the recombinant plasmid pMG36e-nsr was electroporated into L.delbrueckii subsp. bulgaricus L6032 competent cells. When the medium contained a maximum of 400 μg/ml nisin, the construct carrying pMG36e-nsr showed the same growth curve as that cultured in medium containing 10 μg/ml erythromycin. Conclusions: The nsr gene was cloned and successfully expressed in L.delbrueckii subsp. bulgaricus L6032. This study suggests that nsr gene could be used as a potential food-grade selective marker to replace antibiotic resistant markers in future.