Restriction fragment differential display of pediocin-resistant Listeria monocytogenes 412 mutants shows consistent overexpression of a putative {beta}-glucoside-specific PTS system

Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark 1 Display Systems Biotech, Lersø Parkallé 40, DK-2100 Copenhagen, Denmark 2 Author for correspondence: Anne Gravesen. Tel: +45 3528 3272. Fax: +45 3528 3231. e-mail: alg{at}kvl.dk Pediocin PA-1, which is a bacteriocin produced by lactic acid bacteria, has potential as a biopreservative of food. However, such use may lead to the development of resistance in the target organism. Gene expression in two independent pediocin-resistant mutants of Listeria monocytogenes 412 was compared to the original isolate by restriction fragment differential display PCR (RFDD-PCR). This method amplifies cDNA restriction fragments under stringent PCR conditions, enabled by the use of specific primers complementary to ligated adaptor sequences. RFDD-PCR was very well suited for analysis of listerial gene expression, giving reproducible PCR product profiles. Three gene fragments having increased expression in both resistant mutants were identified. All three had homology to components of ß-glucoside-specific phosphoenolpyruvate-dependent phosphotransferase systems (PTS), one fragment having homology to enzyme II permeases, and the two others to phospho-ß-glucosidases. Overexpression of the putative PTS system was consistently observed in 10 additional pediocin-resistant mutants, isolated at different pH, salt content and temperature. The results suggest that RFDD-PCR is a strong approach for the analysis of prokaryotic gene expression and that the putative ß-glucoside-specific PTS system is involved in mediating pediocin resistance. Keywords: restriction fragment differential display, Listeria monocytogenes , pediocin resistance, phosphoenolpyruvate-dependent phosphotransferase system, bacteriocin Abbreviations: DD, differential display; PTS, phosphoenolpyruvate-dependent phosphotransferase system; RFDD, restriction fragment differential display The EMBL accession numbers for the sequences reported in this paper are AJ251202, AJ251203 and AJ251204.