Restriction fragment differential display of pediocin-resistant Listeria monocytogenes 412 mutants shows consistent overexpression of a putative beta-glucoside-specific PTS system.
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 beta-glucoside-specific phosphoenolpyruvate-dependent phosphotransferase systems (PTS), one fragment having homology to enzyme II permeases, and the two others to phospho-beta-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 beta-glucoside-specific PTS system is involved in mediating pediocin resistance.
Gravesen
Anne
A
Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark1.
Warthoe
Peter
P
Display Systems Biotech, Lersø Parkallé 40, DK-2100 Copenhagen, Denmark2.
Knøchel
Susanne
S
Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark1.
Thirstrup
Kenneth
K
Display Systems Biotech, Lersø Parkallé 40, DK-2100 Copenhagen, Denmark2.
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005519:Food Preservation; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D008089:Listeria monocytogenes / Q000187:drug effects* / Q000235:genetics* / Q000472:pathogenicity; D008969:Molecular Sequence Data; D009154:Mutation; D000070017:Pediocins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017386:Sequence Homology, Amino Acid
D001430:Bacteriocins; D017931:DNA Primers; D000070017:Pediocins; C074288:pediocin PA-1; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System