Nisin Immunity and Food-Grade Transformation in Lactic Acid Bacteria
نویسندگان
چکیده
................................................................................................................................ 7 Tiivistelmä (abstract in Finnish) .......................................................................................... 8 Introduction .......................................................................................................................... 9 1. Nisin ............................................................................................................................... 9 1.1. Mode of action ................................................................................................... 10 1.2. Biosynthesis and transcriptional regulation ....................................................... 11 1.3. Immunity ............................................................................................................ 12 2. Food-grade cloning and expression systems for lactic acid bacteria ............................. 13 2.1. Conjugation ........................................................................................................ 14 2.2. Cotransformation ............................................................................................... 15 2.3. Food-grade plasmid vectors and markers .......................................................... 15 2.4. Dominant selection markers .............................................................................. 16 2.4.1. Nisin resistance ................................................................................ 16 2.4.2. Resistance to other bacteriocins ....................................................... 17 2.4.3. Resistance to metal ions ................................................................... 17 2.4.4. Carbohydrate utilization ...................................................................18 2.4.5. Resistance to heat shock .................................................................. 18 2.5. Integrative expression systems .......................................................................... 19 2.5.1. Homologous recombination ............................................................ 19 2.5.2. Insertion sequences .......................................................................... 20 2.5.3. Site-specific integration ................................................................... 21 2.5.4. Group II introns ............................................................................... 22 2.6. Complementary selection markers ..................................................................... 22 2.6.1. Lactose genes ................................................................................... 22 2.6.2. Nonsense suppression ...................................................................... 23 2.6.3. Thymidylate synthase ...................................................................... 23 2.6.4. Alanine racemase.............................................................................. 23 Aims of the study .................................................................................................................. 24 Materials and methods .......................................................................................................... 25 Plasmids ............................................................................................................................. 25 Bacterial strains ................................................................................................................. 26 PCR-primers ...................................................................................................................... 27 Methods ............................................................................................................................. 28 Results and discussion .......................................................................................................... 29 1. Nisin immunity .............................................................................................................. 29 1.1. Lipid-free NisI ................................................................................................... 29 1.1.1. Effect of externally added LF-NisI on nisin immunity ................... 29 1.1.2. Effect of LF-NisI secretion on nisin immunity ............................... 30 1.2. Interaction between nisin and NisI .................................................................... 30 1.3. Nisin immunity mechanism ............................................................................... 31
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