B Kaur, P Balgir
curing, enterococcus faecalis, listeria monocytogenes, pediocin, pediococcus acidilactici, plasmid
B Kaur, P Balgir. Pediocin CP2GENE Localisation To Plasmid PCP289 Of Pediococcus Acidilactici MTCC 5101. The Internet Journal of Microbiology. 2006 Volume 3 Number 2.
Indian Council Of Medical Research and All India Council of Technical Education, New Delhi.
Food technologists started their pioneering work in 1970's to unravel the relationship of plasmid encoded food fermentation capabilities and bacteriocin production trait associated with some strains of
Pediocin PA-1 produced by
Pediocin PA-1 structural gene i.e. pedA encodes a 62 amino acid precursor peptide which is posttranslationally modified (by removal of the 18 amino acid leader peptide) into an active 44 amino acid mature peptide. Precursor peptides are having a conserved processing site 'Gly-Gly' at positions -1 and –2 (Horn et al., 1998). PedB gene product (112 amino acid) is involved in the immunity of producer strain against its own secreted bacteriocin. PedC (174 amino acid) and PedD (724 amino acid), both of these gene products are required for the translocation and secretion of the active molecules outside the cell (Marugg et al., 1992 and Venema et al., 1997).
PCR and plasmid profiles of the producer strains have already linked plasmid-encoded secretion of pediocin AcH (Bhunia et al, 1994). PCR based detection of pediocin biosynthesis genes in certain strains of
Pediocin PA-1 and pediocin AcH producing
A Bac +
Materials And Methods
The material for the present study comprised of a standard plasmid molecular weight marker
Isolation of plasmid DNA was carried out using overnight grown broth cultures of
Plasmid curing was accomplished through a growth medium containing ethidium bromide. A 1% (v/v) inoculum of
Agarose gel electrophoresis of plasmid preparations of both Bac +
PCR was carried out to characterize the thermostable and anti-listerial pediocin CP2 produced by
PCR was carried out in 50«l reaction volumes in a sterile 200µl PCR tubes. DNA amplification core kit supplied by Bangalore Genei Pvt. Ltd. India, was used. The PCR reaction mixture comprised of 500ng plasmid DNA, 50 M of each dNTPs, primerG1 and A2 at 500pM concentrations, and 1.5 units of Taq DNA polymerase. Standard protocol of Rodriguez et al. (1997) with slight modifications was used to amplify pedA-pedB gene fragment from pCP289. It consisted of an initialization step (1 cycle) at 95°C for 3 min; PCR amplification (29 cycles) at 92°C for 2 min, 45°C for 2 min and 72°C for 2 min; and final extension (1 cycle) of the growing fragments at 92°C for 2 min, 45° C for 2min, and 72°C for 10 min respectively. Amplified ssDNA was allowed to form dsDNA by complementary base pairing by cooling at 4°C for 5 min.
Amplification was carried out in techne thermal cycler (programmed as described above) and visualized in 1% agarose gels. The gels were electrophoresed at 100v for 1h using 5 µl Φx174 dna/hae iii digest as a standard molecular weight marker. On completion of the run, the gel was stained with ethidium bromide, and visualized.
Results And Discussion
Currently, pediocin pa-1 is one of the well-studied lab bacteriocins. After nisin, pediocin pa-1 will probably be the second lab bacteriocin to find practical applications as a biopreservative in the food industry, especially because of its attractive inhibitory spectrum. Pcr based molecular method employed in the study may rapidly detect lab strains associated with different food substrates that has the potential to produce pediocin pa-1. The plasmid profiles of
Loss of selected phenotype i.e. Bac + character was invariably reported in the test culture
These observations are consistent with findings of several other workers who also reported loss of Bac + activity after curing of selected plasmids in
Pediocin PA-1 specific primers used in the present study amplified 711 bp DNA containing ped A and ped B genes from plasmid DNA preparation of the ped +
This investigation is supported in part by AICTE, New Delhi Major Research Project No. 8019/RDII/BIO (138) 2000-01 and ICMR, New Delhi (SRF awarded to Kaur B., award No. 45/12/2001-BMS).