Highly Specific Culture-Independent Detection of YGNGV Motif-Containing Pediocin-Producing Strains

Probiotics and Antimicrobial Proteins
S K SoodPuja Lather

Abstract

A novel method based on (1) initial microbiological screening and (2) a highly specific PCR is described for selection of strains expressing YGNGV motif-containing pediocin. Initial screening is carried out using spot on the lawn assay for selection of acid-free, hydrogen peroxide (H2O2)-free and secreted heat-stable inhibitory activity producing strains. This is followed by highly specific PCR for amplification of 406-bp fragment using forward primer: 5'-tggccaatatcattggtggt-3' targeting signal peptide sequence of pediocin structural gene and reverse primer: 5'-ctactaacgcttggctggca-3' encoding N-terminus of immunity gene. The assay was validated with Pediococcus pentosaceus NCDC273 and Pediococcus acidilactici NCDC252 using (1) digestion of amplified 406-bp fragment with HindIII restriction enzyme-producing two restriction fragments of expected sizes (227 and 179 bp), (2) nucleotide sequencing of 406-bp fragment from both strains found these pediocins identical to pediocin PA-1/AcH and (3) identification of both pediocins as pediocin PA-1 at protein level using RP-HPLC. The assay was used for screening six strains (3 pediococci, 2 lactobacilli and an Enterococcus faecium) producing acid-free, hydrogen peroxide (H2O2)-free and ...Continue Reading

References

Aug 11, 2010·FEMS Microbiology Reviews·Hikmate AbriouelAntonio Gálvez
Sep 1, 2007·Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology·Duongdearn SuwanjindaWatanalai Panbangred

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