A novel approach for rapid identification and sequencing of different bacteriocins.

Bacteriocins--inhibitory proteins--produced by lactic acid bacteria (LAB) are considered natural antimicrobials that may enhance the safety of foods. With numerous claims of new bacteriocins appearing from time to time, it's necessary to find new techniques for identifying unique bacteriocins or those that have not been previously characterized.

Scientists at Oklahoma State University wanted to develop rapid microbial and molecular methods that can readily identify different immunity classes of bacteriocins of LAB that would have complementary inhibitory activity. They also wanted to obtain structural gene sequence information for those bacteriocins.

Bacteriocin-resistant strains of L. monocytogenes were made so that the researchers could select LAB bacteriocins. The bacteriocin-resistant Listeria were then used as indicators to find additional bacteriocin-producing strains in retail foods. The researchers repeated this cycle three consecutive times. Bacteriocin-producing strains of interest were subjected to a SYBR green-based real-time PCR array using sets of primers made from the coding sequence for all known LAB bacteriocin structural genes and sequenced at the researchers' university facility. You may recall that SYBR Green I is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology.

The bacteriocins were considered to be of the same immunity class if L. monocytogenes that was bacteriocin-resistant to a particular bacteriocin became cross-resistant to other bacteriocins. These bacteria-resistant L. monocytogenes were again used as indicators for other LAB bacteriocins that also showed inhibition. This cycle was repeated until three successive classes of bacteriocins were obtained based on immunity or the inhibition of single or multiple bacteriocin-resistant strains of L. monocytogenes.

The PCR array allowed the researchers to obtain successful amplification and DNA sequence information for every bacteriocin tested, enabling them to find new bacteriocins whose sequence was not identical to those already residing in the GenBank[R]--the National Institutes of Health genetic sequence database, an annotated collection of all publicly available DNA sequences. The methods allowed the scientists to identify different functional immunity classes based on bacteriocin-resistance screening. A PCR array provided quick amplification and sequence identity of the various structural genes to determine if these bacteriocins were previously studied.

Further information. Peter Muriana, Oklahoma State University, Food and Agricultural Products, Research and Technology Center, Room 109, Stillwater, OK 74078; phone: [phone]; fax: [phone]; email:[e-mail].