Identification of an operon involved in the production of Lactacin B, a bacteriocin produced by Lactobacillus acidophilus

by 1979- Dobson, Alleson Elaine

DOBSON, ALLESON ELAINE. Identification of an operon involved in the production of Lactacin B, a bacteriocin produced by Lactobacillus acidophilus. (Under the direction of Professor T.R. Klaenhammer.) Lactacin B is a class II bacteriocin produced by Lactobacillus acidophilus NCFM (Barefoot and Klaenhammer 1983). Mutational, nucleotide sequence, and transcriptional analyses revealed that the genetic determinants responsible for lactacin B regulation and production are located on a 9.5 kb polycistronic region (LBA1803-LBA1791) of the L. acidophilus NCFM chromosome. The lab operon comprised 12 putative open reading frames (ORFs) organized into three clusters: a production and regulation cluster encoding putative proteins that resemble two component signal transduction systems of the AgrC- AgrA type; an export cluster encoding putative proteins that resemble ATP-binding cassette (ABC) transporters and accessory proteins, and the final cluster composed of three putative proteins of unknown functionality. Each cluster was separated by an intrinsic terminator, the strongest terminators flanked the entire lactacin B region (?G = -13.4kcal/mol and ?G = -17.0kcal/mol respectively). A total of 7 genes with unknown functionality were situated in this region, each containing a double-glycine leader motif characteristic of bacteriocin structural genes and their precursors. Insertional inactivation of the gene believed to encode an ABC transporter (labT) completely abolished bacteriocin activity, implicating this region in lactacin B production. Cloning of the first four genes within this region (LBA1803-LBA1800) onto a high copy number plasmid resulted in markedly higher levels of lactacin B activity compared to the control. These ORFs encoded proteins typical to bacteriocin peptides; small, cationic peptides, each with an N-terminal double glycine leader motif. Experiments with chemically synthesized peptides revealed that LBA1800 was not inhibitory, but induced lactacin B production in broth cultures. The genetic organization of the region indicates that lactacin B production is regulated through the three component regulatory system common to many class II bacteriocin systems.