CLONING AND NUCLEOTIDE-SEQUENCE OF A GENE FROM LACTOBACILLUS-SAKE LB706 NECESSARY FOR SAKACIN-A PRODUCTION AND IMMUNITY

Sakacin A is an antilisterial bacteriocin produced by Lactobacillus sa ke Lb706. In order to identify genes involved in sakacin A production and immunity, the plasmid fraction of L. sake Lb706 was shotgun cloned directly into a sakacin A-nonproducing and -sensitive variant, L. sak e Lb706-B, by using the broad-host-range vector pVS2. Two clones that produced sakacin A and were immune to the bacteriocin were obtained. A DNA fragment of approximately 1.8 kb, derived from a 60-kb plasmid of strain Lb706 and present in the inserts of both clones, was necessary for restoration of sakacin A production and immunity in strain Lb706- B. The sequence of the 1.8-kb fragment from one of the clones was dete rmined. It contained one large open reading frame, designated sakB, po tentially encoding a protein of 430 amino acid residues. Hybridization and nucleotide sequence analyses revealed that the cloned sakB comple mented a mutated copy of sakB present in strain Lb706-B. The sakB gene mapped 1.6 kb from the previously cloned structural gene for sakacin A (sakA) on the 60-kb plasmid. The putative SakB protein shared 22% am ino acid sequence identity (51% similarity if conservative changes are considered) to AgrB, the deduced amino acid sequence of the Staphyloc occus aureus gene agrB. The polycistronic agr (accessory gene regulato r) locus is involved in the regulation of exoprotein synthesis in S. a ureus. Similar to the AgrB protein, SakB had some features in common w ith a family of transmembrane histidine protein kinases, involved in v arious adaptive response systems of bacteria. This similarity included transmembrane regions in the N-terminal half of the protein and certa in conserved amino acid residues.