The genes involved in production of and immunity to sakacin A, a bacteriocin from Lactobacillus sake Lb706

Abstract

Sakacin A is a small, heat-stable, antilisterial bacteriocin produced by Lactobacillus sake Lb706. The nucleotide sequence of a 8,668-bp fragment, shown to contain all information necessary for sakacin A production and immunity, was determined. The sequence revealed the presence of two divergently transcribed operons. The first encompassed the structural gene sapA (previously designated sakA) and saiA, which encoded a putative peptide of 90 amino acid residues. The second encompassed sapK (previously designated sakB), sapR, sapT, and sapE. sapK and sapR presumably encoded a histidine kinase and a response regulator with marked similarities to the AgrB/AgrA type of two-component signal-transducing systems. The putative SapT and SapE proteins shared similarity with the Escherichia coli hemolysin A-like signal sequence-independent transport systems. SapT was the HlyB analog with homology to bacterial ATP-binding cassette exporters implicated in bacteriocin transport. Frameshift mutations and deletion analyses showed that sapK and sapR were necessary for both production and immunity, whereas sapT and sapE were necessary for production but not for immunity. The putative SaiA peptide was shown to be involved in the immunity to sakacin A. The region between the operons contained IS1163, a recently described L. sake insertion element. IS1163 did not appear to be involved in expression of the sap genes. Northern (RNA) blot analysis revealed that the putative SapK/SapR system probably acts as a transcriptional activator on both operons. A 35-bp sequence, present upstream of the putative sapA promoter, and a similar sequence (30 of 35 nucleotides identical) upstream of sapK were shown to be necessary for proper expression and could thus be possible targets for transcriptional activation.