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The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons,  as-48ABC  (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural  as-48A  gene) and  as-48C 1 DD 1 EFGH . The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the  mCherry  gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter P X  as a reference. Our results revealed that only three promoters P A , P 2(2)  and P D1  were recognized in  Enterococcus faecalis ,  Lactococcus lactis  and  Escherichia coli , in the conditions tested. The maximal fluorescence was obtained with P X  in all the strains, followed by the P 2(2)  promoter, which level of fluorescence was 2-fold compared to P A  and 4-fold compared to P D1 . Analysis of putative factors influencing the promoter activity in single and double transformants in  E. faecalis  JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P 2(2)  promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the  as-48  cluster, while the pH seems to affect differently the  as-48  promoter expression.

Analysis of the Promoters Involved in Enterocin AS-48 Expression