The genetic switch for the regulatory pathway of Lactobacillus plantarum phage φgle:: characterization of the promoter PL, the repressor gene cpg, and the cpg-encoded protein Cpg in Escherichia coli

The structural and functional features of the similar to 530 bp P-L/Gb5-Gb6-cpg-Gb7 region (P-L overlaps Gb5) for the lysogenic pathway of L. plantarum phage phi gle were investigated using the cat gene of E. coli plasmid pKK232-8 as a reporter. In E. coli XL1-Blue, a recombinant plasmid pKPL2 (cat under P-L/Gb5-Gb6) exhibited distinct CAT activity, whereas the activity of pKPLCP1 (cat under P-L/Gb5-Gb6-cpg) was only marginal. When pKPL2 was coexistent with a compatible derivative of plasmid pACYC177 carrying P-L/Gb5-Gb6-cpg, the CAT activity was declined to the level of pKPLCP1. On the other hand, the cpg-encoded protein Cpg was overproduced in E. coli under P-T7. The molecular mass of the purified Cpg (14.5 kDa on a SDS gel) corresponded well with that (15.1 kDa) predicted from the DNA sequence. Gel-shift and footprinting assays demonstrated that Cpg selectively binds to about 25 bp bases centered around the GATAC-box (from 1 to 7). Moreover, protein crosslinking experiments using glutaraldehyde showed that Cpg most likely functions as a dimeric form. Thus, the present results indicate that Cpg probably represses P-L through binding to the operator GATAC-box(es), and the P-L/cpg region might participate in the lysogenic pathway. (C) 2000 Elsevier Science B.V. All rights reserved.