The genetic switch for the regulatory pathway of Lactobacillus plantarum phage (phi)g1e: characterization of the promoter P(L), the repressor gene cpg, and the cpg-encoded protein Cpg in Escherichia coli.
The structural and functional features of the approximately 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.
Kakikawa
M
M
Molecular Biology Group, Faculty of Engineering, Toyama University, Japan.
Ohkubo
S
S
Syama
M
M
Taketo
A
A
Kodaira
K I
KI
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D001667:Binding, Competitive; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral / Q000235:genetics; D007778:Lactobacillus / Q000821:virology*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase