Characterization of the repressor gene product (c) and the immunity region of Mu-like transposable bacteriophage D108

Transposable elements are discrete segments of DNA capable of moving, or transposing, from one location to another in host cell DNA. The Escherichia coli, temperate, generalized transducing bacteriophages D108 and Mu are highly homologous, and their linear, double-stranded, 37 Kilobase pair genomes are propagated by multiple rounds of DNA transposition. They are, however, non-homologous in the region of their genomes that regulate transposition. We have cloned both the wild-type $(c sp+)$ and thermo-sensitive (c ts10) D108 repressor genes and the thermo-sensitive (c ts62) Mu repressor gene under the control of the lac UV5 promotor. We localized the functional D108 repressor gene and found that the wild-type and thermo-sensitive D108 repressor gene products have apparent molecular weights that are smaller (19 Kilodaltons (KDa)) than the thermo-sensitive Mu repressor gene product (22.5 KDa). Using gel electrophoresis band-retardation assays, we found that the D108 and Mu thermo-sensitive repressors display different DNA-binding properties. The D108 repressor protects a single 77 bp region from exonuclease III hydrolysis and DNase I cleavage. We mapped the sites of transcription initiation for both the D108 repressor gene and the D108 early region operon. Finally, we determined that the host protein, Integration Host Factor (IHF), is required for D108 lytic growth and that it protects from DNase I cleavage, a sequence that lies within the D108 repressor operator. Our results suggest that the role of the D108 repressor may be to repress early operon expression, and thus lytic growth, by preventing the binding of the transcriptional activator protein IHF