Genetic analysis of the DNA recognition sequence of the P2 Cox protein

Abstract

The Cox protein of temperate Escherichia coli phage P2 is involved in three important biological processes: (i) excision of the integrated prophage genome (G. Lindahl and M. Sunshine, Virology 49:180-187, 1972), (ii) transcriptional repression of the P2 Pc promoter, which controls the expression of the immunity repressor C and the integrase (S. Saha, E. Haggård-Ljungquist, and K. Nordström, EMBO J. 6:3191-3199, 1987), and (iii) transcriptional activation of the late PII promoter of the unrelated satellite phage P4 (S. Saha, E. Haggård-Ljungquist, and K. Nordström, Proc. Natl. Acad. Sci. USA 86:3973-3977, 1989). A comparison of the DNA regions protected by Cox from DNaseI degradation has revealed a presumptive Cox recognition sequence (Saha et al., Proc. Natl. Acad. Sci. USA). The binding region of Cox in the P2 Pc promoter contains three presumptive recognition sequences, "Cox boxes," located in tandem. P2 vir3 and P2 vir24 are virulent deletion mutants unable to plate on Cox-producing strains, most likely because the deletions locate the new early promoters too close to the Cox-binding region (Saha et al., EMBO J.). In this report, spontaneous P2 vir3 and vir24 mutants, no longer sensitive to repression by the Cox protein, have been isolated. These mutants plate with equal efficiency on strains with or without a Cox-producing plasmid, and they have been named cor for cox resistance. Three types are recognized; the four P2 vir3 cor mutants have a 1-base deletion in the first Cox box, while the P2 vir24 cor mutants were of two types; four have a base substitution in the first Cox box, and one has a base substitution in the second Cox box. The effect of the Cox protein on the mutated P2 vir3 and vir24 promoters was analyzed in vivo by using fusions to a promoterless cat (chloramphenicol acetyltransferase) gene. The activities of the P2 vir3 and vir24 early promoters, as opposed to the wild-type early Pe promoter, are drastically reduced by the Cox protein, and the cor mutation renders them as resistant to Cox as the wild-type Pe promoter. Thus, at least the first two Cox boxes are essential for binding of the Cox protein.