A comparative analysis of the bifunctional Cox proteins of two heteroimmune P2-like phages with different host integration sites

Abstract

The Cox protein of the coliphage P2 is multifunctional; it acts as a transcriptional repressor of the Pc promoter, as a transcriptional activator of the P(LL) promoter of satellite phage P4, and as a directionality factor for site-specific recombination. The Cox proteins constitute a unique group of directionality factors since they couple the developmental switch with the integration or excision of the phage genome. In this work, the DNA binding characteristics of the Cox protein of WPhi, a P2-related phage, are compared with those of P2 Cox. P2 Cox has been shown to recognize a 9 bp sequence, repeated at least 6 times in different targets. In contrast to P2 Cox, WPhi Cox binds with a strong affinity to the early control region that contains an imperfect direct repeat of 12 nucleotides. The removal of one of the repeats has drastic effects on the capacity of WPhi to bind to the Pe-Pc region. Again in contrast to P2 Cox, WPhi Cox has a lower affinity to attP compared to the Pe-Pc region, and a repeat of 9 bp can be found that has 5 bp in common with the repeat in the Pe-Pc region. WPhi Cox, however, is essential for excisive recombination in vitro. WPhi Cox, like P2 Cox, binds cooperatively with integrase to attP. Both Cox proteins induce a strong bend in their DNA targets upon binding.

DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels

“…Phage P4 depends on P2 for lytic growth. Cox from WΦ (90 aa), on the other hand, has been shown to repress the WΦ Pc promotor and to promote excision in vitro (7), but does not activate the P4 satellite phage (8). …”

“…P2 Cox binds, in a cooperative manner, to so called cox-boxes on the DNA, which has a sequence of TTAAA(G/C)NC(A/C) (7). These cox-boxes are directional, and basically provide a footprint to where, and in which direction, P2 Cox should bind to the DNA.…”

“…At higher protein concentrations, P2 Cox also binds to non-specific nucleotide sequences, likely also here in a cooperative manner (9). WΦ Cox does not bind to those cox-boxes, but instead recognizes a direct repeat of 12 nucleotides that is found in the WΦ Pe-Pc region (7). We recently determined the structure of P2 Cox (10), which showed that it oligomerizes in a helical fashion, forming a left-handed filament with a diameter of ∼65Å and a rise of ∼34Å, with its DNA binding helix and wing pointing outward (Figure 1).…”

“…This indicates a possibility for the Cox oligomer to bind DNA in a way highly similar to how histone particles wind up DNA around themselves in nucleosomes. From previous work it was clear that both P2 Cox and WΦ Cox strongly bend DNA upon binding and that they protect large stretches of DNA from DNAse I cleavage (4,5,7). Based on the structure, together with previous biochemical and genetical work, we postulated that P2 Cox achieves its different functions by wrapping the DNA around its own oligomer in a helical fashion (Figure 1) (10).…”

“…From sequence alignment it is also clear that WΦ Cox has the same C-terminal organization as P2 Cox, indicating that WΦ Cox oligomerizes in a similar fashion as P2 Cox. Furthermore, it has been suggested that at increased WΦ Cox concentrations, it binds cooperatively also to non-specific nucleotide sequences, which might indicate the formation of a filament (7). Based on this it is therefore likely that the function and DNA binding of WΦ Cox is similar to that of P2 Cox.…”