Bacillus subtilis phage Phi29 protein p6 is required for DNA replication and promotes the switch from early to late transcription. In vivo it binds all along the viral linear DNA, which suggests a global role as an architectural protein; in contrast, binding to bacterial DNA is negligible. This specificity could be due to the p6 binding preference for less negatively supercoiled DNA, as is presumably the case with viral (with respect to bacterial) DNA. Here we demonstrate that p6 binding to Phi29 DNA is greatly increased when negative supercoiling is decreased by novobiocin; in addition, gyrase is required for DNA replication. This indicates that, although non-covalently closed, the viral genome is topologically constrained in vivo. We also show that the p6 binding to different Phi29 DNA regions is modulated by the structural properties of their nucleotide sequences. The higher affinity for DNA ends is possibly related to the presence of sequences in which their bendability properties favor the formation of the p6-DNA complex, whereas the lower affinity for the transcription control region is most probably due to the presence of a rigid intrinsic DNA curvature.