Protein p6 from Bacillus subtilis phage Ø29 (M r ) 11 800) binds in Vitro to DNA forming a large nucleoprotein complex in which the DNA wraps a multimeric protein core. The high intracellular abundance of protein p6 together with its ability to bind the whole Ø29 DNA in Vitro strongly suggests that it plays a role in viral genome organization. We have determined by sedimentation equilibrium analysis that protein p6 (1-100 µM range), in the absence of DNA, is in a monomer-dimer equilibrium, with an association constant (K 2 ) of ∼2 × 10 5 M -1 . The intracellular concentration of protein p6 (∼1 mM) was estimated measuring the number of copies per cell (7 × 10 5 ) and the cell volume (1 × 10 -15 L). At concentrations around 1 mM, protein p6 associates into oligomers. This self-association behavior is compatible with a dimer-hexamer model (K 2,6 ) 3.2 × 10 8 M -2 ) or with an isodesmic association of the dimer (K ) 950 M -1 ), because the apparent weight-average molecular mass (M w,a ) does not reach saturation at the highest protein concentrations. The sedimentation coefficients of protein p6 monomer and dimer were 1.4 and 2.0, respectively, compatible with translational frictional ratios (f/f o ) of 1.15 and 1.30, which slightly deviate from the hydrodynamics of a rigid globular protein. Taking together these results and considering the structure of the nucleoprotein complex, we speculate that the observed oligomers of protein p6 could mimic a scaffold on which DNA folds to form the nucleoprotein complex in ViVo.The genomes of prokaryotic organisms are organized in higher order nucleoprotein complexes that, besides a packaging role, mediate fundamental processes of DNA such as replication, transcription, recombination, and transposition (Echols, 1990). Some of these complexes are assembled by architectural elements that by analogy to eukaryotic systems are referred to as histone-like proteins. In Escherichia coli, the major constituents of bacterial nucleoid are the HU heterodimer [reviewed in Drlica and Rouviere-Yaniv (1987)] and H-NS [reviewed in Ussery et al. (1994) and Atlung and Ingmer (1997)]. These proteins are small, very abundant, and bind DNA with little or no sequence specificity, usually through the minor groove. The pleiotropic nature of mutations in their corresponding genes indicates that these proteins have multiple functions. HU is reported to be involved in replication, transposition, and transcriptional control, while H-NS has been suggested to be a modulator of regulated gene expression.Protein p6 from Bacillus subtilis phage Ø29 has some features resembling those expected for a histone-like protein.Protein p6 is the most abundant protein in Ø29-infected cells and binds in Vitro to the viral DNA forming multiple complexes spread virtually throughout the entire genome, of sizes ranging from ∼100 base pairs (bp) up to ∼2 kilobases (kb) (Gutiérrez et al., 1994). Protein p6 binds to DNA through the minor groove, and it does not recognize a specific sequence, but rather a DNA structural f...

Protein p6 of Bacillus subtilis phage Ø29 is involved in the initiation of viral DNA replication and transcription by forming a multimeric nucleoprotein complex with the phage DNA. Based on this, together with its abundance and its capacity to bind to the whole viral genome, it has been proposed to be a viral histone-like protein. Protein p6 is in a monomer-dimer-oligomer equilibrium association. We have identified protein p6 mutants deficient in self-association by testing random mutants obtained by degenerated polymerase chain reaction in an in vivo assay for dimer formation. The mutations were mainly clustered in two regions located at the N terminus, and the central part of the protein.Site-directed single mutants, corresponding to those found in vivo, have been constructed and purified. Mutant p6A44V, located at the central part of the protein, showed an impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of Ø29 DNA replication. Mutant p6I8T has at least 10-fold reduced self-association capacity, does not bind DNA nor activate Ø29 DNA initiation of replication. Cterminal deletion mutants showed an enhanced dimer formation capacity. The highly acidic tail, removed in these mutants, is proposed to modulate the protein p6 self-association.

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