Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1parFamily of Plasmid Partition Elements

Abstract

The P1, P7, and pMT1 par systems are members of the P1 par family of plasmid partition elements. Each has a ParA ATPase and a ParB protein that recognizes the parS partition site of its own plasmid type to promote the active segregation of the plasmid DNA to daughter cells. ParB contacts two parS motifs known as BoxA and BoxB, the latter of which determines species specificity. We found that the substitution of a single orthologous amino acid in ParB for that of a different species has major effects on the specificity of recognition. A single change in ParB can cause a complete switch in recognition specificity to that of another species or can abolish specificity. Specificity changes do not necessarily correlate with changes in the gross DNA binding properties of the protein. Molecular modeling suggests that species specificity is determined by the capacity to form a hydrogen bond between ParB residue 288 and the second base in the BoxB sequence. As changes in just one ParB residue and one BoxB base can alter species specificity, plasmids may use such simple changes to evolve new species rapidly.The P1 plasmid partition system involves interactions between three proteins (P1 ParA and ParB and the host integration host factor [IHF]) and a ca. 80-bp DNA site (P1 parS), which results in the physical movement of the plasmid DNA molecules in a coordinated fashion (13). Similar elements are found in a variety of plasmid types from various bacterial species (9). Although very similar, the P1 par, pMT1 par, and P7 par members each show unique partition specificities: the Par proteins of one species fail to function with the parS sites of the other two. These different par species are compatible with each other; they do not promote the incompatibility seen between pairs of plasmids that carry identical par elements (4).The Par proteins of the P1 plasmid will not support P7 plasmid partition and vice versa. A key interaction in determining this species specificity proved to be an interaction between the ParB protein and the two BoxB repeat sequences that form the boundaries of the parS site. When the sequences of these P1 parS boxes were changed to their P7 equivalents, the site switched specificity and responded only to the P7 proteins (11). This observation was extended to show that the BoxB sequence also determined species specificity in the pMT1 parS site and showed that the specificity of a parS site could be changed to that of another species by as little as a single BoxB base change (4). Domain-swapping experiments showed that the critical protein sequence for specificity, a 16-amino-acid stretch termed the discriminator recognition sequence (DRS), is located near the carboxyl terminus of the ParB protein (19). This forms a secondary contact between ParB and the parS site, the primary contact involving a helix-turn-helix motif in the central region of ParB and the BoxA sequences in parS (22). The primary interactions are essentially the same in each of the plasmid species.

One evolutionarily selected amino acid variation is sufficient to provide functional specificity in the cold shock protein paralogs of Staphylococcus aureus

“…In agreement with our results, it has also been shown that single amino acid orthologous substitutions provide functional differences among DNA‐binding proteins (Dabrazhynetskaya, Brendler, Ji, & Austin, ; Fukuto et al, ; Hudson et al, ; Treisman, Gönczy, Vashishtha, Harris, & Desplan, ). For example, a substitution of a single orthologous amino acid (Asp288) in the ParB protein that recognizes parS to promote segregation of the plasmid DNA to daughter cells, is sufficient to change or abolish plasmid specificity (Dabrazhynetskaya et al, ). Amino acid variations in position 215 of PhoP cause changes in its activity and have probably contributed to an increased virulence in Y. pestis (Fukuto et al, ).…”

Evidence for Two Different Regulatory Mechanisms Linking Replication and Segregation of Vibrio cholerae Chromosome II

“…ParB proteins of plasmids are known to be plasmid-specific and to bind to their cognate sites [47]. This helps to avoid segregation-mediated incompatibility if different plasmids happen to be present in the same host.…”

A Model for the Evolution of Biological Specificity: a Cross-Reacting DNA-Binding Protein Causes Plasmid Incompatibility

“…Nevertheless, the ability of a mutation that prevents DNA binding to block the repressive activity of ParR pB171 with respect to both parC PB171 and parC pCP301 demonstrates that DNA binding is required for the biological activity of ParR. Interestingly, the discriminator contacts that define the segregation specificity of plasmids encoding the type I Par operon, such as PI, have been mapped to an interaction between two hexameric sequences in the parS DNA and a 16-amino-acid stretch in the C terminus of the ParB protein (13,14,(31)(32)(33). For the P1 plasmid, both these regions are dispensable for parS and ParB binding but are essential for mediating P1 segregation specificity (13).…”