Protein diversity confers specificity in plasmid segregation

Abstract

The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.

FIG. 1.

Organization of the TP228, pVS1, pTAR, pVT745, and pB171 partition systems. (A) Genetic structures of the partition cassettes. Each cassette includes a parF homolog (open arrow) and a flanking gene downstream (18). The parG and parG_VS1 genes are homologs (14, 18). In contrast, the parB_TAR, aa15, and parB_B171 genes are unrelated to parG or to each other (18). Each cassette is flanked by repeat sequences (arrowheads) that differ in number, location, and sequence between each plasmid (4, 8, 12). (B) The consensus sequences of the repeat sequences in TP228 (4), pVS1, pTAR (12), and pB171 (8) are shown. The pVT745 region contains an AT-rich (∼80%) region that includes a number of repeat sequences.

FIG. 2.

Bacterial two-hybrid analysis of protein interactions among ParG analogs in the TP228, pVS1, pTAR, pVT745, and pB171 partition systems. Plasmids carrying fusions of parG analogs to T25- and T18-encoding fragments were cotransformed into E. coli SP850. Transformants were streaked onto MacConkey-maltose plates and incubated at 30°C for 36 h. The strains in the plate sectors contain the following plasmids: A, pT18-ParG plus pT25-ParG (1), pT25-ParB_B171 (2), pT25-ParB_TAR (3), pT25-AA15 (4), or pT25-ParG_VS1 (5); B, pT18-ParG_VS1 plus pT25-ParG_VS1 (1), pT25-ParB_B171 (2), pT25-ParB_TAR (3), pT25-AA15 (4), or pT25-ParG (5); C, pT18-ParB_TAR plus pT25-ParB_TAR (1), pT25-ParB_B171 (2), pT25-AA15 (3), pT25-ParG_VS1 (4), or pT25-ParG (5); D, pT18-AA15 plus pT25-AA15 (1), pT25-ParB_B171 (2), pT25-ParB_TAR (3), pT25-ParG_VS1 (4), or pT25-ParG (5); E, pT18-ParB_B171 plus pT25-ParB_B171 (1), pT25-ParB_TAR (2), pT25-AA15 (3), pT25-ParG_VS1 (4), or pT25-ParG (5).

FIG. 3.

Analysis of the solution oligomeric state of the ParG_VS1, ParB_TAR, AA15, and ParB_B171 proteins. The left panels show time course reactions of cross-linking with DMP for each protein. The positions of monomeric (1^m) and dimeric (2^m) species are indicated on the left; molecular mass markers (kDa) are shown on the right. The right panels show sedimentation velocity profiles of the four proteins. Proteins were subjected to ultracentrifugation at a speed of 60,000 rpm (Optima XL-1 ultracentrifuge; Beckman) and a temperature of 23°C. The data were analyzed as described in Materials and Methods. In each case, a predominant species at the dimeric position is evident.

FIG. 4.

Circular dichroism spectroscopy of the ParG_VS1, ParB_TAR, AA15, and ParB_B171 proteins. (A) Superimposed far-UV spectra of the four proteins. (B) The deconvoluted data in panel A were used to make estimations of the secondary structure content of each protein with the CONTIN prediction program. Analysis with the CDSSTR program yielded comparable results (data not shown).

FIG. 5.

Protein-DNA interactions of the ParG_VS1, ParB_TAR, AA15, and ParB_B171 proteins. The indicated proteins were used in binding reactions with the biotinylated DNA sequences (2 nM) located upstream of their cognate parF genes and analyzed as described in Materials and Methods. These sequences include the repeat motifs shown in Fig. 1. The micromolar protein concentrations used were as follows. ParG lanes: 1, 0.0; 2, 0.05; 3, 0.10; 4, 0.15; 5, 0.26; 6, 0.52. ParG_VS1 lanes: 1, 0.0; 2, 2.5; 3, 5.0; 4, 6.0; 5, 7.0; 6, 8.0; 7, 9.0; 8, 10.0. ParB_TAR lanes: 1, 0.0; 2, 0.2; 3, 0.3; 4, 0.4; 5, 0.5; 6, 0.6; 7, 0.7; 8, 0.8; 9, 0.9; 10, 1.0. AA15 lanes: 1, 0.0; 2, 2.0; 3, 3.0; 4, 4.0; 5, 5.0; 6, 6.0; 7, 7.0; 8, 8.0; 9, 9.0; 10, 10.0. ParB_B171 lanes: 1, 0.0; 2, 0.4; 3, 0.6; 4, 0.8; 5, 1.0; 6, 1.2; 7, 1.4; 8, 1.6; 9, 1.8; 10, 2.0. The interaction of ParG and ParB_TAR with their cognate target sites has been demonstrated previously (4, 23), although the ratio of ParB_TAR to DNA at which nucleoprotein complex formation was observed is slightly different from that previously described (23).

FIG. 6.

DNA interaction specificity of the ParG, ParG_VS1, ParB_TAR, AA15, and ParB_B171 proteins. The indicated proteins were used in binding reactions with the biotinylated DNA sequences (2 nM) located upstream of the cognate parF genes and analyzed as described in Materials and Methods. The substrate DNAs illustrated beneath each panel were derived from TP228 (A), pVS1 (B), pTAR (C), pVT745 (D), and pB171 (E). In each panel, the proteins were used at the following concentrations, which are sufficient to convert the free substrate into fully bound complex by the cognate DNA-binding protein: A, 0.5 μM; B, 20 μM; C, 1.0 μM; D, 20 μM; E, 1.0 μM. −, no protein.