Recruitment of the ParG segregation protein to different affinity DNA sites

Abstract

The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. In addition to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228 is a transcriptional repressor of the parFG partition genes. ParG is a homodimeric DNA binding protein, with C-terminal regions that interlock into a ribbon-helix-helix fold. Antiparallel beta-strands in this fold are presumed to insert into the O(F) operator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The O(F) locus comprises eight degenerate tetramer boxes arranged in a combination of direct and inverted orientation. Each tetramer motif likely recruits one ParG dimer, implying that the fully bound operator is cooperatively coated by up to eight dimers. O(F) was subdivided experimentally into four overlapping 20-bp sites (A to D), each of which comprises two tetramer boxes separated by AT-rich spacers. Extensive interaction studies demonstrated that sites A to D individually are bound with different affinities by ParG (C > A approximately B >> D). Moreover, comprehensive scanning mutagenesis revealed the contribution of each position in the site core and flanking sequences to ParG binding. Natural variations in the tetramer box motifs and in the interbox spacers, as well as in flanking sequences, each influence ParG binding. The O(F) operator apparently has evolved with sites that bind ParG dissimilarly to produce a nucleoprotein complex fine-tuned for optimal interaction with the transcription machinery. The association of other ribbon-helix-helix proteins with complex recognition sites similarly may be modulated by natural sequence variations between subsites.

FIG. 1.

DNase I footprinting of the O_F locus. (A) Footprinting reactions were performed as outlined in Materials and Methods using PCR fragments biotinylated at the 5′ ends of either top or bottom strands. ParG concentrations (μM monomer, left to right): 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0. The locations of sites A to D are marked by open arrows. Tetramer boxes within the sites are indicated by filled arrows. Shaded boxes denote the regions protected from DNase I digestion by ParG. The adjoining hatched box indicates the segment of site D that is only partially protected from DNase I digestion by ParG. The black line denotes the position of the parF translation start codon. A+G, Maxam-Gilbert sequencing reactions. The relative dispositions on the top and bottom strands of the parFG promoter-operator region that are protected from DNase I digestion are illustrated in the bottom panel. Putative −10 and −35 promoter motifs are boxed. (B) Alignment (5′ to 3′) of the top strands of sites A and C and the bottom strands of sites B and D. The degenerate 5′-ACTC-3′ tetramer motifs are boxed. The inverted motif in site C is shaded.

FIG. 2.

Repression analysis of the wild-type parFG promoter-operator region (ABCD) and the same region bearing disruptions of one of the sites within the O_F operator (XBCD, AXCD, ABXD, and ABCX) assessed by CDO activity of parF-xylE transcriptional fusions. The top panel shows CDO activities relative to the levels of the unrepressed wild-type region for each fusion in the absence (filled bars) and presence (open bars) of ParG provided in trans. The inset shows a schematic representation of the parF-xylE constructs. The bottom panel illustrates the repression by ParG of each transcriptional fusion relative to its unrepressed levels.

FIG. 3.

EMSA of the full-length operator O_F (ABCD) and variants in which one site was replaced by a random sequence (designated X and marked by a gray horizontal box). Biotinylated oligonucleotides (98 bp) (see Table S1 in the supplemental material) were tested as described in Materials and Methods using ParG concentrations (μM monomer, left to right): 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6. Unbound DNAs and nucleoprotein complexes are indicated by open arrows and gray vertical bars, respectively. A second species that migrates close to the unbound DNA in some blots in this and other figures is likely to be the same fragment with an atypical secondary structure.

FIG. 4.

EMSA of isolated O_F sites. Biotinylated oligonucleotides (50 bp) (see Table S1 in the supplemental material) were tested as described in Materials and Methods using ParG concentrations (μM monomer, left to right): 0, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0, except for site D from which the 0.75 μM concentration was omitted The oligonucleotides include the 12-bp site cores flanked on either side by 4 bp from the site's normal context (Fig. 1). Unbound DNAs and nucleoprotein complexes are indicated by open and filled arrows, respectively.

FIG. 5.

DNase I footprinting of isolated O_F sites. Footprinting reactions were performed as outlined in Materials and Methods using PCR fragments biotinylated at the 5′ ends of the bottom strands. ParG concentrations (μM monomer, left to right): 0.0, 0.2, 0.3, 0.4, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.5, 4.0, and 5.0. The locations of sites A to D are marked by arrows. Positions that are hypersensitive to DNase I cleavage in the presence of ParG are highlighted by the stars.

FIG. 6.

SPR analysis of isolated O_F sites. Biotinylated oligonucleotides (50 bp) (see Table S1 in the supplemental material) were tested as described in Materials and Methods. The oligonucleotides include the 12-bp site cores flanked on either side by 4 bp from the site's normal context (Fig. 1). Representative sensorgrams of ParG binding (100 nM monomer) to the sites are shown. The bottom panel shows an alignment of sites A and C and variant sites. The degenerate 5′-ACTC-3′ motifs are boxed. Nucleotide differences between wild-type and variant sites are highlighted with dots.

FIG. 7.

Mutational analysis of regions flanking the 12-bp core in site B. The sequence of the bottom strand of site B with 4 bp of flanking nucleotides on either side is shown at the top. The 5′-ACTC-3′ motifs are boxed. Mutated sites are illustrated underneath with mutated positions shaded. Oligonucleotides (50 bp) bearing these sites were tested in EMSA as described in Materials and Methods using ParG concentrations (μM monomer, left to right): 0, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0. Unbound DNAs and nucleoprotein complexes are indicated by open and filled arrows, respectively. The ParG titration shown for site B is the same as that shown in Fig. 4.

FIG. 8.

Scanning mutagenesis of the A site flanked either by randomized sequences (A) or by site B (B). Randomized nucleotides are underlined. Purine-to-pyrimidine and pyrimidine-to-purine substitution mutations at each position within the 12-bp site A, as well as at two flanking base pairs, were studied by EMSA using 48-bp oligonucleotides in both contexts. ParG concentrations up to 8 and 2 μM were used in panels A and B, respectively. Data quantitation using Gel-Pro Analyzer 3.1 (Media Cybernetics) software is shown. The results shown are representative of experiments performed at least in duplicate. WT, wild type.

FIG. 9.

Symmetrical scanning mutagenesis of the AB sites. Complementary purine-to-pyrimidine and pyrimidine-to-purine substitution mutations at each position within the two sites, as well as at flanking base pairs, were studied by EMSA using 48-bp oligonucleotides. ParG concentrations up to 2 μM were used. Data quantitation using Gel-Pro Analyzer 3.1 (Media Cybernetics) software is shown. The results shown are representative of experiments performed at least in duplicate. WT, wild type.