P1 ParB Domain Structure Includes Two Independent Multimerization Domains

Tryptic digestion of ParB and His-ParB. ParB (lanes P) and His-ParB (lanes H) were treated with trypsin at a protein/protease ratio of 1,000:1 (wt/wt) at 20°C for the indicated times. Digestion was stopped with 1% acetic acid. Proteolytic fragments were separated by electrophoresis in SDS–15% polyacrylamide gels and were visualized with Coomassie blue. Undigested ParB and His-ParB migrate with 44- and 50-kDa proteins, respectively. The arrows at the right indicate the major tryptic fragments identified in Table 3. Lane M, size markers.

Chymotrypsin and trypsin digestion of His-ParB. (Left) Five micrograms of protein was incubated with increasing amounts of protease for 2 h at room temperature. Protein-to-protease ratios (wt/wt) were 100:1 (lanes a and i), 500:1 (lanes b and h), 1,000:1 (lanes c and g), and 5,000:1 (lanes d and f). Lane e, no protease. Arrows indicate the fragments whose N termini were sequenced. (Right) Tryptic digest (protein/protease ratio of 1,000:1) performed at 30°C for 7.5 h, illustrating an additional band (E) that was also sequenced. The positions of size markers are indicated beside each gel. Tryp, trypsin.

Tryptic digestion of His-ParB, two C-terminal fragments (His-47-333 ParB and His-67-333 ParB), and two N-terminal fragments (His-1-293 ParB and His-1-274 ParB). For each time course, the protein-to-protease ratio was 1,000:1 (wt/wt), and the digestions were performed at room temperature. Arrows at the left indicate the major proteolytic fragments of His-ParB. M, size markers.

Examples of filter tests to determine β-galactosidase activity in the yeast two-hybrid system. The light patches were white and the grey patches were blue on the filters. Each panel (A and B) is a separate filter, but the patches within each panel are from the same filter. The particular interactions tested are indicated at the right of each filter. Various ParB and ParB fragment interactions (A and B) and ParA-ParB interactions (B) are shown. GAL4-ACT is the GAL4 activation domain alone encoded by pACTII and represents one of the negative controls for these assays.

Summary of dimerization assays with C-terminal fragments of ParB. In yeast two-hybrid analysis, the ParB fragments shown in the diagram were fused to the GAL4 activation domain and were tested against FL-ParB, 30-333 ParB, and 275-333 ParB (in the columns) that were fused to the GAL4 DNA binding domain. For the cross-linking experiments, ParB fragments fused to a polyhistidine tag (Table 2) were purified and examined in vitro (Materials and Methods). The results from the yeast two-hybrid experiments were categorized as follows: −, no color development on filter tests or no growth on plates without histidine; +, moderate color development and moderate growth in the absence of histidine; ++, dark blue color and good growth in the absence of histidine. ND, not determined. The DSP cross-linking results were similarly categorized: −, no cross-linking; +, some cross-linking activity; ++, strong cross-linking, often to completion. Neither set of categories is intended to imply relative strengths of the interactions, which are presumably dependent on the assay. The N termini of the tryptic proteolytic fragments are indicated above the schematic of ParB.

Cross-linking of His-ParB and His-ParB fragments with DSP. Ten micrograms of protein was incubated with DSP at room temperature for the indicated times and analyzed by electrophoresis as described in Materials and Methods. Arrows indicate the positions of monomers and the brackets indicate the positions of cross-linked products (XL). (A) Cross-linking of His-ParB compared with that of His-47-333 ParB on an SDS–10% gel. His-ParB cross-links efficiently to a dimer-size and possibly a tetramer-size smear. Lane M, size markers. (B) Cross-linking of two smaller C-terminal fragments in the presence of DSP. His-87-333 ParB and His-275-333 were treated with DSP and analyzed on a 10% polyacrylamide gel (left) and a 12% polyacrylamide gel (right), respectively. (C) Cross-linking reactions of two N-terminal fragments of ParB, His-1-293 ParB, and His-1-177 ParB, in a 12% polyacrylamide gel.

Summary of dimerization assays with N-terminal fragments of ParB. In yeast two-hybrid analysis, the ParB fragments shown in the diagram were fused to the GAL4 activation domain and were tested against FL-ParB and 30-333 ParB (in the columns) fused to the GAL4 DNA binding domain. For the cross-linking experiments, polyhistidine-tagged protein fusions were purified and tested in vitro. The categories are described in the legend to Fig. 6. ND, not determined.