Z-Ring-Associated Proteins Regulate Clustering of the Replication Terminus-Binding Protein ZapT in Caulobacter crescentus

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Bacterial strains and DNA. The strains, plasmids, and primers used in this study are listed in Tables 1,

2, and 3, respectively. Caulobacter strains were grown at 30°C in peptone-yeast extract (PYE) supplemented

with appropriate antibiotics, as described previously (60, 61). When necessary, cumate (1 m M) or vanillate

(1 mM) was added to the culture medium as indicated. Detailed procedures for construction of the strains

and plasmids are described in the supplemental material (Text S1).

Recombinant proteins. Recombinant proteins were expressed in E. coli and purified as described in

the supplemental material (Text S1).

Size exclusion chromatography. The size exclusion chromatography assay was performed essentially as described previously (57, 62). Briefly, proteins were loaded onto a Superdex 200 PC3.2/30 column (2.4-ml column volume) equilibrated with SEC buffer (25 mM Tris-HCl [pH 7.5], 300 mM sodium

chloride, and 20% sucrose) and fractionated at a flow rate of 20 m l/min, followed by SDS–15% PAGE and

Coomassie brilliant blue staining. When a Superdex 75 HR 10/30 column was used, proteins were separated at a flow rate of 0.2 ml/min.

ChIP sequencing and ChIP-qPCR. ChIP was performed as described previously (24). Briefly, exponentially growing cells (200 ml in PYE) were fixed for 10 min using 1% formaldehyde solution, washed

thoroughly, resuspended in buffer, and lysed through two passages in a French press. After DNA shearing with sonication, the cell debris was removed by ultracentrifugation, and the cleared cell lysate was

incubated with anti-FLAG M2 magnetic beads (Wako or Sigma). After the beads were washed, the bound

materials were incubated at 65°C overnight to reverse cross-linking. The resultant DNA samples were

purified using a DNA cleanup kit (Zymo Research). When SHQ247 (3F-ZauP) cells (100 ml in PYE) were

analyzed, cross-linking was carried out for 5 min in 3.6% formaldehyde instead of 1% formaldehyde.

For deep sequencing, samples were indexed using a NEBNext Ultrall DNA library prep kit and analyzed on an Illumina HiSeq 2500 instrument (single-end).

For qPCR, samples were analyzed by a standard percent input method using TB green premix

ExTaqII and Thermal Cycler Dice TP800 (TaKaRa). Locus-specific primers are listed in Table 3.

Microscopy. Differential interference contrast (DIC), phase-contrast, and fluorescence microscopy

analyses were performed using a Nikon Eclipse 80i microscope equipped with an X-Cite TURBO multiwavelength LED illumination system and an Andor Zyla 4.2 sCMOS camera, as described previously (24).

Quantitative image analyses were performed using the Oufti and MicrobeJ software packages (63, 64).

Western blotting. The Western blot assay was performed as described previously (24). The antimNeonGreen and anti-Flag antibodies were purchased from Chromotek and Thermo, respectively.

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