FtsK is critical for the assembly of the unique divisome complex of the FtsZ-less Chlamydia trachomatis: blots

# FtsK is critical for the assembly of the unique divisome complex of the FtsZ-less *Chlamydia trachomatis*: blots

[https://doi.org/10.5061/dryad.1ns1rn94w](https://doi.org/10.5061/dryad.1ns1rn94w)

## Description of the data and file structure

**Figure 1, figure supplement 1A-Source data 1**: Unlabeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE). Lysates were prepared from uninfected HeLa cells and HeLa cells infected with *Ct* L2. At 21 hpi, lysates were prepared and characterized by immunoblotting with FtsK-specific antibodies. 

**Figure 1, figure supplement 1A-Source data 2**: Labeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE). Lysates were prepared from uninfected HeLa cells and HeLa cells infected with *Ct* L2. At 21 hpi, lysates were prepared and characterized by immunoblotting with FtsK-specific antibodies.

**Figure 1, figure supplement 1B-Source data 3**: Unlabeled, ..., Chlamydia trachomatis serovar L2 (Ct), an obligate intracellular bacterium that does not encode FtsZ, divides by a polarized budding process. In the absence of FtsZ, we show that FtsK, a chromosomal translocase, is critical for divisome assembly in Ct. Chlamydial FtsK forms discrete foci at the septum and at the base of the progenitor mother cell, and our data indicate that FtsK foci at the base of the mother cell mark the location of nascent divisome complexes that form at the site where a daughter cell will emerge in the next round of division. The divisome in Ct has a hybrid composition, containing elements of the divisome and elongasome from other bacteria, and FtsK is recruited to nascent divisomes prior to the other chlamydial divisome proteins assayed, including the PBP2 and PBP3 transpeptidases, and MreB and MreC. Knocking down FtsK prevents divisome assembly in Ct and inhibits cell division and septal peptidoglycan synthesis. We further show that M..., Cell Culture
HeLa cells (ATCC, Manassas, VA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Waltham, MA) containing 10% fetal bovine serum (FBS, Hyclone, Logan, UT) at 37°C in a humidified chamber with 5% CO2. HeLa cells were infected with Ct serovar L2 434/Bu in the same media. Infections of HeLa cells with chlamydial transformants were performed in DMEM containing 10% FBS and 0.36 U/mL penicillin G (Sigma-Aldrich).
Cloning
The plasmids and primers used for generating mCherry fusions of FtsK, PBP2, PBP3, and MreC are listed in Supp. Table S1. The chlamydial ftsK, pbp2, pbp3, and mreC genes were amplified by PCR with Phusion DNA polymerase (NEB, Ipswich, MA) using 10 ng C. trachomatis serovar L2 genomic DNA as a template. The PCR products were purified using a PCR purification kit (Qiagen) and inserted into the pBOMB4-Tet (-GFP) plasmid, which confers resistance to β-lactam antibiotics. The plasmid was cut at the No..., ,