DOI: 10.5061/dryad.1ns1rn94w
title: FtsK is critical for the assembly of the unique divisome complex of the FtsZ-less Chlamydia trachomatis: blots

description: # 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, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE). HeLa cells were infected with *Ct* transformed with FtsK-mCherry (molecular mass - 114,664 Da). The fusion was induced with 10nM aTc at 17 hpi. HeLa cells were harvested at 21 hpi, and a lysate was prepared and characterized by immunoblotting analysis with a rabbit polyclonal mCherry antibody. **Figure 1, figure supplement 1B-Source data 4**: Labeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE). HeLa cells were infected with *Ct* transformed with FtsK-mCherry (molecular mass - 114,664 Da). The fusion was induced with 10nM aTc at 17 hpi. HeLa cells were harvested at 21 hpi, and a lysate was prepared and characterized by immunoblotting analysis with a rabbit polyclonal mCherry antibody. **Figure 2, figure supplement 1A-Source data 1**: Unlabeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE).  HeLa cells were infected with *Ct* transformed with mCherry-PBP2 (molecular mass - 150,842 Da), or mCherry-PBP3 (molecular mass - 100,150 Da). The fusions were induced with 10nM aTc at 17 hpi. HeLa cells were harvested at 21 hpi, and lysates were prepared and characterized by immunoblotting analysis with a rabbit polyclonal mCherry antibody. **Figure 2, figure supplement 1A-Source data 2**: Labeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE).  HeLa cells were infected with *Ct* transformed with mCherry-PBP2 (molecular mass - 150,842 Da), or mCherry-PBP3 (molecular mass - 100,150 Da). The fusions were induced with 10nM aTc at 17 hpi. HeLa cells were harvested at 21 hpi, and lysates were prepared and characterized by immunoblotting analysis with a rabbit polyclonal mCherry antibody. **Figure 2, figure supplement 1A-Source data 3**: Unlabeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE).  HeLa cells were infected with *Ct* transformed with mCherry-MreC (molecular mass - 63,906 Da). The fusion was induced with 10nM aTc at 17 hpi. HeLa cells were harvested at 21 hpi, and lysates were prepared and characterized by immunoblotting analysis with a rabbit polyclonal mCherry antibody. **Figure 2, figure supplement 1A-Source data 4**: Labeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE).  HeLa cells were infected with *Ct* transformed with mCherry-MreC (molecular mass - 63,906 Da). The fusion was induced with 10nM aTc at 17 hpi. HeLa cells were harvested at 21 hpi, and lysates were prepared and characterized by immunoblotting analysis with a rabbit polyclonal mCherry antibody. **Figure 2, figure supplement 2A-Source data 1**: Unlabeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE).  HeLa cells were infected with *Ct* transformed with mCherry-PBP2. The fusion was induced (+aTc) at 17 hpi or was uninduced. The induced and uninduced cells were harvested at 21 hpi, and lysates were prepared and characterized by immunoblotting analysis with rabbit antibodies raised against peptides derived from chlamydial PBP2. The PBP2 antibody primarily detects a species of ~120kD in the induced sample.  **Figure 2, figure supplement 2A-Source data 2**: Labeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE).  HeLa cells were infected with *Ct* transformed with mCherry-PBP2. The fusion was induced (+aTc) at 17 hpi or was uninduced. The induced and uninduced cells were harvested at 21 hpi, and lysates were prepared and characterized by immunoblotting analysis with rabbit antibodies raised against peptides derived from chlamydial PBP2. The PBP2 antibody primarily detects a species of ~120kD in the induced sample. **Figure 2, figure supplement 2A-Source data 3**: Unlabeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE).  HeLa cells were infected with *Ct* transformed with mCherry-PBP3. The fusion was induced (+aTc) at 17 hpi or was uninduced. The induced and uninduced cells were harvested at 21 hpi, and lysates were prepared and characterized by immunoblotting analysis with rabbit antibodies raised against peptides derived from chlamydial PBP3. The PBP3 antibody primarily detects a single species with the predicted molecular mass of mCherry-PBP3 in the induced sample. **Figure 2, figure supplement 2A-Source data 4**: Labeled, uncropped Western Blot .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE).  HeLa cells were infected with Ct transformed with mCherry-PBP3. The fusion was induced (+aTc) at 17 hpi or was uninduced. The induced and uninduced cells were harvested at 21 hpi, and lysates were prepared and characterized by immunoblotting analysis with rabbit antibodies raised against peptides derived from chlamydial PBP3. The PBP3 antibody primarily detects a single species with the predicted molecular mass of mCherry-PBP3 in the induced sample. ## Code/software .zip file obtained from LICOR Odyssey imaging system (LICOR, Lincoln, NE)
description: 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 MreB does not function like FtsZ and serves as a scaffold for the
 assembly of the Ct divisome. Rather, MreB is one of the last
 proteins recruited to the chlamydial divisome, and it is necessary for the
 formation of septal peptidoglycan rings. Our studies illustrate the
 critical role of chlamydial FtsK in coordinating divisome assembly and
 peptidoglycan synthesis in this obligate intracellular bacterial pathogen.
description: 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 <em>Ct</em> 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 <em>ftsK</em>, <em>pbp2, pbp3</em>, and <em>mreC</em> genes were amplified by PCR with Phusion DNA polymerase (NEB, Ipswich, MA) using 10 ng <em>C</em>. <em>trachomatis</em> 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 NotI (FtsK-mCherry) or the KpnI (mCherry-PBP2, mCherry-PBP3, mCherry-MreC) site, and the chlamydial genes were inserted into the cut plasmid using the HiFi DNA Assembly kit (NEB) according to the manufacturer’s instructions. The products of the HiFi reaction were transformed into NEB-5αI<sup>q</sup> competent cells (NEB), and transformants were selected by growth on plates containing ampicillin. DNA from individual colonies was isolated using a mini-prep DNA isolation kit (Qiagen), and plasmids were initially characterized by restriction digestion to verify that the inserts were the correct size. Clones containing inserts of the correct size were DNA sequenced prior to use. DNA and RNA purification and RT-qPCR Total nucleic acids were extracted from HeLa cells infected with <em>Ct</em> plated in 6-well dishes as described previously (Ouellette 2015, Ouellette, Blay et al. 2021). For RNA isolation, cells were rinsed once with PBS, then lysed with 1 mL TRIzol (Invitrogen) per well. Total RNA was extracted from the aqueous layer after mixing with 200 μL per sample of chloroform following the manufacturer’s instructions. Total RNA was precipitated with isopropanol and treated with DNase (Ambion) according to the manufacturer’s guidelines prior to cDNA synthesis using SuperScript III (Invitrogen). For DNA, infected cells were rinsed once with PBS, trypsinized, and pelleted before resuspending each pellet in 500 μL of PBS. Each sample was split in half, and genomic DNA was isolated from each duplicate sample using the DNeasy extraction kit (Qiagen) according to the manufacturer’s guidelines. Quantitative PCR was used to measure <em>C</em>. <em>trachomatis</em> genomic DNA (gDNA) levels using an <em>euo</em> primer set. 150ng of each sample was used in 25μL reactions using standard amplification cycles on a QuantStudio3 thermal cycler (Applied Biosystems) followed by a melting curve analysis. <em>ftsK, pbp2, euo</em>, and <em>omcB</em> transcript levels were determined by RT-qPCR using SYBR Green as described previously (Ouellette SP 2021) (see Supp. Table S2 for primers used for measuring gDNA levels and RT-qPCR). Transcript levels were normalized to genomes and expressed as ng cDNA/gDNA. Transformation of <em>Ct</em> <em>Ct</em> was transformed as described previously (Wang 2011). Briefly, HeLa cells were plated in a 10cm plate at a density of 5 × 10<sup>6</sup> cells the day before beginning the transformation procedure. <em>Ct</em> lacking its endogenous plasmid (-pL2) was incubated with 10μg of plasmid DNA in Tris-CaCl<sub>2</sub> buffer (10 mM Tris-Cl pH 7.5, 50 mM CaCl<sub>2</sub>) for 30 min at room temperature. HeLa cells were trypsinized, washed with 8 mL of 1x DPBS (Gibco), and pelleted. The pellet was resuspended in 300μL of the Tris-CaCl<sub>2</sub> buffer. <em>Ct</em> was mixed with the HeLa cells and incubated at room temperature for an additional 20 min. The mixture was added to 10 mL of DMEM containing 10% FBS and 10 μg/mL gentamicin and transferred to a 10cm plate. At 48 hpi, the HeLa cells were harvested, and <em>Ct</em> in the population was used to infect a new HeLa cell monolayer in media containing 0.36 U/ml of penicillin G to select for transformants. The plate was incubated at 37°C for 48 hours. These harvest and re-infection steps were repeated every 48 hours until inclusions were observed. Immunofluorescence Microscopy HeLa cells were seeded in 10cm plates at a density of 5 × 10<sup>6</sup> cells per well the day before the infection. <em>Ct</em> L2 or chlamydial strains transformed with plasmids encoding FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, or mCherry-MreC or with plasmids that direct the constitutive expression of the crRNAs targeting the <em>pbp2</em> or <em>ftsK</em> promoters were used to infect HeLa cells in DMEM. For experiments with the transformants, aTc was added to the media of infected cells at the indicated concentration and time. At 21 hpi, cells were detached from the 10cm plate by scraping and pelleted by centrifugation for 30 seconds. The pellet was resuspended in 1 mL of 0.1x PBS (Gibco) and transferred to a 2 mL tube containing 0.5mm glass beads (ThermoFisher Scientific). Cells were vortexed for 3 minutes, then centrifuged at 800 rpm for 2 minutes. in a microfuge. 20μLs of the supernatant was mixed with 20μLs of 2x fixing solution (6.4% formaldehyde and 0.044% glutaraldehyde) and incubated on a glass slide for 10 min at room temperature. Cells were washed 3 times with PBS, and the cells were permeabilized by incubation with PBS containing 0.1% Triton X-100 for 1 min. Cells were washed with PBS twice. For experiments with <em>Ct</em> L2, the cells were incubated with a goat primary antibody against the major outer-membrane protein (MOMP; Meridian, Memphis, TN), and the mouse primary antibody that recognizes endogenous FtsK raised against recombinant CT739 protein (https://doi.org/10.1099/mic.0.047746-0), or with rabbit antibodies raised against peptides derived from PBP 2 or PBP3 (Ouellette SP 2012). Briefly, chlamydial antigens or peptides emulsified with Freund’s incomplete adjuvant were used to immunize animals via intramuscular injections three times with an interval of 2 weeks. Antisera were collected from the immunized animals 2 to 4 weeks after the final immunization as the primary antibodies. After the primary antibody labeling, the cells were then rinsed with PBS and incubated with donkey anti-goat IgG (Alexa 488) and donkey anti-mouse IgG (Alexa 594) or donkey-anti-rabbit IgG (Alexa 594) secondary antibodies (Invitrogen). Experiments in which we visualized the distribution of the various mCherry fusions, the localization of the mCherry fluorescence was compared to the distribution of MOMP. In some experiments, we determined the distribution of the MreB_6x His fusion by staining cells expressing the fusion with a rabbit anti-6x His antibody (Abcam, Cambridge, MA) and the goat anti-MOMP antibody, followed by the appropriate secondary antibodies. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. During image acquisition, 0.3μm xy-slices were collected that extended above and below the cell. The images were collected such that the brightest spot in the image was saturated. The images were deconvolved using the nearest neighbor algorithm in the Zeiss Axiovision 4.7 software. Deconvolved images were viewed and assembled using Zeiss Zen-Blue software. For each experiment, three independent replicates were performed, and the values shown for localization are the average of the 3 experiments. In some instances, 3D projections of the acquired xy slices were generated using the Zeiss Zen-Blue software. Peptidoglycan (PG) labeling PG was labeled by incubating cells with 4mM ethylene-D-alanine-D-alanine (E-DA-DA) as described (Cox 2020). The incorporated E-DA-DA was fluorescently labeled using the Click & Go<sup>™</sup> labeling kit (Vector Laboratories). The distribution of fluorescently labeled PG was compared to the distribution of MOMP and endogenous FtsK or the distribution of mCherry-PBP3. Three independent replicates were performed, and the values shown are the average of the 3 experiments. Inclusion forming unit assay HeLa cells were infected with <em>Ct</em> (-pL2) transformed with the pBOMB4 Tet (-GFP) plasmid encoding the indicated aTc-inducible gene. At 8hpi, aTc was added to the culture media at the indicated concentration. Control cells were not induced. At 48hpi, the HeLa cells were dislodged from the culture dishes by scraping and collected by centrifugation. The pellet was resuspended in 1 mL of 0.1x PBS (Gibco) and transferred to a 2 mL tube containing 0.5mm glass bead tubes (ThermoFisher Scientific). Cells were vortexed for 3 min. followed by centrifugation at 800 rpm for 2 min. The supernatants were mixed with an equal volume of a 2x sucrose-phosphate (2SP) solution (ref) and frozen at −80°C. At the time of the secondary infection, the chlamydiae were thawed on ice and vortexed. Cell debris was pelleted by centrifugation for 5 min at 1k x g at 4°C. The EBs in the resulting supernatant were serially diluted and used to infect a monolayer of HeLa cells in a 24-well plate. The secondary infections were allowed to grow at 37°C for 24 hrs before they were fixed and labeled for immunofluorescence microscopy by incubating with a goat anti-MOMP antibody followed by a secondary donkey anti-goat antibody (Alexa Fluor 594). The cells were rinsed in PBS, and inclusions were imaged using an EVOS imaging system (Invitrogen). The number of inclusions was counted in 5 fields of view and averaged. Three independent replicates were performed, and the values from the replicates were averaged to determine the number of inclusion forming units. Chi-squared analysis was used to compare IFUs in induced and uninduced samples. Effect of A22 and mecillinam on the profile of division intermediates and on PG and divisome protein localization in <em>Ct</em> HeLa cells were infected with <em>Ct</em> transformed with the pBOMB4-Tet (-GFP) plasmid encoding FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, mCherry-MreC, or MreB-6xHis. The fusions were induced at 20hpi with 10 nM aTc for 1 hour in the absence or presence of 75 μM A22. At 22 hpi, cells were harvested and prepared for staining as described above. Three independent replicates were performed, and the values shown for localization are the average of the 3 experiments. HeLa cells were infected with <em>Ct</em> L2, and 20μM mecillinam (Sigma) was added to the media of infected cells at 17 hpi. Control cells were untreated. At 22 hpi, infected cells were harvested and RBs were prepared and stained with MOMP, FtsK, PBP2, or PBP3 antibodies as described above. Alternatively, cells were incubated with 4 mM EDA-DA at 17 hpi in the presence or absence of 20μM mecillinam. The cells were harvested at 22 hpi, and RBs were prepared, and PG was click-labeled, and its distribution was visualized in MOMP-stained cells as described above. Three independent replicates were performed, and the values shown for localization are the average of the 3 experiments. Immunoblotting HeLa cells infected with <em>Ct</em> L2 were harvested by scraping the infected cells from the plate at 24 hpi. Uninfected HeLa cells were included as a control. The HeLa cells were pelleted by centrifugation, resuspended in SDS sample buffer, and electrophoresed on a 10% SDS polyacrylamide gel. The gel was electrophoretically transferred to nitrocellulose (Schleicher and Schuell), and the filter was incubated with mouse polyclonal antibodies raised against chlamydial FtsK. The filter was rinsed and incubated with 800 donkey anti-mouse IgG secondary antibodies (LICOR, Lincoln, NE) and imaged using a LICOR Odyssey imaging system. HeLa cells were infected with <em>Ct</em> transformed with plasmids that inducibly express FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, or mCherry-MreC. The fusions were induced by the addition of 10nM aTc to the media of infected cells at 17 hpi. The cells were harvested and pelleted at 21 hpi. The cell pellet was resuspended in 1 mL of 0.1x PBS (Gibco) and transferred to a 2 mL tube containing 0.5mm glass beads (ThermoFisher Scientific). Cells were vortexed for 3 min. followed by centrifugation at 800 rpm for 2 min. The supernatant was collected and centrifuged for 3 min at 13,000 rpm, and the pellet containing <em>Ct</em> was resuspended in TBS containing 1% TX-100, 1X protease inhibitor cocktail (Sigma), and 5μM lactacystin. The suspension was sonicated 3 times on ice and centrifuged at 13,000 rpm for 3 mins. The supernatant was collected and mixed with SDS sample buffer. The samples were boiled and electrophoresed on a 10% SDS polyacrylamide gel, and the gel was electrophoretically transferred to nitrocellulose. The blots from these analyses were probed with a rabbit anti-mCherry primary antibody (Invitrogen) and an 800 donkey anti-rabbit IgG secondary antibody (LICOR, Lincoln, NE). The filters were imaged using a LICOR Odyssey imaging system. HeLa cells were infected with <em>Ct</em> transformed with the pBOMB4-Tet (-GFP) plasmid encoding mCherry-PBP2 or mCherry-PBP3. The fusions were induced with 10nM aTc at 17 hpi. Uninduced cells were included as a control. The cells were harvested at 21 hpi, and samples were processed for immunoblotting as described above. The blots were probed with rabbit polyclonal antibodies raised against peptides derived from chlamydial PBP2 or PBP3 (Ouellette SP 2012). The blots were then rinsed and incubated with 800 donkey anti-rabbit IgG secondary antibodies (LICOR, Lincoln, NE). The filters were imaged using a LICOR Odyssey imaging system. 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 <em>Ct</em> 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 <em>ftsK</em>, <em>pbp2, pbp3</em>, and <em>mreC</em> genes were amplified by PCR with Phusion DNA polymerase (NEB, Ipswich, MA) using 10 ng <em>C</em>. <em>trachomatis</em> 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 NotI (FtsK-mCherry) or the KpnI (mCherry-PBP2, mCherry-PBP3, mCherry-MreC) site, and the chlamydial genes were inserted into the cut plasmid using the HiFi DNA Assembly kit (NEB) according to the manufacturer’s instructions. The products of the HiFi reaction were transformed into NEB-5αI<sup>q</sup> competent cells (NEB), and transformants were selected by growth on plates containing ampicillin. DNA from individual colonies was isolated using a mini-prep DNA isolation kit (Qiagen), and plasmids were initially characterized by restriction digestion to verify that the inserts were the correct size. Clones containing inserts of the correct size were DNA sequenced prior to use. DNA and RNA purification and RT-qPCR Total nucleic acids were extracted from HeLa cells infected with <em>Ct</em> plated in 6-well dishes as described previously (Ouellette 2015, Ouellette, Blay et al. 2021). For RNA isolation, cells were rinsed once with PBS, then lysed with 1 mL TRIzol (Invitrogen) per well. Total RNA was extracted from the aqueous layer after mixing with 200 μL per sample of chloroform following the manufacturer’s instructions. Total RNA was precipitated with isopropanol and treated with DNase (Ambion) according to the manufacturer’s guidelines prior to cDNA synthesis using SuperScript III (Invitrogen). For DNA, infected cells were rinsed once with PBS, trypsinized, and pelleted before resuspending each pellet in 500 μL of PBS. Each sample was split in half, and genomic DNA was isolated from each duplicate sample using the DNeasy extraction kit (Qiagen) according to the manufacturer’s guidelines. Quantitative PCR was used to measure <em>C</em>. <em>trachomatis</em> genomic DNA (gDNA) levels using an <em>euo</em> primer set. 150ng of each sample was used in 25μL reactions using standard amplification cycles on a QuantStudio3 thermal cycler (Applied Biosystems) followed by a melting curve analysis. <em>ftsK, pbp2, euo</em>, and <em>omcB</em> transcript levels were determined by RT-qPCR using SYBR Green as described previously (Ouellette SP 2021) (see Supp. Table S2 for primers used for measuring gDNA levels and RT-qPCR). Transcript levels were normalized to genomes and expressed as ng cDNA/gDNA. Transformation of <em>Ct</em> <em>Ct</em> was transformed as described previously (Wang 2011). Briefly, HeLa cells were plated in a 10cm plate at a density of 5 × 10<sup>6</sup> cells the day before beginning the transformation procedure. <em>Ct</em> lacking its endogenous plasmid (-pL2) was incubated with 10μg of plasmid DNA in Tris-CaCl<sub>2</sub> buffer (10 mM Tris-Cl pH 7.5, 50 mM CaCl<sub>2</sub>) for 30 min at room temperature. HeLa cells were trypsinized, washed with 8 mL of 1x DPBS (Gibco), and pelleted. The pellet was resuspended in 300μL of the Tris-CaCl<sub>2</sub> buffer. <em>Ct</em> was mixed with the HeLa cells and incubated at room temperature for an additional 20 min. The mixture was added to 10 mL of DMEM containing 10% FBS and 10 μg/mL gentamicin and transferred to a 10cm plate. At 48hpi, the HeLa cells were harvested, and <em>Ct</em> in the population was used to infect a new HeLa cell monolayer in media containing 0.36 U/ml of penicillin G to select for transformants. The plate was incubated at 37°C for 48 hours. These harvest and re-infection steps were repeated every 48 hours until inclusions were observed. Immunofluorescence Microscopy HeLa cells were seeded in 10cm plates at a density of 5 × 10<sup>6</sup> cells per well the day before the infection. <em>Ct</em> L2 or chlamydial strains transformed with plasmids encoding FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, or mCherry-MreC or with plasmids that direct the constitutive expression of the crRNAs targeting the <em>pbp2</em> or <em>ftsK</em> promoters were used to infect HeLa cells in DMEM. For experiments with the transformants, aTc was added to the media of infected cells at the indicated concentration and time. At 21 hpi, cells were detached from the 10cm plate by scraping and pelleted by centrifugation for 30 seconds. The pellet was resuspended in 1 mL of 0.1x PBS (Gibco) and transferred to a 2 mL tube containing 0.5mm glass beads (ThermoFisher Scientific). Cells were vortexed for 3 minutes, then centrifuged at 800 rpm for 2 minutes. in a microfuge. 20μLs of the supernatant was mixed with 20μLs of 2x fixing solution (6.4% formaldehyde and 0.044% glutaraldehyde) and incubated on a glass slide for 10 min at room temperature. Cells were washed 3 times with PBS, and the cells were permeabilized by incubation with PBS containing 0.1% Triton X-100 for 1 min. Cells were washed with PBS twice. For experiments with <em>Ct</em> L2, the cells were incubated with a goat primary antibody against the major outer-membrane protein (MOMP; Meridian, Memphis, TN), and the mouse primary antibody that recognizes endogenous FtsK raised against recombinant CT739 protein (https://doi.org/10.1099/mic.0.047746-0), or with rabbit antibodies raised against peptides derived from PBP 2 or PBP3 (Ouellette SP 2012). Briefly, chlamydial antigens or peptides emulsified with Freund’s incomplete adjuvant were used to immunize animals via intramuscular injections three times with an interval of 2 weeks. Antisera were collected from the immunized animals 2 to 4 weeks after the final immunization as the primary antibodies. After the primary antibody labeling, the cells were then rinsed with PBS and incubated with donkey anti-goat IgG (Alexa 488) and donkey anti-mouse IgG (Alexa 594) or donkey-anti-rabbit IgG (Alexa 594) secondary antibodies (Invitrogen). Experiments in which we visualized the distribution of the various mCherry fusions, the localization of the mCherry fluorescence was compared to the distribution of MOMP. In some experiments, we determined the distribution of the MreB_6x His fusion by staining cells expressing the fusion with a rabbit anti-6x His antibody (Abcam, Cambridge, MA) and the goat anti-MOMP antibody, followed by the appropriate secondary antibodies. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. During image acquisition, 0.3μm xy-slices were collected that extended above and below the cell. The images were collected such that the brightest spot in the image was saturated. The images were deconvolved using the nearest neighbor algorithm in the Zeiss Axiovision 4.7 software. Deconvolved images were viewed and assembled using Zeiss Zen-Blue software. For each experiment, three independent replicates were performed, and the values shown for localization are the average of the 3 experiments. In some instances, 3D projections of the acquired xy slices were generated using the Zeiss Zen-Blue software. Peptidoglycan (PG) labeling PG was labeled by incubating cells with 4mM ethylene-D-alanine-D-alanine (E-DA-DA) as described (Cox 2020). The incorporated E-DA-DA was fluorescently labeled using the Click & Go<sup>™</sup> labeling kit (Vector Laboratories). The distribution of fluorescently labeled PG was compared to the distribution of MOMP and endogenous FtsK or the distribution of mCherry-PBP3. Three independent replicates were performed, and the values shown are the average of the 3 experiments. Inclusion forming unit assay HeLa cells were infected with <em>Ct</em> (-pL2) transformed with the pBOMB4 Tet (-GFP) plasmid encoding the indicated aTc-inducible gene. At 8hpi, aTc was added to the culture media at the indicated concentration. Control cells were not induced. At 48 hpi, the HeLa cells were dislodged from the culture dishes by scraping and collected by centrifugation. The pellet was resuspended in 1 mL of 0.1x PBS (Gibco) and transferred to a 2 mL tube containing 0.5mm glass bead tubes (ThermoFisher Scientific). Cells were vortexed for 3 min. followed by centrifugation at 800 rpm for 2 min. The supernatants were mixed with an equal volume of a 2x sucrose-phosphate (2SP) solution (ref) and frozen at −80°C. At the time of the secondary infection, the chlamydiae were thawed on ice and vortexed. Cell debris was pelleted by centrifugation for 5 min at 1k x g at 4°C. The EBs in the resulting supernatant were serially diluted and used to infect a monolayer of HeLa cells in a 24-well plate. The secondary infections were allowed to grow at 37°C for 24 hrs before they were fixed and labeled for immunofluorescence microscopy by incubating with a goat anti-MOMP antibody followed by a secondary donkey anti-goat antibody (Alexa Fluor 594). The cells were rinsed in PBS, and inclusions were imaged using an EVOS imaging system (Invitrogen). The number of inclusions was counted in 5 fields of view and averaged. Three independent replicates were performed, and the values from the replicates were averaged to determine the number of inclusion forming units. Chi-squared analysis was used to compare IFUs in induced and uninduced samples. Effect of A22 and mecillinam on the profile of division intermediates and on PG and divisome protein localization in <em>Ct</em> HeLa cells were infected with <em>Ct</em> transformed with the pBOMB4-Tet (-GFP) plasmid encoding FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, mCherry-MreC, or MreB-6xHis. The fusions were induced at 20 hpi with 10 nM aTc for 1 hour in the absence or presence of 75 μM A22. At 22 hpi, cells were harvested and prepared for staining as described above. Three independent replicates were performed, and the values shown for localization are the average of the 3 experiments. HeLa cells were infected with <em>Ct</em> L2, and 20μM mecillinam (Sigma) was added to the media of infected cells at 17 hpi. Control cells were untreated. At 22 hpi, infected cells were harvested and RBs were prepared and stained with MOMP, FtsK, PBP2, or PBP3 antibodies as described above. Alternatively, cells were incubated with 4 mM EDA-DA at 17 hpi in the presence or absence of 20μM mecillinam. The cells were harvested at 22 hpi, and RBs were prepared, and PG was click-labeled, and its distribution was visualized in MOMP-stained cells as described above. Three independent replicates were performed, and the values shown for localization are the average of the 3 experiments. Immunoblotting HeLa cells infected with <em>Ct</em> L2 were harvested by scraping the infected cells from the plate at 24 hpi. Uninfected HeLa cells were included as a control. The HeLa cells were pelleted by centrifugation, resuspended in SDS sample buffer, and electrophoresed on a 10% SDS polyacrylamide gel. The gel was electrophoretically transferred to nitrocellulose (Schleicher and Schuell), and the filter was incubated with mouse polyclonal antibodies raised against chlamydial FtsK. The filter was rinsed and incubated with 800 donkey anti-mouse IgG secondary antibodies (LICOR, Lincoln, NE) and imaged using a LICOR Odyssey imaging system. HeLa cells were infected with <em>Ct</em> transformed with plasmids that inducibly express FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, or mCherry-MreC. The fusions were induced by the addition of 10nM aTc to the media of infected cells at 17 hpi. The cells were harvested and pelleted at 21 hpi. The cell pellet was resuspended in 1 mL of 0.1x PBS (Gibco) and transferred to a 2 mL tube containing 0.5mm glass beads (ThermoFisher Scientific). Cells were vortexed for 3 min. followed by centrifugation at 800 rpm for 2 min. The supernatant was collected and centrifuged for 3 min at 13,000 rpm, and the pellet containing <em>Ct</em> was resuspended in TBS containing 1% TX-100, 1X protease inhibitor cocktail (Sigma), and 5μM lactacystin. The suspension was sonicated 3 times on ice and centrifuged at 13,000 rpm for 3 mins. The supernatant was collected and mixed with SDS sample buffer. The samples were boiled and electrophoresed on a 10% SDS polyacrylamide gel, and the gel was electrophoretically transferred to nitrocellulose. The blots from these analyses were probed with a rabbit anti-mCherry primary antibody (Invitrogen) and an 800 donkey anti-rabbit IgG secondary antibody (LICOR, Lincoln, NE). The filters were imaged using a LICOR Odyssey imaging system. HeLa cells were infected with <em>Ct</em> transformed with the pBOMB4-Tet (-GFP) plasmid encoding mCherry-PBP2 or mCherry-PBP3. The fusions were induced with 10nM aTc at 17 hpi. Uninduced cells were included as a control. The cells were harvested at 21 hpi, and samples were processed for immunoblotting as described above. The blots were probed with rabbit polyclonal antibodies raised against peptides derived from chlamydial PBP2 or PBP3 (Ouellette SP 2012). The blots were then rinsed and incubated with 800 donkey anti-rabbit IgG secondary antibodies (LICOR, Lincoln, NE). The filters were imaged using a LICOR Odyssey imaging system.