(A) The linear divisome assembly pathway of E. coli is shown. Ct encodes the divisome proteins boxed in red. HeLa cells were infected with Ct L2 and RBs were prepared at 21hpi. The cells were fixed and stained with MOMP (green) and FtsK (red) antibodies. The distribution of FtsK in (B) coccoid cells and in (C) cell division intermediates that had not initiated secondary bud formation is shown. Bars are 1 μm. HeLa cells were infected with Ct transformed with the pBOMB4 -Tet (-GFP) plasmid encoding FtsK-mCherry. The fusion was induced with 10nM aTc for 1 hr. and RBs were prepared from infected HeLa cells at 21hpi and stained with MOMP antibodies (green). The distribution of MOMP relative to the mCherry fluorescence (D) in coccoid cells and in (E) cell division intermediates that had not initiated secondary bud formation is shown. Bars are 1μm. Arrowheads in C and E denote foci of FtsK above or below the MOMP-stained septum. (F) HeLa cells were infected with Ct L2. At 21hpi, the cells were harvested and RBs were stained with MOMP antibodies. The number of dividing cells that had initiated secondary bud formation was quantified in 150 cells. Three independent replicates were performed, and the values shown are the average of the 3 replicates. (G) Endogenous FtsK and FtsK-mCherry accumulate in foci at the septum of secondary buds (marked with arrowheads).

HeLa cells were infected with Ct transformed with PBP2, PBP3, or MreC with an N-terminal mCherry tag, or with Ct transformed with an MreB_6xHis fusion (Lee 2020). Each of the fusions was induced by adding 10nM aTc to the media at 17hpi. Lysates were prepared at 21hpi and the cells were fixed and stained with a MOMP antibody. The distribution of the mCherry fluorescence in (A) coccoid cells and in (B) dividing cells that had not initiated secondary bud formation is shown. The MreB_6x His fusion was stained with rabbit anti-6x his antibody (red) and MOMP antibodies (green). Dividing cells with foci at the septum, foci at the septum and foci at the base of the mother cell, or foci at the base alone are shown for each of the fusions. Arrowheads in B denote foci of the divisome proteins above or below the plane of the MOMP-stained septum. (C) HeLa cells were infected with Ct L2 or with Ct that inducibly express FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, mCherry-MreC, or MreB_6xHis. The cells were fixed at 21hpi and the distribution of endogenous FtsK, or the mCherry fluorescence in cells inducibly expressing the mCherry fusions, or the distribution of MreB in cells where the MreB_6xHis fusion was inducibly expressed were compared to the distribution of MOMP. The localization profiles were quantified in 100 cells. Three independent replicates were performed, and the values shown are the average of the 3 replicates. Chi-squared analysis revealed that the localization profiles of endogenous FtsK and FtsK-mCherry are not statistically different from each other, but they are statistically different than the PBP2, PBP3, MreC and MreB localization profiles (* – p<0.009). The localization profile of the MreB fusion is also statistically different than the localization profiles of the mCherry fusions of PBP2 and PBP3 (#-p = 0.05). (D) Hela cells were infected with Ct transformed with PBP2, PBP3, or MreC with a N-terminal mCherry tag, or with Ct transformed with an MreB_6xHis fusion (Lee 2020). The fusions were induced by adding 10 nM aTc to the media at 17hpi. The cells were harvested at 21hpi and Ct were harvested and stained with FtsK and MOMP antibodies. The cells expressing the MreB fusion were stained with FtsK, MOMP, and 6xHis antibodies. Imaging analyses revealed that FtsK was present in foci at the septum and in foci at the base in these cells, while each of the fusions was only detected at the septum where they overlapped the distribution of septal FtsK (Bars are1μM).

(A) HeLa cells infected with Ct L2 were treated with 75μM A22 for 1 hour. Control cells were not treated with A22. Lysates were prepared at 21hpi and the number of coccoid and dividing cells in the population were quantified in 100 cells. Three independent replicates were performed, and the values shown are the average of the 3 replicates. (B-E) Alternatively, HeLa cells were infected with Ct transformed with plasmids encoding FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, mCherry-MreC, or MreB-6xHis. The fusions were induced at 20hpi with 10nM aTc for 1hr in the absence (B and D) or presence (C and E) of 75μM A22. Coccoid cells prepared from the infected cells at 21hpi were stained with MOMP antibodies (green). The MreB-6xHis fusion was also stained with 6xHis antibodies (red). Panel B shows the distribution of the fusions in untreated coccoid cells. Panel C illustrates the effect of A22 on the localization of the fusions in coccoid cells. Bars in B and C are 1μm. The distribution of FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, mCherry-MreC, and MreB-6xHis was quantified in (D) control coccoid cells and in (E) A22-treated cells coccoid cells (n=50) is shown. Three replicates were performed, and the values shown in D and E are the averages of the 3 replicates. Student T-test indicated that A22 had a statistically significant effect on the localization of MreB and MreC (* – p<0.01).

HeLa cells were infected with Ct transformed with the pBOMBL-12CRia plasmid, which constitutively expresses a ftsK crRNA or pbp2 crRNA and encodes dCas12 under the control of an aTc-inducible promoter. dCas12 was induced at 17hpi by adding 5nM aTc to the media. In a control infection, the expression of dCas12 was not induced. Cells were harvested at 24hpi and the morphology of Ct in induced and uninduced control cells was assessed in 250 cells (A and B). 3 replicates were performed, and the values shown are the averages of the 3 replicates. The localization of endogenous FtsK, endogenous PBP2, and endogenous PBP3 was assessed in cells transformed with the pBOMBL-12CRia plasmid that targets ftsK or pbp2. The localization is shown in coccoid cells where dCas12 expression was (C) uninduced or (D) induced. White bars are 1μm. The localization profiles of FtsK, PBP2, and PBP3 were quantified in (E) uninduced and (F) induced cells. 3 replicates were performed, and the values shown are the averages of the 3 replicates.

Effect of mecillinam on endogenous FtsK, PBP2, and PBP3 localization. (A) HeLa cells were infected with Ct and 20μM mecillinam was added to the media at 17hpi. Untreated coccoid cells were included as a control. The cells were harvested at 21hpi and the morphology of MOMP-stained cell was assessed in 200 cells. 3 replicates were performed, and the values shown are the averages of the 3 replicates. (B and C) The localization of endogenous FtsK, endogenous PBP2, and endogenous PBP3 in untreated coccoid or in mecillinam-treated coccoid cells is shown. Bars are1μM. (D and E) Localization of FtsK, PBP2, and PBP3 in untreated and mecillinam-treated coccoid cells was quantified in 50 cells. Three replicates were performed, and the values shown are the averages of the 3 replicates.

PG distribution in Ct. HeLa cells were infected with Ct L2. At 17hpi, 4mM ethylene-DA-DA (EDA-DA) was added to the media, the cells were harvested at 21hpi, and the EDA-DA was click labeled and compared to the distribution of MOMP. (A) Imaging revealed that PG organization can vary at the septum and base of dividing cells (B) The localization of click-labeled PG was compared to the localization of endogenous FtsK and mCherry-PBP3 at the septum of dividing cells. 3D projections revealed that multiple foci of each fusion are associated with PG intermediates. (C) PG organization in untreated coccoid cells. (D) Quantification of PG organization in untreated coccoid cells, A22-treated coccoid cells, mecillinam-treated coccoid cells, and in coccoid cells resulting from the inducible knockdown of ftsk. Fifty cells were counted for each condition. Three replicates were performed and the average from the 3 replicates is shown. (E) PG organization in A22-treated and mecillinam-treated coccoid cells, and in coccoid cells resulting from the inducible knockdown of ftsk is shown. Bars are 1μM. (F) Putative Ct divisome assembly pathway in is shown. Proteins characterized in this study are bolded. The ordering of the remaining proteins is based on the assembly of the divisome and elongasome in E. coli (Du 2017; Liu 2020).

(A) Lysates were prepared from uninfected HeLa cells and HeLa cells infected with Ct L2. At 21hpi, lysates were prepared and characterized by immunoblotting with FtsK-specific antibodies. (B) HeLa cells were infected with Ct transformed with mCherry fusions of FtsK, PBP2, PBP3, or MreC. The fusions were induced with 10nM aTc at 17hpi. HeLa cells were harvested at 21hpi and lysates were prepared and characterized by immunoblotting analysis with a rabbit polyclonal mCherry antibodies. (C) HeLa cells were infected with Ct transformed N-terminal fusions of PBP2 or PBP3. The fusions were induced (+aTc) at17hpi. Controls were not induced (-aTc). The cells were harvested at 21hpi and lysates were prepared as described in the Methods and characterized by immunoblotting analysis with rabbit antibodies raised against peptides derived from chlamydial PBP2 or PBP3. The PBP3 antibody primarily detects a single species with the predicted molecular mass of mCherry-PBP3 in the induced sample, The PBP2 antibody primarily detects a single species of 120kD in the induced sample, which is smaller than the predicted molecular mass of mCherry-PBP2 (∼150kD). The failure to detect full length mCherry-PBP2 may be due to the masking of the epitope recognized by the PBP2 antibody by the N-terminal mCherry tag in the full-length protein. (D) HeLa cells were infected with Ct transformed with mCherry-PBP2 or mCherry-PBP3. The fusions were induced by the addition of 10nM aTc to the media of the infected cells at 19hpi. Infected cells were harvested at 21hpi and lysates were prepared and stained with the PBP2 or PBP3 antibodies. The staining with the PBP2 and PBP3 antibodies completely overlaps the mCherry fluorescence from the mCherry-PBP2 and mCherry-PBP3 fusions (Bars are 3μM)

(A) HeLa cells were infected with Ct transformed with FtsK-mCherry, mCherry-PBP2, mCherry-PBP3, and mCherry-MreC. The fusions were uninduced or induced by the addition of varying amounts of aTc to the media of the infected cells at 8hpi. The cells were harvested at 48hpi and Ct were isolated. The number of infectious Ct in the lysates was measured by an IFU assay. Chi-squared analysis revealed that induction of the fusions did not have a statistically significant effect on the growth of Ct and the production of infectious EBs. (B) Each of the mCherry fusions accumulate in foci at the septum and in foci at the base (marked with arrowheads) of dividing cells with secondary buds.

(A) Localization analyses with rabbit polyclonal antibodies that recognize endogenous PBP2 or PBP3. These analyses revealed that endogenous PBP2 and PBP3 accumulate in foci in coccoid cells, and in foci at the septum, foci at the septum and base, or in foci at the base alone in cell division intermediates in Ct. PBP2 and PBP3 foci are also detected at the base of secondary buds. Bars are1μM (B) Quantification revealed that the localization profiles of endogenous PBP2 and PBP3 were not statistically different than the localization profiles of the mCherry-PBP2 and mCherry-PBP3 fusions shown in Fig. 2C.

HeLa cells were infected with Ct transformed with the pBOMBL12CRia plasmid that constitutively expresses ftsK or pbp2-targeting crRNAs. dCas12 expression was induced by the addition of 5nM aTc to the media of infected cells at 8hpi. Control cells were not induced. Nucleic acids were isolated from induced cells and from uninduced controls at various times post-infection, and RT-qPCR was used to measure ftsK or pbp2 transcript levels. (A) The induction of dCas12 resulted in ∼10-fold reduction in ftsK transcript levels in cells expressing the ftsK-targeting crRNA, (B) and ∼8-fold reduction in pbp2 transcript levels in cells expressing the pbp2-targeting crRNA, while these crRNAs had minimal or no effect on chlamydial euo and omcB transcript levels. HeLa cells were infected with Ct transformed pBOMBL12CRia plasmid that constitutively expresses a (C) ftsK or (D) pbp2-targeting crRNA. dCas12 expression was induced by the addition of 5nM aTc to the media of infected cells at 8hpi. Control cells were not induced. The infected cells were fixed at 24hpi and stained with MOMP and Cas12 antibodies. Ct morphology was normal and dCas12 was undetectable in the inclusions of uninduced control cells. Foci of dCas12 were observed in induced cells, and Ct in the inclusion exhibited an enlarged aberrant morphology. Bars in C and D are 2μm. (E) HeLa cells were infected with Ct transformed with the pBOMBL12CRia plasmid that constitutively expresses a ftsK or pbp2-targeting crRNA. dCas12 was induced at 17hpi by the addition of 10nM aTc to the media. Control cells were not induced. The cells were harvested at 21hpi, and Ct were prepared and stained with antibodies that recognize that endogenous FtsK or PBP2. Quantification shows that polarized foci of FtsK and PBP2 were almost undetectable when ftsK or pbp2 were transiently knocked down.

MreC rings in (A) dividing Ct and in (B) coccoid Ct.

List of primers and plasmids used for cloning mCherry fusions of FtsK, PBP2, PBP3, MreB and MreC.

List of primers used for RT-qPCR.