Source data: FtsK is critical for the assembly of the unique divisome complex of the FtsZ-less Chlamydia trachomatis figures 1-7

# Source data: FtsK is critical for the assembly of the unique divisome
complex of the FtsZ-less *Chlamydia trachomatis* figures 1-7
[https://doi.org/10.5061/dryad.4qrfj6qnh](https://doi.org/10.5061/dryad.4qrfj6qnh) ## Description of the data and file structure ### Files and variables ### **Figure 1 Source Data:** #### File: Figure\_1B\_endogenous\_FtsK\_coccoid.zvi **Description:** Figure 1-Source Data 1. Original .zvi file showing endogenous FtsK (red fluorescence) distribution in a coccoid cell at 21 hpi. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera.  #### File: Figure\_1D\_FtsK\_mcherry\_coccoid.zvi **Description:** Figure 1-Source Data 5. Original .zvi file showing FtsK-mCherry (red - mCherry fluorescence) localization in a coccoid cell at 21 hpi. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_1C\_endogenous\_FtsK\_septum.zvi **Description:** Figure 1-Source Data 2. Original .zvi file showing endogenous FtsK (red fluorescence) localized to the septum of a dividing cell at 21 hpi. Stages of division were determined by the distribution of the major outer membrane (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_1E\_FtsKmcherry\_septum.zvi **Description:** Figure 1-Source Data 6. Original .zvi file showing FtsK-mCherry (red - mCherry fluorescence) localized to the septum of a dividing cell at 21 hpi. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure1C.\_endogenous\_FtsK\_septum\_and\_base.zvi **Description:** Figure 1-Source Data 3. Original .zvi file showing endogenous FtsK (red fluorescence) localized to the septum and the base of a dividing cell at 21 hpi. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_1E\_FtsKmcherry\_septum\_and\_base.zvi **Description:** Figure 1-Source Data 7. Original .zvi file showing FtsK-mCherry (red - mCherry fluorescence) localized to the septum and base of a dividing cell at 21 hpi. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera.   #### File: Figure\_1C\_endog\_K\_base.zvi **Description:** Figure 1-Source Data 4. Original .zvi file showing endogenous FtsK (red fluorescence) localized to the base of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_1E\_FtsKmcherry\_base.zvi **Description:** Figure 1-Source Data 8. Original .zvi file showing FtsK-mCherry (red - mCherry fluorescence) localized to the base of a dividing cell at 21 hpi. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_1G\_endogenous\_K\_multibud.zvi **Description:** Figure 1-Source Data 9. Original .zvi file showing endogenous FtsK (red fluorescence) localized to the septum and base in a cell containing a secondary bud site at the base. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera.   #### File: Figure\_1G\_FtsKmchery\_multibud.zvi **Description:** Figure 1-Source Data 10. Original .zvi file showing FtsK-mCherry (red - mCherry fluorescence) localized to the septum and base in a cell containing a secondary bud site at the base. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. ### **Figure 2 Source Data:** #### File: Figure\_2A\_PBP2\_coccoid.zvi **Description:** Figure 2-Source Data 1. Original .zvi file showing mCherry-PBP2 (red - mCherry fluorescence) localization in a coccoid cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2A\_PBP3\_coccoid\_.zvi \**Description: **Figure 2-Source Data 2. Original .zvi file showing mCherry-PBP3 (red - mCherry fluorescence) localization in a coccoid cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2A\_MreB\_coccoid.zvi **Description:** Figure 2-Source Data 3. Original .zvi file showing MreB_6xHis (red fluorescence) localization in a coccoid cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2A\_MreC\_coccoid.zvi **Description:** Figure 2-Source Data 4. Original .zvi file showing mCherry-MreC (red - mCherry fluorescence) localization in a coccoid cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2B\_PBP2\_septum.zvi **Description:** Figure 2-Source Data 5. Original .zvi file showing mCherry-PBP2 (red - mCherry fluorescence) localized to the septum of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2B\_PBP3\_septum.zvi **Description:** Figure 2-Source Data 6. Original .zvi file showing mCherry-PBP3 (red - mCherry fluorescence) localized to the septum of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera.  #### File: Figure\_2B\_MreB\_septum.zvi **Description:** Figure 2-Source Data 7. Original .zvi file showing MreB_6xHis (red fluorescence) localized to the septum of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2B\_MreC\_septum.zvi **Description:** Figure 2-Source Data 8. Original .zvi file showing mCherry-MreC (red - mCherry fluorescence) localized to the septum of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera.   #### File: Figure\_2B\_PBP2\_septum\_and\_base.zvi **Description:** Figure 2-Source Data 9. Original .zvi file showing mCherry-PBP2 (red - mCherry fluorescence) localized to the septum and base of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2B\_PBP3\_septum\_and\_base.zvi **Description:** Figure 2-Source Data 10. Original .zvi file showing mCherry-PBP3 (red - mCherry fluorescence) localized to the septum and base of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2B\_MreB\_septum\_and\_base.zvi **Description:** Figure 2-Source Data 11. Original .zvi file showing MreB_6xHis (red fluorescence) localized to the septum and base of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2B\_MreC\_septum\_and\_base\_(ds\_with\_K).zvi **Description:** Figure 2-Source Data 12. Original .zvi file showing mCherry-MreC (red - mCherry fluorescence) localized to the septum and base of a dividing cell. The stage of division was determined by the distribution of the major outer membrane protein (MOMP - green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. The cell shown was also stained with FtsK antibodies (blue). #### File: Figure\_2B\_PBP2\_base.zvi **Description:** Figure 2-Source Data 13. Original .zvi file showing mCherry-PBP2 (red - mCherry fluorescence) localized to the base of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera.   #### File: Figure\_2B\_PBP3\_base.zvi **Description:** Figure 2-Source Data 14. Original .zvi file showing mCherry-PBP3 (red - mCherry fluorescence) localized to the base of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. ####  File: Figure\_2B\_MreB\_base.zvi **Description:** Figure 2-Source Data 15. Original .zvi file showing MreB_6xHis (red fluorescence) localized to the base of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2B\_MreC\_base.zvi **Description:** Figure 2-Source Data 16. Original .zvi file showing mCherry-MreC (red – mCherry fluorescence) localized to the base of a dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2D\_FtsK\_and\_PBP2.zvi **Description:** Figure 2-source Data 17. Original .zvi file showing mCherry-PBP2 (red - mCherry fluorescence) localized to the septum of the dividing cell. The cell was also stained with antibodies that recognize endogenous FtsK (blue), which accumulated at the septum and base of the dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2D\_FtsK\_and\_PBP3.zvi **Description:** Figure 2-source Data 18. Original .zvi file showing mCherry-PBP3 (red - mCherry fluorescence) localized to the septum of the dividing cell. The cell was also stained with antibodies that recognize endogenous FtsK (blue), which accumulated at the septum and base of the dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2D\_FtsK\_and\_MreB.zvi **Description:** Figure 2-source Data 19. Original .zvi file showing MreB_6xHis (red fluorescence) localized to the septum of the dividing cell. The cell was also stained with antibodies that recognize endogenous FtsK (blue), which accumulated at the septum and base of the dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_2D\_FtsK\_and\_MreC.zvi **Description:** Figure 2-source Data 20. Original .zvi file showing mCherry-MreC (red - mCherry fluorescence) localized to the septum of the dividing cell. The cell was also stained with antibodies that recognize endogenous FtsK (blue), which accumulated at the septum and base of the dividing cell. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3B\_FtsK\_UTD.zvi **Description:** Figure 3-Source Data 1. Original .zvi file showing FtsK-mCherry (red – mCherry fluorescence) localization in a coccoid cell (no treatment with A22). Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3B\_PBP2\_UTD.zvi **Description:** Figure 3-Source Data 2. Original .zvi file showing mCherry-PBP2 (red - mcherry fluorescence) localization in a coccoid cell (no treatment with A22). Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3B\_PBP3\_UTD.zvi **Description:** Figure 3-Source Data 3. Original .zvi file showing mCherry-PBP3 (red - mCherry fluorescence) localization in a coccoid cell (no treatment with A22). Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3B\_MreB\_UTD.zvi **Description:** Figure 3-Source Data 4. Original .zvi file showing MreB_6xHis (red fluorescence) localization in a coccoid cell (no treatment with A22). Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3B\_MreC\_UTD.zvi **Description:** Figure 3-Source Data 5. Original .zvi file showing mCherry-MreC (red - mCherry fluorescence) localization in a coccoid cell (no treatment with A22). Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3C\_FtsK\_A22.zvi **Description:** Figure 3-Source Data 6. Original .zvi file showing FtsK-mCherry (red – mCherry fluorescence) localization in a coccoid cell (treated with A22). Stages of division are determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3C\_PBP2\_A22.zvi **Description:** Figure 3-Source Data 7. Original .zvi file showing mCherry-PBP2 (red - mCherry fluorescence) localization in a coccoid cell (treated with A22). Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3C\_PBP3\_A22.zvi **Description:** Figure 3-Source Data 8. Original .zvi file showing mCherry-PBP3 (red - mCherry fluorescence) localization in a coccoid cell (treated with A22). Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3C\_MreB\_A22.zvi **Description:** Figure 3-Source Data 9. Original .zvi file showing MreB_6xHis (red fluorescence) localization in a coccoid cell (treated with A22). Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_3C\_MreC\_A22.zvi **Description:** Figure 3-Source Data 10. Original .zvi file showing mCherry-MreC (red – mCherry fluorescence) localization in a coccoid cell (treated with A22). Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: 05\_ftsK-KD\_24hpi\_UI\_dCas12594\_MOMP488.czi **Description:** Figure 4-Source Data 1. Original .czi file showing inclusions of Hela cells infected with *Chlamydia trachomatis* that constitutively express the crRNA targeting *ftsK. *dCas12 expression was not inducible in these cells. The infected cells were fixed at 24 hpi and stained with an antibody against the major outer membrane protein (green fluorescence) and an antibody against Cas12 (red fluorescence). Cells were imaged using a Zeiss Apotome microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: 04\_ftsK-KD\_24hpi\_20hIn\_5nMaTc\_dCas12594\_MOMP488.czi **Description:** Figure 4-Source Data 2. Original .czi file showing inclusions of Hela cells infected with *Chlamydia trachomatis* that constitutively express the crRNA targeting *ftsK. *dCas12 expression was inducible in *Chlamydia trachomatis* by the addition of 5nM anahydrotetracycline to the media at 8hpi. The infected cells were fixed at 24 hpi and stained with an antibody against the major outer membrane protein (green fluorescence) and an antibody against Cas12 (red fluorescence). Cells were imaged using a Zeiss Apotome microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: 08\_pbp2-KD\_24hpi\_UI\_dCas12594\_MOMP488.czi **Description:** Figure 4-Source Data 3. Original .czi file showing inclusions of Hela cells infected with *Chlamydia trachomatis* that constitutively express the crRNA targeting *pbp2. *dCas12 expression was not inducible in these cells. The infected cells were fixed at 24 hpi and stained with an antibody against the major outer membrane protein (green fluorescence) and an antibody against Cas12 (red fluorescence). Cells were imaged using a Zeiss Apotome microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: 06\_pbp2-KD\_24hpi\_20hIn\_5nMaTc\_dCas12594\_MOMP488.czi **Description:** Figure 4-Source Data 4. Original .czi file showing inclusions of Hela cells infected with *Chlamydia trachomatis* that constitutively express the crRNA targeting *pbp2. *dCas12 expression was inducible in *Chlamydia trachomatis* by the addition of 5nM anahydrotetracycline to the media at 8hpi. The infected cells were fixed at 24 hpi and stained with an antibody against the major outer membrane protein (green fluorescence) and an antibody against Cas12 (red fluorescence). Cells were imaged using a Zeiss Apotome microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_5C\_endog\_PBP2\_FtsK\_iKD\_uninduced.zvi **Description:** Figure 5-Source Data 1. Original .zvi file showing endogenous PBP2 distribution in a coccoid cell. HeLa cells were infected with the chlamydial strain constitutively expressing the crRNA targeting *ftsK. dCas12 *expression was not inducible. Cells were harvested at 24 hpi and stained with antibodies that detect PBP2 (red fluorescence) and the major outer membrane protein (MOMP – green fluorescence). The stage of division was determined by the distribution of MOMP. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_5C\_endog\_PBP3\_FtsK\_iKD\_uninduced.zvi **Description:** Figure 5-Source Data 2. Original .zvi file showing endogenous PBP3 distribution in a coccoid cell. HeLa cells were infected with the chlamydial strain constitutively expressing the crRNA targeting *ftsK. dCas12 *expression was not inducible. Cells were harvested at 24 hpi and stained with antibodies that detect PBP3 (red fluorescence) and the major outer membrane protein (MOMP - green fluorescence). The stage of division was determined by the distribution of MOMP. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_5C\_endog\_FtsK\_PBP2\_iKD\_uninduced.zvi **Description:** Figure 5-Source Data 3. Original .zvi file showing endogenous FtsK distribution in a coccoid cell. HeLa cells were infected with the chlamydial strain constitutively expressing the crRNA targeting *pbp2. dCas12 *expression was not inducible. Cells were harvested at 24 hpi and stained with antibodies that detect FtsK (red fluorescence) and the major outer membrane protein (MOMP - green fluorescence). The stage of division was determined by the distribution of MOMP. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_5C\_endog\_PBP3\_PBP2\_iKD\_uninduced.zvi **Description:** Figure 5-Source Data 4. Original .zvi file showing endogenous PBP3 distribution in a coccoid cell. HeLa cells were infected with the chlamydial strain constitutively expressing the crRNA targeting *pbp2. dCas12 *expression was not inducible. Cells were harvested at 24 hpi and stained with antibodies that detect PBP3 (red fluorescence) and the major outer membrane protein (MOMP – green fluorescence). The stage of division was determined by the distribution of MOMP. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. ####  File: Figure\_5D\_endog\_PBP2\_FtsK\_iKD\_.zvi **Description:** Figure 5-Source Data 5. Original .zvi file showing endogenous PBP2 distribution in a coccoid cell. HeLa cells were infected with the chlamydial strain constitutively expressing the crRNA targeting *ftsK. dCas12 *expression was inducible by the addition of 5 nM anahydrotetracycline to the media at 17 hpi. Cells were harvested and fixed at 24 hpi and stained with PBP2 (red fluorescence) and major outer membrane protein antibodies (MOMP – green fluorescence). The stage of division was determined by the distribution of MOMP. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_5D\_endog\_PBP3\_FtsK\_iKD.zvi **Description:** Figure 5-Source Data 6. Original .zvi file showing endogenous PBP3 localization in a coccoid cell from an induced *ftsK* iKD strain. Cells were induced with aTc at 17 hpi. Cells were harvested and fixed at 24 hpi and stained with PBP3 (red fluorescence) and major outer membrane protein antibodies (MOMP - green fluorescence). The stage of division was determined by the distribution of MOMP. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera.. #### File: Figure\_5D\_endog\_FtsK\_PBP2\_iKD.zvi **Description:** Figure 5-Source Data 7. Original .zvi file showing endogenous FtsK localization in a coccoid cell from an induced *pbp2* iKD strain. Cells were induced with aTc at 17 hpi. Cells were harvested and fixed at 24 hpi and stained with FtsK (red fluorescence) and major outer membrane protein antibodies (MOMP – green fluorescence). The stage of division was determined by the distribution of MOMP.  The stage of division was determined by the distribution of MOMP. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_5D\_endog\_PBP3\_PBP2\_iKD.zvi **Description:** Figure 5-Source Data 8. Original .zvi file showing endogenous PBP3 localization in a coccoid cell from an induced *pbp2* iKD strain. Cells were induced with aTc at 17 hpi. Cells were harvested and fixed at 24 hpi and stained with PBP3 (red fluorescence) and major outer membrane protein antibodies (MOMP – green fluorescence). The stage of division was determined by the distribution of MOMP. Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_6C\_endog\_FtsK\_UTD.zvi **Description:** Figure 6-Source Data 1. Original .zvi file showing endogenous FtsK (red fluorescence) localization in a coccoid cell that was not treated with mecillinam. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_6B\_endog\_PBP2\_UTD.zvi **Description:** Figure 6-Source Data 2. Original .zvi file showing endogenous PBP2 (red fluorescence) localization in a coccoid cell that was not treated with mecillinam. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_6B\_endog\_PBP3\_UTD.zvi **Description:** Figure 6-Source Data 3. Original .zvi file showing endogenous PBP3 (red fluorescence) localization in a coccoid cell that was not treated with mecillinam. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_6C\_endog\_FtsK\_mec.zvi **Description:** Figure 6-Source Data 4. Original .zvi file showing endogenous FtsK (red fluorescence) localization in a coccoid cell that was treated with mecillinam. Stages of division were determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_6C\_endog\_PBP2\_mec.zvi **Description:** Figure 6-Source Data 5. Original .zvi file showing endogenous PBP2 (red fluorescence) localization in a coccoid cell that was treated with mecillinam. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_6C\_endog\_PBP3\_mec.zvi **Description:** Figure 6-Source Data 6. Original .zvi file showing endogenous PBP3 (red fluorescence) localization in a coccoid cell that was treated with mecillinam. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_7A\_foci\_and\_foci\_PG.zvi **Description:** Figure 7-Source Data 1. Original .zvi file showing EDA-DA click-labeled peptidoglycan (red fluorescence) localized in foci at the septum and at the base of a dividing cell. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_7A\_foci\_and\_ring\_PG.zvi **Description:** Figure 7-Source Data 2. Original .zvi file showing EDA-DA click-labeled peptidoglycan (red fluorescence) localized in a focus at the septum and a ring at the base of a dividing cell. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_7B\_PG\_and\_FtsK..zvi **Description:** Figure 7-Source Data 3. Original .zvi file showing EDA-DA click-labeled peptidoglycan (blue fluorescence) localized as a ring at the septum of a dividing cell. Endogenous FtsK (red fluorescence) accumulates in multiple foci that overlap the distribution of the peptidoglycan ring. The stage of division was determined by the distribution of the major outer membrane (green fluorescence). 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. A 3D projection of the xy slices is shown. #### File: Figure\_7B\_PG\_and\_PBP3.zvi **Description:** Figure 7-Source Data 4. Original .zvi file showing EDA-DA click-labeled peptidoglycan (blue fluorescence) localized as a ring at the septum of a dividing cell. mCherry-PBP3 (red – mCherry fluorescence) accumulates in multiple foci that overlap the distribution of the peptidoglycan ring. The stage of division was determined by the distribution of the major outer membrane (green fluorescence). 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. A 3D projection of the xy slices is shown. #### File: Figure\_7C\_foci\_coccoid.zvi **Description:** Figure 7-Source Data 5. Original .zvi file showing EDA-DA click-labeled peptidoglycan (red fluorescence) localized in a focus in the membrane of a coccoid cell. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. File: Figure_7D_bar_coccoid.zvi #### File: Figure\_7C\_bar\_coccoid.zvi **Description:** Figure 7-Source Data 6. Original .zvi file showing EDA-DA click-labeled peptidoglycan (red fluorescence) localized as a bar in the membrane of a coccoid cell. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_7C\_ring\_coccoid.zvi **Description:** Figure 7-Source Data 7. Original .zvi file showing EDA-DA click-labeled peptidoglycan (red fluorescence) localized as a ring in the membrane of a coccoid cell. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_7E\_A22\_treated\_cells\_PG.zvi **Description:** Figure 7-Source Data 8. Original .zvi file showing EDA-DA click-labeled peptidoglycan (red fluorescence) distribution in a coccoid cell treated with A22. The stage of division was determined by the distribution of the major outer membrane (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_7E\_mec\_treated\_cells\_PG.zvi **Description:** Figure 7-Source Data 9. Original .zvi file showing EDA-DA click-labeled peptidoglycan (red fluorescence) distribution in a coccoid cell treated with mecillinam. Stages of division were determined by the distribution of the major outer membrane (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. #### File: Figure\_7E\_FtsK\_iKD\_induced\_cells\_PG.zvi **Description:** Figure 7-Source Data 10. Original .zvi file showing EDA-DA click-labeled peptidoglycan (red fluorescence) distribution in a coccoid cell in an induced *ftsK* iKD strain. The stage of division was determined by the distribution of the major outer membrane protein (green fluorescence). Cells were imaged using a Zeiss AxioImager2 microscope equipped with a 100x oil immersion PlanApochromat objective and a CCD camera. ## Code/software All .zvi and .czi files can be viewed using Zeiss Zen Blue software, which is available as a free download at Zeiss. It can also be viewed in Fiji. Arrows depict cells of interest. ## Access information NA