PomX, a ParA/MinD ATPase activating protein, is a triple regulator of cell division in Myxococcus xanthus
- Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch, Germany
- Arnold Sommerfeld Center for Theoretical Physics and Center for NanoScience, Department of Physics, Ludwig-Maximilians-Universität München, Germany
Figures
Figure 1 with 1 supplement
PomX consists of two domains that are both required for function.
(A) Similarity and identity analysis of PomX, PomY, and PomZ homologs. The three Myxococcales suborders are indicated. An open box indicates that a homolog is not present. (B) Similarity and …
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Figure 1—source data 1
Source Data for Figure 1D.
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Figure 1—figure supplement 1
PomX variants accumulate in M.xanthus.
(A) Schematic of pomXYZ locus (upper panel) and the construct used for ectopic expression of mCh-pomX and its variants from the attB site (lower panel). The brown region upstream of pomZ was used as …
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1B.
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Figure 2 with 1 supplement
PomXC interacts with PomX and PomY while PomXN stimulates PomZ ATPase activity.
(A) BACTH analysis of interactions between Pom proteins. The indicated protein fragments were fused to T18 and T25 as indicated. Blue colony indicates an interaction, white no interaction. Positive …
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Figure 2—source data 1
Source data for Figure 2A.
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Figure 2—source data 2
Source data for Figure 2B.
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Figure 2—source data 3
Source data for Figure 2C.
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Figure 2—source data 4
Source data for Figure 2D.
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Figure 2—source data 5
Source data for Figure 2E.
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Figure 2—source data 6
Source data for Figure 2F.
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Figure 2—source data 7
Source data for Figure 2G.
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Figure 2—source data 8
Source data for Figure 2H.
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Figure 2—source data 9
Source data for Figure 2I.
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Figure 2—figure supplement 1
Purification and analysis of Pom proteins.
(A) SDS-PAGE analysis of purified proteins used in this study. Molecular size markers are shown on the left and the purified proteins including calculated MW on the right. Two µg per protein was …
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Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1A.
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Figure 2—figure supplement 1—source data 2
Source data for Figure 2—figure supplement 1B.
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Figure 2—figure supplement 1—source data 3
Source data for Figure 2—figure supplement 1C.
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Figure 2—figure supplement 1—source data 4
Source data for Figure 2—figure supplement 1D.
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Figure 2—figure supplement 1—source data 5
Source data for Figure 2—figure supplement 1E.
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Figure 2—figure supplement 1—source data 6
Source data for Figure 2—figure supplement 1F.
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Figure 3 with 1 supplement
PomXN harbors a conserved N-terminal peptide crucial for cell division site positioning at midcell.
(A) Multiple sequence alignment of the conserved PomX N-terminus. Black background indicates similar amino acids. Positively and negatively charged residues are indicated in blue and red, …
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Figure 3—source data 1
Source data for Figure 3B.
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Figure 3—source data 2
Source data for Figure 3D.
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Figure 3—figure supplement 1
PomX homologs are highly conserved.
Alignment of PomX homologs from other fully sequenced genomes of Myxobacteria. Sequences were aligned with MUSCLE and color-coded by homology using Bioedit. Black and white backgrounds indicate …
Figure 4 with 2 supplements
PomXK13AR15A forms clusters and interacts with PomY but not with PomZ in vivo.
(A-C) Fluorescence microscopy of cells of indicated genotypes. Phase-contrast (PH) images and/or overlays of fluorescence images and PH of representative cells. Red arrows indicate division …
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Figure 4—source data 1
Source data for Figure 4A.
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Figure 4—source data 2
Source data for Figure 4B.
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Figure 4—source data 3
Source data for Figure 4C.
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Figure 4—figure supplement 1
The PomXK13AR15A variant is impaired in function.
(A) Western blot analysis of the accumulation of mCh-PomX variants in indicated strains. Protein from the same number of cells was loaded per lane. Molecular mass marker is shown on the left and …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1A.
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Figure 4—figure supplement 1—source data 2
Source data for Figure 4—figure supplement 1C.
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Figure 4—figure supplement 2
Western blot analysis of PomY-mCh and PomZD90A-mCh accumulation.
(A) Western blot analysis of PomZD90A-mCh accumulation in indicated strains. Protein from the same number of cells was loaded per lane. Molecular mass markers are indicated on the left and analyzed …
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Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2A.
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Figure 4—figure supplement 2—source data 2
Source data for Figure 4—figure supplement 2B.
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Figure 5
PomX AAP activity resides in PomXNPEP.
(A) BACTH analysis of interactions between Pom proteins and PomX variants. Experiments were performed in parallel with those in Figure 2A. For presentation purposes, the results for PomXWT and PomXN …
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Figure 5—source data 1
Source data for Figure 5A.
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Figure 5—source data 2
Source data for Figure 5B.
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Figure 5—source data 3
Source data for Figure 5C.
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Figure 5—source data 4
Source data for Figure 5D.
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Figure 5—source data 5
Source data for Figure 5E.
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Figure 6
The PomX/PomZ interaction is important for cluster fission during division.
(A) Fluorescence time-lapse microscopy of mCh-PomX variants in cells of indicated genotypes. Overlays of representative mCh images and PH are shown in 20 min intervals. Stippled lines indicate cell …
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Figure 6—source data 1
Source data for Figure 6B.
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Figure 7
AAPs of MinD/ParA ATPases are diverse but share common features.
Left, domain analysis of known and predicted AAPs of ParA/MinD ATPases with key below. Sequences on the right, N-terminus of indicated proteins. Positively charged amino acids are indicated on blue, …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (M. xanthus) | pomX | NCBI | mxan_0636 new locus tag MXAN_RS03090 | |
| Gene (M. xanthus) | pomY | NCBI | mxan_0634 new locus tag MXAN_RS03080 | |
| Gene (M. xanthus) | pomZ | NCBI | mxan_0635 new locus tag MXAN_RS03085 | |
| Strain, strain background (E. coli) | Arctic Express DE3 RP | Agilent Technologies | E. coli B F– ompT hsdS(rB– mB–) dcm+TetR gal λ(DE3) endA Hte [cpn10 cpn60 GentR] | Used for protein expression |
| Strain, strain background (E. coli) | Rosetta 2 DE3 | Novagen | F- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2 (CamR) | Used for protein expression |
| Strain, strain background (E. coli) | NiCo21(DE3) | New England Biolabs | can::CBD fhuA2 [lon] ompT gal (λ DE3) [dcm] arnA::CBD slyD::CBD glmS6Ala ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 | Used for protein expression |
| Strain, strain background (E. coli) | NEB Turbo | New England Biolabs | F' proA+B+ lacIq(∆lacZM15/fhuA2 ∆(lac-proAB) glnV galK16 galE15 R(zgb-210::Tn10) TetS endA1 thi-1 ∆(hsdS-mcrB)5) | Used for cloning |
| Strain, strain background (M. xanthus) | DK1622 | DOI: 10.1073/pnas.76.11.5952 | Wildtype | |
| Strain, strain background (M. xanthus) | SA3108 | DOI: 10.1111/mmi.12094 | ΔpomZ | Strain with an in-frame deletion in pomZ |
| Strain, strain background (M. xanthus) | SA3146 | DOI: 10.1111/mmi.12094 | ΔpomZ; attB::Pmxan0635 pomZD90A-mCh (pKA43) | Strain expressing PomZD90A-mCh in a ΔpomZ background |
| Strain, strain background (M. xanthus) | SA4223 | https://doi.org/10.1016/j.devcel.2017.04.011 | ΔpomX | Strain with an in-frame deletion in pomX |
| Strain, strain background (M. xanthus) | SA4252 | https://doi.org/10.1016/j.devcel.2017.04.011 | ΔpomX; attB::Pmxan0635 mCh-pomX (pAH53) | Strain expressing mCh-PomX in a ΔpomX background |
| Strain, strain background (M. xanthus) | SA4703 | https://doi.org/10.1016/j.devcel.2017.04.011 | ΔpomY | Strain with an in-frame deletion in pomY |
| Strain, strain background (M. xanthus) | SA4712 | https://doi.org/10.1016/j.devcel.2017.04.011 | ΔpomY; attB::PpilA pomY-mCh (pDS7) | Strain expressing PomY-mCh in a ΔpomY background |
| Strain, strain background (M. xanthus) | SA4797 | https://doi.org/10.1016/j.devcel.2017.04.011 | ΔmglA; ΔpomX; attB::Pmxan0635 mCh-pomX (pAH53) | Strain expressing mCh-PomX in a non-motile ΔpomX background |
| Strain, strain background (M. xanthus) | SA4297 | this study | Wild-type; attB::Pmxan0635 mCh-pomX (pAH53) | Strain expressing mCh-PomX in WT background |
| Strain, strain background (M. xanthus) | SA6100 | this study | pomX::pomXK13AR15A | Strain with a replacement of pomX with the pomXK13AR15A allele |
| Strain, strain background (M. xanthus) | SA7014 | this study | ΔpomX; ΔpomZ; attB::Pmxan0635 pomZD90A-mCh (pKA43) | Strain expressing PomZD90A-mCh in a pomX and pomZ deletion background |
| Strain, strain background (M. xanthus) | SA7061 | this study | ΔmglA; ΔpomZ; ΔpomX; attB::Pmxan0635 mCh-pomX (pAH53) | Strain expressing mCh-PomX in a non-motile pomX and pomZ deletion background |
| Strain, strain background (M. xanthus) | SA7063 | this study | ΔpomZ; ΔpomX; attB::Pmxan0635 mCh-pomX (pAH53) | Strain expressing mCh-PomX in a pomX and pomZ deletion background |
| Strain, strain background (M. xanthus) | SA8240 | this study | pomX::pomXK13AR15A; ΔpomZ; attB::Pmxan0635 pomZD90A-mCh (pKA43) | Strain expressing PomZD90A-mCh in a pomZ deletion background with pomXK13AR15A mutation. |
| Strain, strain background (M. xanthus) | SA8250 | this study | pomX::pomXK13AR15A; ΔpomY; attB::PpilA pomY-mCh (pDS7) | Strain expressing PomZD90A-mCh in a pomXK13AR15A background |
| Strain, strain background (M. xanthus) | SA8268 | this study | pomX::pomXK13AR15A; ΔpomY; ΔpomZ; attB::Pmxan0635 pomZD90A-mCh (pKA43) | Strain expressing PomZD90A-mCh in a pomZ and pomY deletion background with pomXK13AR15A mutation |
| Strain, strain background (M. xanthus) | SA9700 | this study | pomX::pomXE6A | Strain with a replacement of pomX with the pomXE6A allele |
| Strain, strain background (M. xanthus) | SA9701 | this study | pomX::pomXQ7A | Strain with a replacement of pomX with the pomXQ7A allele |
| Strain, strain background (M. xanthus) | SA9702 | this study | pomX::pomXN8A | Strain with a replacement of pomX with the pomXN8A allele |
| Strain, strain background (M. xanthus) | SA9714 | this study | pomX::pomXR11A | Strain with a replacement of pomX with the pomXR11A allele |
| Strain, strain background (M. xanthus) | SA9715 | this study | pomX::pomXK3A | Strain with a replacement of pomX with the pomXK3A allele |
| Strain, strain background (M. xanthus) | SA9716 | this study | pomX::pomXR17A | Strain with a replacement of pomX with the pomXR17A allele |
| Strain, strain background (M. xanthus) | SA9717 | this study | pomX::pomXT22A | Strain with a replacement of pomX with the pomXT22A allele |
| Strain, strain background (M. xanthus) | SA9718 | this study | pomX::pomXK2A | Strain with a replacement of pomX with the pomXK2A allele |
| Strain, strain background (M. xanthus) | SA9719 | this study | pomX::pomXR15A | Strain with a replacement of pomX with the pomXR15A allele |
| Strain, strain background (M. xanthus) | SA9720 | this study | ΔpomY; ΔpomZ; attB::Pmxan0635 pomZD90A-mCh (pKA43) | Strain expressing PomZD90A-mCh in a pomZ and pomY deletion background |
| Strain, strain background (M. xanthus) | SA9721 | this study | ΔpomX; ΔpomY; attB::PpilA pomY-mCh (pDS7) | Strain expressing PomY-mCh in a pomY and pomX deletion background |
| Strain, strain background (M. xanthus) | SA9726 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXN (pDS252) | Strain expressing mCh-PomXN in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9727 | this study | Wild-type; attB::Pmxan0635 mCh-pomXN (pDS252) | Strain expressing mCh-PomXN in a WT background |
| Strain, strain background (M. xanthus) | SA9731 | this study | pomX::pomXK13A | Strain with a replacement of pomX with the pomXK13A allele |
| Strain, strain background (M. xanthus) | SA9732 | this study | pomX::pomXS10A | Strain with a replacement of pomX with the pomXS10A allele |
| Strain, strain background (M. xanthus) | SA9739 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXQ7A (pDS317) | Strain expressing mCh-PomXQ7A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9740 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXN8A (pDS318) | Strain expressing mCh-PomXN8A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9741 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXR17A (pDS323) | Strain expressing mCh-PomXR17A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9742 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXT22A (pDS324) | Strain expressing mCh-PomXT22A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9743 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXS10A (pDS319) | Strain expressing mCh-PomXS10A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9744 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXR11A (pDS320) | Strain expressing mCh-PomXR11A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9747 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXK13A (pDS321) | Strain expressing mCh-PomXK13A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9748 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXR15A (pDS322) | Strain expressing mCh-PomXR15A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9749 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXK2A (pDS314) | Strain expressing mCh-PomXK2A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9750 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXK3A (pDS315) | Strain expressing mCh-PomXK3A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9751 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXE6A (pDS316) | Strain expressing mCh-PomXE6A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9752 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXK13AR15A (pDS325) | Strain expressing mCh-PomXK13AR15A in a pomX deletion background |
| Strain, strain background (M. xanthus) | SA9753 | this study | ΔmglA; ΔpomX; attB::Pmxan0635 mCh-pomXK13AR15A (pDS325) | Strain expressing mCh-PomXK13AR15A in a non-motile pomX deletion background |
| Strain, strain background (M. xanthus) | SA9754 | this study | ΔmglA; ΔpomZ; ΔpomX; Pmxan0635 mCh-pomX (pAH53); mxan18-19::Pmxan0635 pomZD90A (pDS80) | Strain expressing mCh-PomX in a non-motile pomX and pomZ deletion background that expresses PomZD90A. |
| Strain, strain background (M. xanthus) | SA9755 | this study | Wild-type; attB::Pmxan0635 mCh-pomXC (pDS329) | Strain expressing mCh-PomXC in a WT background |
| Strain, strain background (M. xanthus) | SA9756 | this study | ΔpomY; attB::Pmxan0635 mCh-pomXC (pDS329) | Strain expressing mCh-PomXC in a pomY deletion background |
| Strain, strain background (M. xanthus) | SA9757 | this study | ΔpomZ; attB::Pmxan0635 mCh-pomXC (pDS329) | Strain expressing mCh-PomXC in a pomZ deletion background |
| Strain, strain background (M. xanthus) | SA9762 | this study | ΔpomX; attB::Pmxan0635 mCh-pomXC (pDS329) | Strain expressing mCh-PomXC in a pomX deletion background |
| Recombinant DNA reagent | pAH27 (plasmid) | https://doi.org/10.1016/j.devcel.2017.04.011 | Construct for in-frame deletion of pomX, KmR | |
| Recombinant DNA reagent | pAH53 (plasmid) | https://doi.org/10.1016/j.devcel.2017.04.011 | Pmxan0635 mCh-pomX, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS1 (plasmid) | https://doi.org/10.1016/j.devcel.2017.04.011 | Construct for in-frame deletion of pomY, KmR | |
| Recombinant DNA reagent | pDS7 (plasmid) | https://doi.org/10.1016/j.devcel.2017.04.011 | PpilA pomY-mCh, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS16 (plasmid) | https://doi.org/10.1016/j.devcel.2017.04.011 | Construct for in-frame deletion of pomY and pomZ, KmR | |
| Recombinant DNA reagent | pDS80 (plasmid) | https://doi.org/10.1016/j.devcel.2017.04.011 | Pmxan0635 pomZD90A, mxan_18–19 intergenic region, TcR | |
| Recombinant DNA reagent | pEMR3 (plasmid) | https://doi.org/10.1016/j.devcel.2017.04.011 | Overexpression of PomX-His6, KmR | |
| Recombinant DNA reagent | pKA1 (plasmid) | DOI: 10.1111/mmi.12094 | Construct for in-frame deletion of pomZ, KmR | |
| Recombinant DNA reagent | pKA3 (plasmid) | DOI: 10.1111/mmi.12094 | Overexpression of His6-PomZ, KmR | |
| Recombinant DNA reagent | pKA43 (plasmid) | DOI: 10.1111/mmi.12094 | Pmxan0635 pomZD90A-mCh, Mx8 attB, TcR | |
| Recombinant DNA reagent | pMAT12 (plasmid) | https://doi.org/10.1016/j.devcel.2017.04.011 | Construct for in-frame deletion of pomZ and pomX, KmR | |
| Recombinant DNA reagent | pAH152 (plasmid) | this study | Overexpression of PomXC-His6, KmR | |
| Recombinant DNA reagent | pSL16 (plasmid) | DOI: 10.1038/emboj.2011.291 | Construct for in-frame deletion of mglA, KmR | |
| Recombinant DNA reagent | pUT18 (plasmid) | https://doi.org/10.1073/pnas.95.10.5752 | BACTH plasmid | |
| Recombinant DNA reagent | pUT18C (plasmid) | https://doi.org/10.1073/pnas.95.10.5752 | BACTH plasmid | |
| Recombinant DNA reagent | pKT25 (plasmid) | https://doi.org/10.1073/pnas.95.10.5752 | BACTH plasmid | |
| Recombinant DNA reagent | pKNT25 (plasmid) | https://doi.org/10.1073/pnas.95.10.5752 | BACTH plasmid | |
| Recombinant DNA reagent | pAH154 (plasmid) | this study | Pmxan0635 mCh-pomXN, Mx8 attB, KmR | |
| Recombinant DNA reagent | pAH157 (plasmid) | this study | Overexpression of PomXN-His6, KmR | |
| Recombinant DNA reagent | pAH165 (plasmid) | this study | Overexpression of PomXN_K13AR15A-His6, KmR | |
| Recombinant DNA reagent | pDS100 (plasmid) | this study | BACTH plasmid for pomZ (pUT18C), AmpR | |
| Recombinant DNA reagent | pDS103 (plasmid) | this study | BACTH plasmid for pomX (pUT18C), AmpR | |
| Recombinant DNA reagent | pDS106 (plasmid) | this study | BACTH plasmid for pomX (pKT25), KmR | |
| Recombinant DNA reagent | pDS109 (plasmid) | this study | BACTH plasmid for pomZ (pUT18), AmpR | |
| Recombinant DNA reagent | pDS110 (plasmid) | this study | BACTH plasmid for pomX (pUT18), AmpR | |
| Recombinant DNA reagent | pDS114 (plasmid) | this study | BACTH plasmid for pomX (pKNT25) KmR | |
| Recombinant DNA reagent | pDS115 (plasmid) | this study | BACTH plasmid for pomZD90A (pUT18C), AmpR | |
| Recombinant DNA reagent | pDS117 (plasmid) | this study | BACTH plasmid for pomZD90A (pUT18), AmpR | |
| Recombinant DNA reagent | pDS120 (plasmid) | this study | BACTH plasmid for pomY (pUT18C), AmpR | |
| Recombinant DNA reagent | pDS122 (plasmid) | this study | BACTH plasmid for pomY (pUT18), AmpR | |
| Recombinant DNA reagent | pDS184 (plasmid) | this study | BACTH plasmid for pomXΔ2-21 (pUT18), AmpR | |
| Recombinant DNA reagent | pDS185 (plasmid) | this study | BACTH plasmid for pomXΔ2-21 (pUT18C), AmpR | |
| Recombinant DNA reagent | pDS186 (plasmid) | this study | BACTH plasmid for pomXΔ2-21 (pKT25), KmR | |
| Recombinant DNA reagent | pDS187 (plasmid) | this study | BACTH plasmid for pomXΔ2-21 (pKNT25) KmR | |
| Recombinant DNA reagent | pDS188 (plasmid) | this study | BACTH plasmid for pomXC (pUT18), AmpR | |
| Recombinant DNA reagent | pDS189 (plasmid) | this study | BACTH plasmid for pomXC (pUT18C), AmpR | |
| Recombinant DNA reagent | pDS190 (plasmid) | this study | BACTH plasmid for pomXC (pKT25), KmR | |
| Recombinant DNA reagent | pDS191 (plasmid) | this study | BACTH plasmid for pomXC (pKNT25) KmR | |
| Recombinant DNA reagent | pDS192 (plasmid) | this study | BACTH plasmid for pomXN (pUT18), AmpR | |
| Recombinant DNA reagent | pDS193 (plasmid) | this study | BACTH plasmid for pomXN (pUT18C), AmpR | |
| Recombinant DNA reagent | pDS194 (plasmid) | this study | BACTH plasmid for pomXN (pKT25), KmR | |
| Recombinant DNA reagent | pDS195 (plasmid) | this study | BACTH plasmid for pomXN (pKNT25) KmR | |
| Recombinant DNA reagent | pDS232 (plasmid) | this study | Overexpression of PomXN-Strep, KmR | |
| Recombinant DNA reagent | pDS252 (plasmid) | this study | Pmxan0635 mCh-pomXN, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS253 (plasmid) | this study | BACTH plasmid for pomXN_K13AR15A (pUT18), AmpR | |
| Recombinant DNA reagent | pDS254 (plasmid) | this study | BACTH plasmid for pomXN_K13AR15A (pUT18C), AmpR | |
| Recombinant DNA reagent | pDS255 (plasmid) | this study | BACTH plasmid for pomXN_K13AR15A (pKT25), KmR | |
| Recombinant DNA reagent | pDS256 (plasmid) | this study | BACTH plasmid for pomXN_K13AR15A (pKNT25) KmR | |
| Recombinant DNA reagent | pDS257 (plasmid) | this study | BACTH plasmid for pomXN_Δ2-21 (pUT18), AmpR | |
| Recombinant DNA reagent | pDS258 (plasmid) | this study | BACTH plasmid for pomXN_Δ2-21 (pUT18C), AmpR | |
| Recombinant DNA reagent | pDS259 (plasmid) | this study | BACTH plasmid for pomXN_Δ2-21 (pKT25), KmR | |
| Recombinant DNA reagent | pDS260 (plasmid) | this study | BACTH plasmid for pomXN_Δ2-21 (pKNT25) KmR | |
| Recombinant DNA reagent | pDS303 (plasmid) | this study | nat. site codon exchange for pomXK2A, KmR | |
| Recombinant DNA reagent | pDS304 (plasmid) | this study | nat. site codon exchange for pomXK3A, KmR | |
| Recombinant DNA reagent | pDS305 (plasmid) | this study | nat. site codon exchange for pomXE6A, KmR | |
| Recombinant DNA reagent | pDS306 (plasmid) | this study | nat. site codon exchange for pomXQ7A, KmR | |
| Recombinant DNA reagent | pDS307 (plasmid) | this study | nat. site codon exchange for pomXN8A, KmR | |
| Recombinant DNA reagent | pDS308 (plasmid) | this study | nat. site codon exchange for pomXS10A, KmR | |
| Recombinant DNA reagent | pDS309 (plasmid) | this study | nat. site codon exchange for pomXR11A, KmR | |
| Recombinant DNA reagent | pDS310 (plasmid) | this study | nat. site codon exchange for pomXK13A, KmR | |
| Recombinant DNA reagent | pDS311 (plasmid) | this study | nat. site codon exchange for pomXR15A, KmR | |
| Recombinant DNA reagent | pDS312 (plasmid) | this study | nat. site codon exchange for pomXR17A, KmR | |
| Recombinant DNA reagent | pDS313 (plasmid) | this study | nat. site codon exchange for pomXT22A, KmR | |
| Recombinant DNA reagent | pDS314 (plasmid) | this study | Pmxan0635 mCh-pomXK2A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS315 (plasmid) | this study | Pmxan0635 mCh-pomXK3A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS316 (plasmid) | this study | Pmxan0635 mCh-pomXE6A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS317 (plasmid) | this study | Pmxan0635 mCh-pomXQ7A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS318 (plasmid) | this study | Pmxan0635 mCh-pomXN8A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS319 (plasmid) | this study | Pmxan0635 mCh-pomXS10A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS320 (plasmid) | this study | Pmxan0635 mCh-pomXR11A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS321 (plasmid) | this study | Pmxan0635 mCh-pomXK13A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS322 (plasmid) | this study | Pmxan0635 mCh-pomXR15A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS323 (plasmid) | this study | Pmxan0635 mCh-pomXR17A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS324 (plasmid) | this study | Pmxan0635 mCh-pomXT22A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS325 (plasmid) | this study | Pmxan0635 mCh-pomXK13AR15A, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS329 (plasmid) | this study | Pmxan0635 mCh-pomXC, Mx8 attB, KmR | |
| Recombinant DNA reagent | pDS333 (plasmid) | this study | Overexpression of PomXC-Strep, KmR | |
| Recombinant DNA reagent | pEMR1 (plasmid) | this study | Overexpression of PomY-His6, KmR | |
| Recombinant DNA reagent | pSH1 (plasmid) | this study | nat. site codon exchange for pomXK13AR15A, KmR | |
| Recombinant DNA reagent | pSH36 (plasmid) | this study | BACTH plasmid for pomXK13AR15A (pKNT25) KmR | |
| Recombinant DNA reagent | pSH37 (plasmid) | this study | BACTH plasmid for pomXK13AR15A (pKT25), KmR | |
| Recombinant DNA reagent | pSH38 (plasmid) | this study | BACTH plasmid for pomXK13AR15A (pUT18), AmpR | |
| Recombinant DNA reagent | pSH39 (plasmid) | this study | BACTH plasmid for pomXK13AR15A (pUT18C), AmpR | |
| Recombinant DNA reagent | pSH58 (plasmid) | this study | Overexpression of PomXK13AR15A-His6, KmR | |
| Sequence-based reagent | pomX BTH fwd XbaI | this study | PCR primer | 5’-GCGTCTAGAGATGAAGAAAGCCTTTGAAC-3’ |
| Sequence-based reagent | pomX BTH rev KpnI | this study | PCR primer | 5’-GCGGGTACCCGGCGCACCGTGGCCTGAC-3’ |
| Sequence-based reagent | pomY BTH fwd XbaI | this study | PCR primer | 5’-GCGTCTAGAGGTGAGCGACGAGCGTCCG-3’ |
| Sequence-based reagent | pomY BTH rev KpnI | this study | PCR primer | 5’-GCGGGTACCCGAGCGGCGAAGTATTTGTG-3’ |
| Sequence-based reagent | pomZ BTH fwd XbaI | this study | PCR primer | 5’-GCGTCTAGAGATGGAAGCGCCGACGTAC-3’ |
| Sequence-based reagent | pomZ BTH rev KpnI | this study | PCR primer | 5’-GCGGGTACCCGGCCGGCCTGCTGGGTGCC-3’ |
| Sequence-based reagent | pomXΔ2–21 BTH fwd XbaI | this study | PCR primer | 5’-GCGTCTAGAGATGACGGGCCTCGTCGACCCC-3’ |
| Sequence-based reagent | pomXC BTH fwd XbaI | this study | PCR primer | 5’-GCGTCTAGAGATGGCCACCGTGGCGGAGGCG-3’ |
| Sequence-based reagent | pomXN BTH rev KpnI | this study | PCR primer | 5’-GCGGGTACCCGGGGCAGCGGCTCCGGGCG-3’ |
| Sequence-based reagent | 0636 up fwd | this study | PCR primer | 5’-GCGGGATCCGTCACCCCAAGCCATTC-3’ |
| Sequence-based reagent | PomX K2A rev native | this study | PCR primer | 5’-CAAAGGCTTTCGCCATGGTTCTCAG-3’ |
| Sequence-based reagent | PomX K2A fwd native | this study | PCR primer | 5’-CTGAGAACCATGGCGAAAGCCTTTG-3’ |
| Sequence-based reagent | 0636 HindIII rev stop | this study | PCR primer | 5’-GCGAAGCTTTCAGCGCACCGTGGCCTGAC-3’ |
| Sequence-based reagent | PomX K3A rev native | this study | PCR primer | 5’-CTGTTCAAAGGCCGCCTTCATGGTTC-3’ |
| Sequence-based reagent | PomX K3A fwd native | this study | PCR primer | 5’-GAACCATGAAGGCGGCCTTTGAACAG-3’ |
| Sequence-based reagent | PomX E6A rev | this study | PCR primer | 5’-GGACACGTTCTGCGCAAAGGCTTTCTT-3’ |
| Sequence-based reagent | PomX E6A fwd | this study | PCR primer | 5’-AAGAAAGCCTTTGCGCAGAACGTGTCC-3’ |
| Sequence-based reagent | PomX Q7A rev | this study | PCR primer | 5’-GCGGGACACGTTCGCTTCAAAGGCTTT-3’ |
| Sequence-based reagent | PomX Q7A fwd | this study | PCR primer | 5’-AAAGCCTTTGAAGCGAACGTGTCCCGC-3’ |
| Sequence-based reagent | PomX N8A rev | this study | PCR primer | 5’-GGCGCGGGACACCGCCTGTTCAAAGGC-3’ |
| Sequence-based reagent | PomX N8A fwd | this study | PCR primer | 5’-GCCTTTGAACAGGCGGTGTCCCGCGCC-3’ |
| Sequence-based reagent | PomX S10A rev | this study | PCR primer | 5’-CGGCTTGGCGCGCGCCACGTTCTGTTC-3’ |
| Sequence-based reagent | PomX S10A fwd | this study | PCR primer | 5’-GAACAGAACGTGGCGCGCGCCAAGCCG-3’ |
| Sequence-based reagent | PomX R11A rev | this study | PCR primer | 5’-GCGCGGCTTGGCCGCGGACACGTTCTG-3’ |
| Sequence-based reagent | PomX R11A fwd | this study | PCR primer | 5’-CAGAACGTGTCCGCGGCCAAGCCGCGC-3’ |
| Sequence-based reagent | PomX K13A rev | this study | PCR primer | 5’-GCGGAGGCGCGGCGCGGCGCGGGACAC-3’ |
| Sequence-based reagent | PomX K13A fwd | this study | PCR primer | 5’-GTGTCCCGCGCCGCGCCGCGCCTCCGC-3’ |
| Sequence-based reagent | PomX R15A rev | this study | PCR primer | 5’-GCCCAGGCGGAGCGCCGGCTTGGCGCG-3’ |
| Sequence-based reagent | PomX R15A fwd | this study | PCR primer | 5’-CGCGCCAAGCCGGCGCTCCGCCTGGGC-3’ |
| Sequence-based reagent | PomX R17A rev | this study | PCR primer | 5’-CAGCGCGCCCAGCGCGAGGCGCGGCTT-3’ |
| Sequence-based reagent | PomX R17A fwd | this study | PCR primer | 5’-AAGCCGCGCCTCGCGCTGGGCGCGCTG-3’ |
| Sequence-based reagent | PomX T22A rev | this study | PCR primer | 5’-GTCGACGAGGCCCGCCAGCGCGCCCAG-3’ |
| Sequence-based reagent | PomX T22A fwd | this study | PCR primer | 5’-CTGGGCGCGCTGGCGGGCCTCGTCGAC-3’ |
| Sequence-based reagent | PomX K13AR15A rev | this study | PCR primer | 5’-CAGAACGTGTCCCGCGCCGCGCCGGCCCTCCGCCTGGGCGCGCTG-3’ |
| Sequence-based reagent | PomX K13AR15A fwd | this study | PCR primer | 5’-CAGCGCGCCCAGGCGGAGGGCCGGCGCGGCGCGGGACACCTTCTG-3’ |
| Sequence-based reagent | mCherry XbaI fwd | this study | PCR primer | 5’-GCGTCTAGAGTGAGCAAGGGCGAGGAG-3’ |
| Sequence-based reagent | PomX K2A rev | this study | PCR primer | 5’-TTCAAAGGCTTTCGCCATGGCTCCGCC-3’ |
| Sequence-based reagent | PomX K2A fwd | this study | PCR primer | 5’-GGCGGAGCCATGGCGAAAGCCTTTGAA-3’ |
| Sequence-based reagent | KA348 | this study | PCR primer | 5’-GCCAAGCTTTCAGCGCACCGTGGCCTG-3’ |
| Sequence-based reagent | PomX K3A fwd | this study | PCR primer | 5’-GGAGCCATGAAGGCGGCCTTTGAACAG-3’ |
| Sequence-based reagent | PomX K3A rev | this study | PCR primer | 5’-CTGTTCAAAGGCCGCCTTCATGGCTCC-3’ |
| Sequence-based reagent | AH142 | this study | PCR primer | 5’-GGAATTCCATATGGCCACCGTGGCGGAGGCG-3’ |
| Sequence-based reagent | KA346 | this study | PCR primer | 5’-GCCAAGCTTGCGCACCGTGGCCTGACTC-3’ |
| Sequence-based reagent | AH143 | this study | PCR primer | 5’-CCCAAGCTTGGGCAGCGGCTCCGGGCG-3’ |
| Sequence-based reagent | NdeI PomX fwd | this study | PCR primer | 5’-GGAATTCCATATGAAGAAAGCCTTTGAACAG-3’ |
| Sequence-based reagent | AH144 | this study | PCR primer | 5’-GCCAAGCTTTCAGGGCAGCGGCTCCGGGCG-3’ |
| Sequence-based reagent | KA384 | this study | PCR primer | 5’-GCGGGATCCGGCGGAGCCATGAAGAAAGCCTTTGAACAG-3’ |
| Sequence-based reagent | DS276 | this study | PCR primer | 5’-GCGAAGCTTACTTCTCGAACTGTGGGTGACTCCAGCGCACCGTGGCCTGAC-3’ |
| Sequence-based reagent | DS277 | this study | PCR primer | 5’-GCGCCATGGCCACCGTGGCGGAGGCG-3’ |
| Sequence-based reagent | PomX BspHI fwd | this study | PCR primer | 5’-GCGTCATGAAGAAAGCCTTTGAACAGAACG-3’ |
| Sequence-based reagent | PomXN rev strep-tag | this study | PCR primer | 5’-GCGAAGCTTACTTCTCGAACTGTGGGTGACTCCAGGGCAGCGGCTCCGGGCG-3’ |
| Sequence-based reagent | NdeI-PomY fwd | this study | PCR primer | 5’-GGAATTCCATATGAGCGACGAGCGTCCGGAC-3’ |
| Sequence-based reagent | PomY C-term his rev | this study | PCR primer | 5’-CGGAAGCTTAGCGGCGAAGTATTTGTGC-3’ |
| Sequence-based reagent | AH141 | this study | PCR primer | 5’-GCGGGATCCGGCGGAGCCGCCACCGTGGCGGAGGCG-3’ |
| Antibody | α-PomX (rabbit, polyclonal) | https://doi.org/10.1016/j.devcel.2017.04.011 | Western Blot (1:15000) | |
| Antibody | α-PomY (rabbit, polyclonal) | https://doi.org/10.1016/j.devcel.2017.04.011 | Western Blot (1:15000) | |
| Antibody | α-PomZ (rabbit, polyclonal) | DOI: 10.1111/mmi.12094 | Western Blot (1:10000) | |
| Antibody | α-PilC (rabbit, polyclonal) | DOI:10.1111/j.1365–2958.2009.06891.x | Western Blot (1:3000) | |
| Antibody | α-mCherry (rabbit, polyclonal) | Biovision | Cat# 5993 | Western Blot (1:10000) |
| Antibody | horseradish-conjugated α-rabbit immunoglobulin G (goat,polyclonal) | Sigma-Aldrich | Cat# A0545-1ML | Western Blot (1:25000) |
| Peptide, recombinant protein | PomXNPEP | Thermo Scientific | MKKAFEQNVSRAKPRLRLGALT | |
| Peptide, recombinant protein | PomXNPEPK13AR15A | Thermo Scientific | MKKAFEQNVSRAAPALRLGALT | |
| Software, algorithm | Metamorph_v 7.5 | Molecular Devices | ||
| Software, algorithm | Oufti | DOI: 10.1111/mmi.13264 | http://www.oufti.org/ | |
| Software, algorithm | Matlab R2018a | MathWorks | ||
| Commercial assay or kit | Luminata Forte | Fisher scientific | Cat# 10394675 |
