1. Cell Biology
  2. Microbiology and Infectious Disease
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PomX, a ParA/MinD ATPase activating protein, is a triple regulator of cell division in Myxococcus xanthus

  1. Dominik Schumacher
  2. Andrea Harms
  3. Silke Bergeler
  4. Erwin Frey
  5. Lotte Søgaard-Andersen  Is a corresponding author
  1. Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch, Germany
  2. Arnold Sommerfeld Center for Theoretical Physics and Center for NanoScience, Department of Physics, Ludwig-Maximilians-Universität München, Germany
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Cite this article as: eLife 2021;10:e66160 doi: 10.7554/eLife.66160

Abstract

Cell division site positioning is precisely regulated but the underlying mechanisms are incompletely understood. In the social bacterium Myxococcus xanthus, the ~15 MDa tripartite PomX/Y/Z complex associates with and translocates across the nucleoid in a PomZ ATPase-dependent manner to directly position and stimulate formation of the cytokinetic FtsZ-ring at midcell, and then undergoes fission during division. Here, we demonstrate that PomX consists of two functionally distinct domains and has three functions. The N-terminal domain stimulates ATPase activity of the ParA/MinD ATPase PomZ. The C-terminal domain interacts with PomY and forms polymers, which serve as a scaffold for PomX/Y/Z complex formation. Moreover, the PomX/PomZ interaction is important for fission of the PomX/Y/Z complex. These observations together with previous work support that the architecturally diverse ATPase activating proteins of ParA/MinD ATPases are highly modular and use the same mechanism to activate their cognate ATPase via a short positively charged N-terminal extension.

Introduction

Accurate positioning of the cell division site ensures the formation of daughter cells of correct size and shape. In most bacteria, cell division initiates with positioning of the tubulin homolog FtsZ at the incipient division site (Du and Lutkenhaus, 2019). Subsequently, FtsZ polymerizes to form a dynamic ring-like structure, the so-called (Fts)Z-ring, which serves as a scaffold for recruitment of other components of the division machinery (Du and Lutkenhaus, 2019). Accordingly, systems that regulate division site positioning act at the level of Z-ring formation (Eswara and Ramamurthi, 2017). While the core components of the division machinery are conserved, the regulatory systems that ensure Z-ring positioning and, thus, the division site, are surprisingly diverse and also incompletely understood (Eswara and Ramamurthi, 2017). Interestingly, several of these systems have at their core a member of the ParA/MinD superfamily of P-loop ATPases that interacts with system-specific components to generate system-specific dynamic localization patterns that bring about correct Z-ring positioning (Lutkenhaus, 2012). These patterns include pole-to-pole oscillations in the Escherichia coli MinCDE system, bipolar gradient formation in the Caulobacter crescentus MipZ/ParB system, polar localization in the Bacillus subtilis MinCDJ/DivIVA system, and translocation across the nucleoid in the PomXYZ system of Myxococcus xanthus (Treuner-Lange and Søgaard-Andersen, 2014; Schumacher and Søgaard-Andersen, 2017).

ParA/MinD ATPases are key players in orchestrating the subcellular organization of bacterial cells and are not only involved in division site positioning but also in chromosome and plasmid segregation as well as positioning of other macromolecular complexes (Lutkenhaus, 2012). The function of ParA/MinD ATPases critically depends on the ATPase cycle during which they alternate between a monomeric form and a dimeric ATP-bound form (Leonard et al., 2005; Hu et al., 2003; Wu et al., 2011; Scholefield et al., 2011). Generally, ParA/MinD ATPases have a low intrinsic ATPase activity that is stimulated by a single cognate ATPase Activating Protein (AAP) (Lutkenhaus, 2012). While ParA/MinD ATPases share overall sequence conservation, the AAPs are much less conserved. The ParABS systems for chromosome and plasmid segregation and the E. coli MinCDE system have provided fundamental insights into how ParA/MinD ATPase/AAP pairs interact to establish dynamic localization patterns: The AAP ParB stimulates ATPase activity of ATP-bound ParA dimers bound non-specifically to DNA (Leonard et al., 2005; Bouet and Funnell, 1999; Hester and Lutkenhaus, 2007). In chromosome segregation systems, ParB binds to multiple parS sites at the origin of replication, forming a large complex (Lin and Grossman, 1998; Yamaichi et al., 2007; Mohl and Gober, 1997) while ATP-bound ParA dimers bind non-specifically to the nucleoid (Castaing et al., 2008; Hester and Lutkenhaus, 2007; Leonard et al., 2005; Scholefield et al., 2011). Upon replication, one of the duplicated ParB-parS complexes interacts with nucleoid-bound ParA dimers, thereby stimulating ATP hydrolysis and causing the release of ParA monomers from the nucleoid (Bouet and Funnell, 1999; Ptacin et al., 2010; Schofield et al., 2010; Vecchiarelli et al., 2013). Released ParA monomers undergo nucleotide exchange and rebind to the nucleoid (Bouet and Funnell, 1999; Ptacin et al., 2010; Schofield et al., 2010; Vecchiarelli et al., 2013). Repeated interactions between the large ParB-parS complex and nucleoid-bound ParA result in translocation of the ParB-parS complex to the opposite cell half (Vecchiarelli et al., 2014; Lim et al., 2014; Ptacin et al., 2010; Schofield et al., 2010). In the MinCDE system, the AAP MinE, which is non-homologous to ParB, stimulates ATPase activity of membrane- and ATP-bound MinD dimers (Hu and Lutkenhaus, 2001; Lackner et al., 2003; Park et al., 2011; Hu and Lutkenhaus, 2003; Szeto et al., 2002). In vivo, dimeric ATP-bound MinD forms a complex with the inhibitor of Z-ring formation MinC at the membrane (de Boer et al., 1991; Hu and Lutkenhaus, 1999; Hu and Lutkenhaus, 2003; Hu et al., 1999). Upon stimulation of MinD ATPase activity by MinE, the MinD/C complex is released from the membrane (Hu et al., 2002; Hu and Lutkenhaus, 2001; Lackner et al., 2003; Park et al., 2012). Subsequently, MinD undergoes nucleotide exchange and rebinds to the membrane together with MinC. These interactions ultimately result in the coupled pole-to-pole oscillations of the MinC/D complex and MinE (Hu and Lutkenhaus, 1999; Raskin and de Boer, 1999).

In the rod-shaped M. xanthus cells, Z-ring formation and positioning at midcell between two segregated chromosomes are stimulated by the tripartite PomX/Y/Z complex (Schumacher et al., 2017; Treuner-Lange et al., 2013; Harms et al., 2013). PomZ is a ParA/MinD ATPase while PomX and PomY separately have AAP activity and function to synergistically stimulate the low intrinsic ATPase activity of DNA- and ATP-bound dimeric PomZ (Schumacher et al., 2017; Treuner-Lange et al., 2013). PomX and PomY are non-homologous, and share homology with neither ParB nor MinE. The Pom proteins form a dynamically localized complex with an estimated size of ~15 MDa in vivo that is visible as a cluster by epifluorescence microscopy (Schumacher et al., 2017). This complex associates with the nucleoid via PomZ, and early during the cell cycle, it is positioned on the nucleoid away from midcell, that is off-center, close to the new cell pole (Schumacher et al., 2017). Subsequently, the complex translocates by biased random motion on the nucleoid to the midnucleoid, which coincides with midcell. At midnucleoid, the PomXYZ complex undergoes constrained motion and stimulates Z-ring formation. Intriguingly, during cell division, the PomX/Y/Z complex undergoes fission, with the two ‘portions’ segregating to the two daughters (Schumacher et al., 2017).

The Pom proteins interact in all three pairwise combinations in vitro and PomX is essential for cluster formation by PomY and PomZ in vivo (Schumacher et al., 2017): In vitro PomX spontaneously polymerizes in a cofactor-independent manner to form filaments, and alone forms a cluster in vivo. PomY bundles PomX filaments in vitro; in vivo PomY is recruited by PomX to form a PomX/Y complex, which is not associated with the nucleoid and stalled somewhere in cells. ATP- and DNA-bound PomZ dimers, but not monomeric PomZ, are recruited to the PomX/Y complex by interactions with PomX as well as PomY, resulting in the association of the PomX/Y/Z complex to the nucleoid. Due to the AAP activity of PomX and PomY, PomZ is rapidly turned over in the PomX/Y/Z complex and released in a monomeric form to the cytosol. Motion of the PomX/Y/Z complex depends on non-specific DNA binding and ATP hydrolysis by PomZ (Schumacher et al., 2017). Translocation to midnucleoid and constrained motion at midnucleoid arise from the continuous turnover of PomZ in the complex together with the diffusive PomZ flux on the nucleoid into the PomX/Y/Z complex (Schumacher et al., 2017). Finally, while all three Pom proteins are important for Z-ring formation at midcell, PomY and PomZ in the PomX/Y/Z complex are thought to be directly involved in recruiting FtsZ to the division site based on protein-protein interaction analyses (Schumacher et al., 2017).

The Pom system displays unusual spatiotemporal dynamics and is also unusual because it incorporates two AAPs. It remains elusive how PomX and PomY stimulate PomZ ATPase activity. With the long-term goal to understand the spatiotemporal dynamics of the Pom system, we focused on the function of PomX in vivo and in vitro. Here, we show that PomX is a triple regulator of cell division and displays three activities: The AAP activity resides in the N-terminal domain, the C-terminal domain serves as a scaffold for PomX/Y/Z complex formation, and the PomX/Z interaction is important for PomX/Y/Z complex fission during division. Moreover, our findings support the notion that that AAPs of ParA/MinD ATPases use the same mechanism to activate their cognate ATPase.

Results

PomX consists of two functional domains with distinct functions

PomX and PomY co-occur with PomZ in Cystobacterineae (Schumacher et al., 2017). PomZ sequences are highly conserved, whereas PomX and PomY sequences are more divergent (Figure 1A). In M. xanthus PomX, the region from residues 27 to 182 is Ala and Pro-rich (23% Pro; 19% Ala) and the region from residues 222 to 401 contains a coiled-coil domain (Figure 1B). Although PomX homologs are of different length, their overall architecture is similar and with a high level of similarity in the C-terminal regions while the N-terminal regions vary in length and similarity (Figure 1B). To analyze how PomX functions, we divided PomX into two parts. From hereon, we refer to these two parts as the N-terminal domain (PomXN, residues 1–213) and the C-terminal domain (PomXC, residues 214–404) (Figure 1C).

Figure 1 with 1 supplement see all
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 identity of PomX domains in different PomX homologs. Similarity and identity were calculated based on the domains of M. xanthus PomX shown in C. (C) PomX truncations used in this study. Numbers on top indicate the start and stop positions of the truncations relative to full-length PomXWT. (D) Cell length distribution of cells of indicated genotypes. Cells below stippled line are minicells. Numbers indicate mean cell length±STDEV. In the boxplots, boxes include the 25th and the 75th percentile, whiskers data points between the 10% and 90% percentile, outliers are shown as black dots. Black and red lines indicate the median and mean, respectively. Number of analyzed cells is indicated. In the complementation strains, pomX alleles were expressed from plasmids integrated in a single copy at the attB site. (E) Fluorescence microscopy of cells of indicated genotypes. Phase-contrast and fluorescence images of representative cells were overlayed. Numbers indicate fraction of cells with fluorescent clusters. Demographs show fluorescence signals of analyzed cells sorted according to length and with off-center signals to the right. Numbers in upper right indicate number of cells used to create demographs. Scale bar, 5 µm. (F) Fluorescence microscopy of cells of indicated genotypes. Images of representative cells and demographs were created as in (E). Scale bar, 5 µm. For experiments in D, E and F similar results were obtained in two independent experiments.

We fused the two PomX domains to mCherry (mCh) and expressed them ectopically (Figure 1—figure supplement 1A). mCh-PomXN, mCh-PomXC, and full-length mCh-PomXWT accumulated at or above PomXWT levels in ΔpomX and pomX+ cells (Figure 1—figure supplement 1B). WT M. xanthus cells have a cell length of 8.0 ± 1.8 µm (mean ± standard deviation (STDEV)), while ΔpomX cells are filamentous with a length of 13.1 ± 6.1 µm and also generate DNA-free minicells (Figure 1D). mCh-PomXWT complemented the division defect of the ΔpomX mutant in agreement with previous observations (Schumacher et al., 2017), while the truncated variants did not.

mCh-PomXWT formed a single well-defined cluster in 93% and 94% of ΔpomX and WT cells, respectively (Figure 1E). These clusters localized in the off-center position (defined as clusters outside the midcell region at 50 ± 5% of cell length) in short cells and at midcell in long cells (Figure 1E). The truncated mCh-PomX variants displayed diffuse localization in ΔpomX cells (Figure 1E). However, mCh-PomXC but not mCh-PomXN formed a single cluster in 90% of pomX+ cells and localized as mCh-PomXWT (Figure 1E). This cluster formation by mCh-PomXC was independent of PomY and PomZ (Figure 1F and Figure 1—figure supplement 1B), supporting that mCh-PomXC is integrated into the PomX/Y/Z cluster via interaction with PomX. As described (Schumacher et al., 2017), the PomX clusters were more elongated in the absence of PomY (Figure 1F). The incorporation of PomXC into the PomX/Y/Z complex interfered with neither PomX/Y/Z complex formation nor function (Figure 1D,E). We conclude that both PomX domains are essential for function and that mCh-PomXC can integrate into the PomX/Y/Z complex via interaction with PomX while mCh-PomXN cannot.

PomX AAP activity resides in PomXN

We used the bacterial adenylate cyclase two-hybrid system (BACTH) to test for protein-protein interactions involving the two PomX domains. In agreement with previous observations using purified proteins, full-length PomXWT self-interacted and interacted with PomY (Figure 2A). PomXC self-interacted and also interacted with full-length PomX and PomY, while PomXN neither self-interacted nor interacted with PomXWT, PomXC, or PomY. To test for interactions with PomZ, we used the two PomZ variants PomZWT and PomZD90A, the latter of which is locked in the DNA-binding, dimeric, ATP-bound form that interacts strongly with the PomX/PomY cluster in vivo (Schumacher et al., 2017). PomXWT and PomXN interacted with PomZWT and PomZD90A while PomXC did not; also, PomZD90A interacted more strongly with PomXN than PomZWT (Figure 2A).

Figure 2 with 1 supplement see all
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 control in upper left corner, leucine zipper of GCN4 fused to T25 and T18. For negative controls, co-transformations with empty plasmids were performed. Images show representative results and were performed in three independent experiments. (B) TEM images of negatively stained purified proteins. Proteins were applied to the EM grids alone or after mixing in a 1:1 molar ratio as indicated before staining. Scale bar, 200 nm. Images show representative results of several independent experiments. (C, D) In vitro pull-down experiments with purified PomXC-Strep, PomXN-Strep, PomXWT-His6, and PomY-His6. Instant Blue-stained SDS-PAGE shows load (L), flow-through (FL), wash (W), and elution (E) fractions using MagStrep XT beads in pull-down experiments with 10 µM of indicated proteins alone or pre-mixed as indicated on top. Molecular size markers are shown on the left and proteins analyzed on the right together with their calculated MW. Note that PomXWT-His6 (Schumacher et al., 2017) and PomXN-Strep migrate aberrantly and according to a higher MW. All samples in a panel were analyzed on the same gel and black lines are included for clarity. Experiments were repeated in two independent experiments with similar results. (E–I) His6-PomZ ATPase activity. ADP production rate was determined in an NADH-coupled photometric microplate assay in the presence of 1 mM ATP at 32°C. DNA and PomX variants were added as indicated. Spontaneous ATP hydrolysis and NADH consumption was accounted for by subtracting the measurements in the absence of His6-PomZ. Data points show the mean±STDEV calculated from six independent measurements.

Figure 2—source data 1

Source data for Figure 2A.

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Source data for Figure 2B.

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Source data for Figure 2C.

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Source data for Figure 2D.

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Source data for Figure 2E.

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Source data for Figure 2F.

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Source data for Figure 2G.

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Source data for Figure 2H.

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Source data for Figure 2I.

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To test for interactions between the Pom proteins in vitro, we purified tagged variants of PomXWT, PomXN, PomXC, PomY, and PomZ (Figure 2—figure supplement 1A). We confirmed by negative stain transmission electron microscopy (TEM) that PomXWT-His6 formed filaments that were bundled by PomY-His6, while PomY-His6 on its own did not form higher order structures (Figure 2B; Schumacher et al., 2017). Consistently, when analyzed separately in high-speed centrifugation experiments, 93–95% of PomXWT-His6 and 29–35% of PomY-His6 were recovered in the pellet fraction, whereas 71–86% and 48–49%, respectively of PomXWT-His6 and PomY-His6 were in the pellet fraction when mixed in equimolar amounts (Figure 2—figure supplement 1B,C,D).

As noted for PomXWT, PomXN-His6 (molecular weight (MW) of monomer: 24.3 kDa) migrated aberrantly in SDS-PAGE (Figure 2—figure supplement 1A). By size exclusion chromatography (SEC), the majority of PomXN-His6 eluted corresponding to a globular protein with a MW of ~136 kDa and in a smaller peak corresponding to a MW of ~306 kDa (Figure 2—figure supplement 1E). PomXN-His6 neither formed higher-order structures by TEM nor in high-speed centrifugation experiments (Figure 2B and Figure 2—figure supplement 1C,D). Because PomXN does not self-interact in BACTH, migrates aberrantly by SDS-PAGE, and is rich in Ala/Pro residues and, therefore, may not have a globular conformation, it is unclear whether SEC reflects the formation of PomXN oligomers. PomXC-His6 in SDS-PAGE migrated at the expected size (MW of monomer: 22.8 kDa) (Figure 2—figure supplement 1A); however, the protein could not be analyzed by SEC because it did not enter the matrix. Accordingly, PomXC-His6 spontaneously formed filaments visible by TEM and mostly accumulated in the pellet fraction in high-speed centrifugation experiments (Figure 2B and Figure 2—figure supplement 1B,D). PomY-His6 bundled the PomXC-His6 filaments and was enriched in the pellet fraction in centrifugation assays in the presence of PomXC-His6 (Figure 2B and Figure 2—figure supplement 1B). Interactions between PomXN-His6 and PomXWT-His6, PomXC-His6 and PomY-His6 were observed by neither TEM nor high-speed centrifugation (Figure 2B and Figure 2—figure supplement 1C,D). Finally, we confirmed in pull-down experiments using truncated PomX-Strep variants (Figure 2—figure supplement 1A) that PomXWT-His6 and PomY-His6 interact with PomXC-Strep (Figure 2C) but not with PomXN-Strep (Figure 2D) and that PomXC-His6 did not interact with PomXN-Strep (Figure 2—figure supplement 1F).

To test in vitro for interactions between PomX variants and PomZ, we used PomZ ATPase activity as a readout. First, we established a base-line for these analyses. The amount of hydrolyzed ATP increased with His6-PomZ concentration in the absence of DNA (specific activity: 7 ± 1 ATP hr−1, 4 µM His6-PomZ) (Figure 2E). In the presence of increasing concentrations of non-specific herring sperm DNA, His6-PomZ ATP hydrolysis was stimulated, reaching saturation at ~40 µg/ml DNA. At this concentration, His6-PomZ ATP hydrolysis was stimulated twofold (specific activity: ~15 ATP hr−1, 4 µM His6-PomZ) (Figure 2F). In the presence of saturating concentrations of DNA (60 µg/ml), His6-PomZ ATPase activity also increased with concentration (Figure 2E). For comparison, in an average M. xanthus cell with one chromosome, the DNA concentration is ~3400 µg/ml suggesting that PomZ in vivo works under fully DNA-saturating conditions.

PomXWT-His6 only stimulated His6-PomZ ATPase activity in the presence of DNA (Figure 2G). Stimulation increased with increasing PomXWT-His6 concentrations (specific activity: 44 ± 7 ATP hr−1 at 15 µM PomXWT-His6, 4 µM His6-PomZ), and His6-PomZ ATPase activity did not reach a plateau even at the highest PomXWT-His6 concentration (Figure 2G). Importantly, and in agreement with the BACTH analysis, PomXN-His6 stimulated His6-PomZ ATP hydrolysis in the presence of DNA as efficiently as PomXWT-His6 (specific activity: 43 ± 4 ATP hr−1 at 15 µM PomXN-His6, 4 µM His6-PomZ) (Figure 2H). By contrast, PomXC-His6 did not stimulate His6-PomZ ATPase activity (Figure 2I). The observations that PomXN does not spontaneously form filaments in vitro while full-length PomX does and these two PomX variants stimulate PomZ ATPase activity with equal efficiency provide evidence that PomX filament formation is not essential for AAP activity. These observations also strongly support that the PomXN domains in a PomX filament act independently of each other to stimulate PomZ ATPase activity.

Altogether, we conclude that (1) PomX consists of two domains with distinct functions that are both required for PomX activity in vivo; (2) PomXN interacts with PomZ and harbors the entire AAP activity; (3) PomXC is required and sufficient to mediate PomX self-interaction with spontaneous filament formation in vitro and PomX-dependent cluster incorporation in vivo; (4) PomXC interacts with PomY; and, (5) the PomXN domains in a PomX filament function independently of each other to stimulate PomZ ATPase activity.

Two positively charged residues in PomXNPEP are important for division site and PomX/Y/Z cluster positioning at midcell

To define the PomXN region involved in AAP activity, we performed a detailed sequence analysis of the N-terminal domain of PomX homologs. This analysis revealed a stretch of highly conserved amino acids at the N-terminus (residues 1–22, from hereon PomXNPEP) that is enriched in charged amino acids, six of which are positively charged (Figures 1B and 3A and Figure 3—figure supplement 1).

Figure 3 with 1 supplement see all
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, respectively. Weblogo consensus sequence is shown below. (B) Western blot analysis of accumulation of PomX variants. Protein from the same number of cells was loaded per lane. Molecular mass markers are indicated on the left. PilC was used as a loading control. (C) Phase-contrast microscopy of strains of indicated genotypes. Representative cells are shown. Red arrows indicate cell division constrictions. Scale bar, 5 µm. (D) Analysis of cell length distribution and cell division constrictions of cells of indicated genotypes. Left panel, boxplot is as in Figure 1D. Number of cells analyzed is indicated at the top. *p<0.001; **p<0.05 in Mann-Whitney test. Right panel, cell division position in % of cell length is plotted as a function of cell length. Dots represent mean ± STDEV. Numbers below indicate cell division constriction frequency. In B, C, and D, similar results were obtained in two independent experiments.

To probe the role of PomXNPEP, we generated PomX variants lacking residues 2–21 (PomXΔ2-21) with and without mCh; however, none of these variants accumulated in M. xanthus. Therefore, we performed Ala scanning of PomXNPEP in which all charged or hydrophilic residues were replaced by Ala. Then the mutant alleles replaced the pomXWT allele at the pomX locus. All 11 PomX variants except for PomXK3A accumulated similarly to or at slightly lower or higher levels than PomXWT (Figure 3B). Most strains had a cell length similar to WT and cell division constrictions at midcell (Figure 3C,D). As expected, pomXK3A cells were similar to ΔpomX cells (but see also details below about mCh-PomXK3A). More importantly, the pomXK13A and pomXR15A mutants generated filamentous cells and minicells, but the filamentous cells were shorter than ΔpomX cells (Figure 3D left panel). pomXK13A and pomXR15A cells had a cell division constriction frequency similar to WT (Figure 3D right panel), but these constrictions were mostly not at midcell (Figure 3C,D right panel). The pomXK13AR15A double mutant had a more pronounced filamentation phenotype than the single mutants, formed minicells, had a constriction frequency similar to WT, and mostly with the constrictions away from midcell (Figure 3C,D). PomXK13AR15A accumulated similarly to PomXWT (Figure 3B). We conclude that Lys13 and Arg15 in PomXNPEP are important for PomX function. Because the PomXK13A, PomXR15A, and PomXK13AR15A variants caused defects distinct from the ΔpomX mutant, i.e., cells are shorter, and with more constrictions, we conclude that substitution of Lys13 and/or Arg15 does not result in a complete loss of PomX function (see Discussion).

Next, we determined the localization of the PomXNPEP variants with Ala substitutions using mCh fusion proteins ectopically expressed in the ΔpomX mutant. All 11 fusion proteins with a single substitution as well as mCh-PomXK13AR15A accumulated at the same level as mCh-PomXWT in M. xanthus (Figure 4—figure supplement 1A). Most strains including the one expressing mCh-PomXK3A (cell length: 7.9 ± 1.8 µm) had a cell length similar to WT (cell length: 7.7 ± 1.7 µm) and cell division constrictions at midcell (Figure 4—figure supplement 1B) demonstrating that Lys3 is not important for PomX function. More importantly, mCh-PomXK13A, mCh-PomXR15A, and mCh-PomXK13AR15A formed clusters in vivo; however, these were mostly not at midcell and in the case of mCh-PomXK13AR15A ~50% localized in the DNA-free subpolar regions while this localization pattern was not observed for mCh-PomXWT (Figure 4A and Figure 4—figure supplement 1C). The clusters formed by these three mCh-PomX variants, similarly to those of mCh-PomXWT, colocalized with cell division constrictions. Overall, these observations demonstrate that mCh-PomXK13A, mCh-PomXR15A, and mCh-PomXK13AR15A are functional in forming clusters, defining the division site, and stimulating division but cannot correctly position the division site at midcell. All other PomX variants with substitutions in PomXNPEP, including mCh-PomXK3A, localized as mCh-PomXWT (Figure 4—figure supplement 1B).

Figure 4 with 2 supplements see all
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 constrictions. Scale bar, 5 µm. In A, numbers in overlays indicate fraction of cells with a cluster and numbers below indicate localization patterns in % and number of cells analyzed. Demographs are as in Figure 1E. Similar results were observed in two independent experiments.

Because recruitment of the PomX/Y cluster to the nucleoid and PomX/Y/Z cluster localization at midcell depend on PomZ, these observations pointed in the direction that the PomZ and PomX interaction involves Lys13 and Arg15. To test for an interaction between PomZ and PomXNPEP variants in vivo, we explored PomXK13AR15A in more detail and took advantage of the ATP-locked PomZD90A variant. In the presence of PomXWT, PomX/Y/ZD90A clusters are randomly positioned on the nucleoid and rarely at midcell due to lack of PomZ ATPase activity (Schumacher et al., 2017; Figure 4B). We confirmed that in the absence of PomX, PomZD90A-mCh did not form clusters and instead colocalized with the nucleoid, while PomZD90A-mCh still formed clusters in the absence of PomY (Figure 4B and Figure 4—figure supplement 2A). In the presence of PomXK13AR15A, PomZD90A-mCh formed clusters (Figure 4B). Because PomZ also interacts with PomY, we speculated that this cluster incorporation resulted from PomY recruiting PomZD90A-mCh. Indeed, upon additional deletion of pomY, PomZD90A-mCh no longer formed clusters and colocalized with the nucleoid when PomXK13AR15A was the only source of PomX (Figure 4B).

An active PomY-mCh fusion did not form clusters in the absence of PomX but formed clusters in the presence of PomXK13AR15A (Figure 4C and Figure 4—figure supplement 2B). As expected, in the presence of PomXK13AR15A, the PomY-mCh clusters generally localized away from midcell as opposed to clusters in the presence of PomXWT.

Altogether, these observations support that PomXNPEP is required for the interaction between PomX and PomZ but for neither PomX self-interaction nor PomX interaction with PomY. Moreover, they support that the PomX/Y/Z clusters formed in the presence of PomXK13AR15A are proficient in stimulating cell division but deficient in efficiently localizing to midcell, consistent with the PomX/PomZ interaction being perturbed. Finally, they support that PomZ in its ATP-bound dimeric form can be recruited independently by PomX and PomY to the PomX/Y complex.

PomXNPEP is required and sufficient for stimulation of PomZ ATP hydrolysis

We generated PomX constructs for BACTH analysis that were either truncated for PomXNPEP (PomXΔ2-21 and PomXN_Δ2-21) or contained substitutions in PomXNPEP (PomXK13AR15A and PomXN_K13AR15A). PomXK13AR15A and PomXΔ2-21 self-interacted, and also interacted with PomXWT and PomY (Figure 5A). Consistently, in vitro PomXK13AR15A-His6 formed filaments that were bundled by PomY-His6, accumulated in the pellet fraction after high-speed centrifugation, and brought PomY-His6 to the pellet fraction similarly to PomXWT-His6 (Figure 5B,C). As expected, in the BACTH neither PomXN_ Δ2-21 nor PomXN_K13AR15A interacted with PomXWT and PomY (Figure 5A). Importantly, all four PomXNPEP variants (PomXΔ2-21, PomXN_Δ2-21, PomXK13AR15A and PomXN_K13AR15A) were dramatically reduced in interaction with PomZ and PomZD90A (Figure 5A; see also Figure 2A). Altogether, these findings further support that PomXNPEP is specifically important for the PomX/PomZ interaction and not for PomX/PomX and PomX/PomY interactions.

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 T25 fusion proteins and their interaction with PomZ and PomZD90A T18 fusion proteins were not included but are included in Figure 2A. Images show representative results and similar results were obtained in three independent experiments. (B) TEM images of negatively stained purified proteins. Experiments were done as in Figure 2B. Scale bar, 200 nm. Images show representative results of several independent experiments. (C) Sedimentation assays with indicated purified proteins. Proteins were analyzed at a concentration of 3 µM alone or in combination. After high-speed centrifugation, proteins in the supernatant (S) and pellet (P) fractions were separated by SDS-PAGE and stained with Instant Blue. Molecular size markers are shown on the left and analyzed proteins on the right including their calculated MW. Numbers below indicate % of proteins in different fractions. Similar results were obtained in two independent experiments. All samples were analyzed on the same gel; the black line indicates that lanes were removed for presentation purposes. (D, E) His6-PomZ ATPase activity. Experiments were done and analyzed as in Figure 2E–I in the presence or absence of DNA and the indicated proteins and peptides. Data points show the mean±STDEV calculated from six independent measurements. In (D), stippled lines indicate the regression of the ADP production rate in the presence of PomXWT-His6 (left, Figure 2G) and PomXN-His6 (right, Figure 2H). In E, the stippled line indicates the regression of the ADP production rate in the presence of PomXWT-His6.

Next, we tested whether PomXNPEP is important for PomX AAP activity. Because His6-tagged truncated PomXNPEP variants could not be overexpressed in E. coli, we focused on the PomXK13AR15A-His6 and PomXN_K13RAR15A-His6 variants (Figure 2—figure supplement 1A). PomXN_K13AR15A-His6 (calculated MW: 24.1 kDa) behaved similarly to PomXN-His6 in SEC and eluted as a single peak corresponding to a globular protein of ~136 kDa (Figure 2—figure supplement 1E). Remarkably, neither PomXK13AR15A-His6 nor PomXN_K13AR15A-His6 stimulated PomZ ATPase activity (Figure 5D). Consequently, we tested whether a peptide consisting of the 22 PomXNPEP residues alone is sufficient to stimulate His6-PomZ ATPase activity. The PomXNPEP peptide alone stimulated His6-PomZ ATPase activity in the presence of DNA (specific activity: 27 ± 5 ATP hr−1 at 15 µM PomXNPEP, 4 µM His6-PomZ) while a PomXNPEP peptide with the K13AR15A substitutions did not (Figure 5E). We conclude that PomXNPEP is required and sufficient for stimulation of PomZ ATPase activity by PomX.

The PomX/PomZ interaction is important for PomX/Y/Z cluster fission during division

Twenty-four percent of cells containing mCh-PomXK13AR15A lacked a visible cluster compared to only 6% in the presence of mCh-PomXWT (Figure 4A). Tagged and untagged PomXK13AR15A accumulate at the same level as tagged and untagged PomXWT (Figure 3B and Figure 4—figure supplement 1A), suggesting that this difference in cluster formation is not caused by differences in gene expression or protein stability. We, therefore, investigated whether mCh-PomXK13AR15A causes a cluster fission defect during cell division.

In the presence of mCh-PomXWT, ~80% of divisions are accompanied by symmetric or asymmetric cluster fission, with each portion of a divided cluster segregating to a daughter (Figure 6A,B). In the remaining ~20%, cluster splitting did not visibly occur, the undivided cluster segregated to one of the daughters, and ‘empty’ daughter cells eventually regenerated a cluster that was visible after ~2 hr (Figure 6A). mCh-PomXK13AR15A clusters showed the same three patterns during division. However, cluster fission and segregation to daughters occurred in only ~20% of cells (Figure 6A,B).

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 division events. Orange and gray arrows mark mCh-PomX clusters in daughter cells after cell division with cluster fission and without cluster fission, respectively. Scale bar, 5 µm. (B) Quantification of cluster fission during cell division in cells of indicated genotypes. Cell division events were divided into those with (orange) and without (gray) cluster fission. Number of analyzed cell divisions is shown on top. The same results were obtained in two independent experiments.

Because PomXK13AR15A has reduced PomZ AAP activity, we tested cluster fission in ΔpomZ cells and in cells containing PomZD90A. In ΔpomZ cells, division occurs at a reduced frequency but still over the PomX/Y cluster (Schumacher et al., 2017). In these cells, mCh-PomXWT clusters rarely underwent fission and mostly segregated into one daughter (Figure 6A,B). In cells with PomZD90A, divisions also occurred over the cluster, and ~80% of clusters underwent fission during division and segregated to both daughters (Figure 6A,B). Altogether, these observations suggest that the interaction between PomZ and PomX is important for PomX/Y/Z cluster fission, while ATP hydrolysis by PomZ is not.

Discussion

In the present study, we used in vivo and in vitro approaches to functionally dissect the cell division regulatory protein PomX. We demonstrate that PomX is composed of two domains and has three functions in vivo. The N-terminal PomXN domain contains the PomZ AAP activity; the C-terminal PomXC domain is essential for PomX self-interaction, for the interaction with PomY, and functions as a scaffold for PomX/Y/Z cluster formation; and, the PomX/Z interaction, but not PomZ ATPase activity, is important for PomX/Y/Z cluster fission during division.

PomXN, which is Ala/Pro-rich and, therefore, likely unstructured, interacts with PomZ and activates ATPase activity of DNA-bound PomZ in vitro as efficiently as PomXWT. Moreover, a peptide comprising the N-terminal 22 residues of PomX (PomXNPEP) is sufficient to activate PomZ ATPase activity. In PomXNPEP, two positively charged residues (Lys13 and Arg15) are essential for AAP activity. Of note, the AAP activity of PomXNPEP was lower than that of PomXN. Similarly, in the case of the ParB homologs Spo0J of Thermus thermophilus and SopB of plasmid F of E. coli, as well as MinE of Neisseria gonorrhoeae, peptides comprising the N-terminal 20, 52, and 22 residues, respectively are sufficient for AAP activity (Ah-Seng et al., 2009; Ghasriani et al., 2010; Leonard et al., 2005). In the case of the shorter Spo0J and MinE peptides, the AAP activity was also lower than for the full-length proteins (Ghasriani et al., 2010; Leonard et al., 2005), while the longer SopB peptide was as efficient as full-length SopB (Ah-Seng et al., 2009). Because BACTH analyses did not reveal an interaction between PomZ and PomX variants lacking PomXNPEP or containing the K13AR15A substitutions, we speculate that the lower AAP activity of PomXNPEP indicates that the context of PomXNPEP might be important for its AAP activity; however, we cannot rule out that interactions between PomZ and PomXN beyond NPEP may also be important for PomZ ATPase activation.

PomXC with its predicted coiled-coil domain self-interacts and also interacts with PomXWT and PomY. Specifically, PomXC, similarly to PomXWT, formed filaments in vitro that were bundled by PomY. In vivo mCh-tagged PomXC integrated into clusters containing PomXWT but alone was not sufficient to form a cluster. By contrast, PomXN did not form or integrate into a cluster under any conditions tested; moreover, PomXN interacted with neither PomXWT nor PomY in BACTH or in vitro. Because PomXWT alone can form a cluster and is essential for PomX/Y/Z cluster formation in vivo, these observations support a model whereby PomXWT serves as a scaffold protein for cluster formation in vivo and in which PomXC has a key role in this scaffolding function by self-interacting and interacting with PomY. The observation that neither PomXN nor PomXC alone is sufficient to form a cluster in vivo suggests that these two domains may also interact. Our experiments suggest that these interactions are of low affinity because there were not detected by any of the methods used here (BACTH, TEM, high-speed centrifugation, and pull-down experiments). Alternatively, the conformation of the two separated domains could be different from that in PomXWT, and, therefore, no interactions were detected. Altogether, we suggest that PomXWT monomers in vivo interact via their coiled-coil domain in PomXC to spontaneously form a polymeric structure that is stabilized or modified by PomXN; this polymeric structure, in turn, serves as a scaffold to recruit PomY resulting in formation of the PomX/Y complex. PomZ in its ATP-bound dimeric form is recruited to this PomX/Y complex and associates it to the nucleoid. Because PomXWT and the likely unstructured PomXN stimulate PomZ ATPase activity to the same extent, these observations also strongly support that PomXWT monomers in the polymeric structure in vitro and cluster in vivo stimulate PomZ ATPase activity independently of each other. In vitro, purified PomXWT spontaneously polymerizes under all conditions tested. We speculate that this spontaneous polymerization ensures that one, and only one, PomX scaffold is formed per cell in vivo, thus guaranteeing that only one PomX/Y/Z complex is formed per cell as would be required for a complex that defines the site of cell division.

By analyzing PomZ variants, we previously showed that PomZ ATPase activity is essential for PomX/Y/Z cluster translocation and cluster localization to midcell. Because the PomX variants with reduced AAP activity resulted in the formation of PomX/Y/Z clusters that were typically not at midcell, we conclude that the low intrinsic PomZ ATPase activity is not sufficient to fuel translocation of the PomX/Y/Z cluster to midcell and that this translocation is fueled by PomX, and likely also by PomY, stimulated ATP hydrolysis by PomZ. In the PomX AAP mutants, constrictions were formed at WT frequencies over the PomX/Y/Z cluster. Because these clusters were typically not at midcell, these PomX variants resulted in divisions away from midcell giving rise to filamentous cells and minicells. Thus, PomX AAP mutants are fully competent in stimulating cell division but deficient in positioning the PomX/Y/Z cluster at midcell. These observations also support that PomX-stimulated PomZ ATPase activity is not important for stimulation of Z-ring formation. In cells lacking the PomX protein, PomY and PomZ do not form clusters and cell divisions occur at a low frequency (Schumacher et al., 2017); by contrast, the PomX AAP mutants still support PomX/Y/Z cluster formation and cell division. Thus, PomX AAP mutants retain partial PomX activity. Importantly, in mutants lacking PomZ, the PomX/Y cluster is also mostly away from midcell, and Z-rings and constrictions are formed over the cluster away from midcell, but at a much-reduced frequency compared to WT. Thus, PomX AAP mutants and a mutant lacking PomZ phenocopy each other with respect to PomX/Y/Z cluster positioning but not with respect to constriction frequency. We suggest that this difference is the result of different cluster compositions, that is the Pom clusters formed in the AAP mutants contain PomZ as well as PomY, which both interact with FtsZ (Schumacher et al., 2017), while the Pom clusters formed in the absence of PomZ only contain PomX and PomY. Thus, the division defects in Δpom mutants are a consequence of reduced formation and mislocalization of division constrictions, while the PomX AAP mutants are only deficient in cell division localization.

DNA binding by PomZ is important for the low intrinsic as well as PomX-stimulated ATPase activity. This is similar to what has been described for several DNA-binding ParA ATPases and their partner AAPs (Ah-Seng et al., 2009; Kiekebusch et al., 2012; Leonard et al., 2005; Lim et al., 2014; Scholefield et al., 2011; Schumacher et al., 2017). Similarly, MinE-dependent stimulation of ATP hydrolysis by a MinD variant that does not bind the membrane is reduced (Hu and Lutkenhaus, 2003). However, it remains unknown how DNA or membrane binding makes these ATPases competent for ATPase activity. Likewise, the precise molecular mechanism by which AAPs stimulate ATPase activity of their cognate ParA/MinD family ATPase remains unknown. However, in the case of the E. coli MinD/MinE system, it has been suggested that MinD ATPase activation by MinE involves the asymmetric interaction of the N-terminus of a MinE monomer with the ATP-bound MinD dimer (Park et al., 2012). Our data support that activation of many ParA/MinD family ATPases by their partner AAP may involve a shared mechanism: In agreement with the findings here that PomXNPEP is enriched in positively charged residues and that Lys13 and Arg15 are essential for PomX AAP activity, it has previously been noted that ParB-type AAPs, MinE-type AAPs and the non-homologous AAP ParG of plasmid TP228 that functions together with the ParA ATPase ParF contain a stretch of N-terminal residues rich in positively charged amino acids (Barillà et al., 2007; Leonard et al., 2005; Ah-Seng et al., 2009; Ghasriani et al., 2010; Figure 7). For several of these AAPs, it has been shown that one or more of the positively charged residues are important for AAP activity (Figure 7; Leonard et al., 2005; Scholefield et al., 2011; Barillà et al., 2007; Ah-Seng et al., 2009; Park et al., 2012; Hu and Lutkenhaus, 2001; Ghasriani et al., 2010). Also, as noted above, in the case of Spo0J of T. thermophilus, plasmid F SopB, and MinE of N. gonorrhoeae, 20, 52, and 22 N-terminal residues are sufficient for AAP activity. Thus, PomX is the fourth type of ParA/MinD AAP that displays this characteristic N-terminus and in which positively charged residues are important for AAP activity. Intriguingly, TlpT, which is the suggested AAP of the ParA-like ATPase PpfA involved in translocation and positioning of the large cytoplasmic chemoreceptor cluster in Rhodobacter sphaeroides (Roberts et al., 2012), and McdB, which is the AAP of the ParA-like ATPase McdA important for carboxysome translocation and positioning in Synechococcus elongatus (MacCready et al., 2018) both have an N-terminal extension enriched in positively charged residues (Figure 7). Altogether, these findings lend further support to the notion that many AAPs of ParA/MinD ATPases use the same mechanism to stimulate ATPase activity (Leonard et al., 2005; Park et al., 2011; Zhang and Schumacher, 2017; Barillà et al., 2007). Moreover, they support that ParA/MinD AAPs display remarkable plasticity and modularity in which a stretch of N-terminal amino acids enriched in positively charged residues grafted onto an interaction domain, which is involved in protein-protein, protein-DNA, or protein-membrane interaction, can generate an AAP. Interestingly, PomY, the second AAP of PomZ (Schumacher et al., 2017), does not have an N-terminus enriched in positively charged residues suggesting that its mode of action could be different from that of previously described AAPs.

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, and positively charged residues experimentally demonstrated to be important for AAP activity on red. Underlined sequences indicate peptides experimentally demonstrated to have AAP activity. Spo0J of T. thermophilus and B. subtilis, and SopB of plasmid F are ParB homologs.

In addition to similarities at the biochemical level, systems incorporating a DNA-binding ParA ATPase and an AAP(s) also share similarities in their translocation. The PomX/Y complex, ParB-parS complexes, cytoplasmic chemoreceptor clusters and carboxysomes are all large structures that are translocated as cargo on the nucleoid in a mechanism that depends on an AAP(s) stimulating ATP hydrolysis by the cognate DNA-binding ParA ATPase. Importantly, the AAP(s) is an integral part of the translocated cargo. We speculate that the integration of the AAP activity into a large cargo structure provides an elegant solution to guarantee that ATPase activation is spatially restricted to the transported cargo.

In addition to serving as a scaffold for PomX/Y/Z cluster formation and as a PomZ AAP, we report that PomX has a third important function in PomX/Y/Z cluster fission during division. In the presence of PomXWT, the cluster visibly undergoes fission during ~80% of divisions in otherwise WT cells, and those cells that do not receive a cluster slowly rebuild a cluster [here; (Schumacher et al., 2017)]. As a result, most WT cells contain a visible PomX/Y/Z cluster. By contrast, a PomX AAP mutant did not support fission as efficiently as PomXWT and fewer cells contain a cluster. We speculate that the reduced frequency of cluster fission events contributes to the cell division defect in the PomX AAP mutants. In cells lacking PomZ, the PomX/Y cluster also does not split efficiently during division, while the ATP-locked dimeric PomZD90A stimulates fission as efficiently as PomZWT. In all these mutants, division occurs over the Pom cluster. Additionally, in cells treated with cephalexin, which blocks cell division, cluster fission also does not occur (Schumacher et al., 2017; Treuner-Lange et al., 2013). Because cephalexin-treated cells still segregate their chromosomes (Schumacher et al., 2017; Treuner-Lange et al., 2013), these observations support that cell division is essential for fission, chromosome segregation is not sufficient, and the PomX/Z interaction is important while PomZ ATPase activity is not. This is in contrast to the SopB/SopA system for plasmid F segregation. For this system, it has been shown that SopB-stimulated ATPase activity of SopA is important to split plasmid clusters (Ah-Seng et al., 2013) suggesting that the splitting of plasmid clusters and PomX/Y/Z cluster fission may occur by different mechanisms. In the future, it will be interesting to establish the mechanism for PomX/Y/Z cluster fission in detail.

Materials and methods

M. xanthus and E. coli strains and growth

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Strains, plasmids, and primers are listed in the Key Resources Table. M. xanthus strains are derivatives of DK1622 WT (Kaiser, 1979). M. xanthus strains were cultivated in 1% CTT medium (1% casitone, 10 mM Tris-HCl pH 7.6, 1 mM KPO4 pH 7.6, 8 mM MgSO4) or on 1% CTT 1.5% agar plates (Hodgkin and Kaiser, 1977). Kanamycin, oxytetracycline, and gentamycin were added at concentrations of 50 µg/ml, 10 µg/ml, and 10 µg/ml, respectively. Growth was measured as an increase in optical density (OD) at 550 nm. M. xanthus cells were transformed by electroporation. In-frame deletions were generated as described (Shi et al., 2008). Plasmids were integrated by site-specific recombination at the Mx8 attB locus or by homologous recombination at the endogenous site. All plasmids were verified by sequencing. All strains were verified by PCR. Non-motile strains were generated to allow time-lapse microscopy for several hours by deletion of mglA (Miertzschke et al., 2011; Schumacher and Søgaard-Andersen, 2018). E. coli strains were grown in LB or 2xYT medium in the presence of relevant antibiotics or on LB plates containing 1.5% agar (Sambrook and Russell, 2001). Plasmids were propagated in E. coli NEB Turbo cells (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). Growth of E. coli was measured as an increase in OD at 600 nm.

Fluorescence microscopy and live cell imaging

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Fluorescence microscopy was performed as described (Schumacher et al., 2017). Briefly, exponentially growing cells were transferred to slides with a thin 1.0% agarose pad (SeaKem LE agarose, Cambrex) with TPM buffer (10 mM Tris-HCl pH 7.6, 1 mM KH2PO4 pH 7.6, 8 mM MgSO4), covered with a coverslip and imaged using a temperature-controlled Leica DMi6000B inverted microscope with an HCX PL FLUOTAR objective at 32°C. Phase-contrast and fluorescence images were recorded with a Hamamatsu ORCA-flash 4.0 sCMOS camera using the LASX software (Leica Microsystems). Time-lapse microscopy was performed as described (Schumacher and Søgaard-Andersen, 2018). Briefly, cells were transferred to a coverslip mounted on a metallic microscopy slide and covered with a pre-warmed 1% agarose pad supplemented with 0.2% casitone in TPM buffer. Slides were covered with parafilm to retain humidity of the agarose. Live-cell imaging was performed at 32°C. For DNA staining, cells were incubated with 1 mg/ml 2-(4-Amidinophenyl)−6-indolcarbamidine-dihydrochloride (DAPI) for 10 min at 32°C before microscopy. Image processing was performed with Metamorph_v 7.5 (Molecular Devices). For image analysis, cellular outlines were obtained from phase-contrast images using Oufti and manually corrected if necessary (Paintdakhi et al., 2016). Fluorescence microscopy image analysis was performed with a custom-made Matlab script (Matlab R2018a, MathWorks) available at https://github.com/SBergeler/ImageAnalysisMyxo copy archived at 500 cells from a dataset (unless otherwise stated) and calculating the length of the cells' centerlines (using meshes obtained from Oufti). Next, the fluorescence intensity of each cell was corrected by the background fluorescence locally around each cell. Cells were ordered by length and oriented according to the cell segment with the brightest intensity. The brightest 3% of the intensity values of all cells were set to the maximal intensity value, which is scaled to one, to be able to visualize fluorescence intensity variations inside the cells despite very bright clusters.

Bacterial two hybrid assay

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BACTH experiments were performed as described (Karimova et al., 1998). Relevant genes were cloned into the appropriate vectors to construct N-terminal and C-terminal fusions with the 25 kDa N-terminal or the 18 kDa C-terminal adenylate cyclase fragments of B. pertussis. Restoration of cAMP production was observed by the formation of blue color on LB agar supplemented with 80 µg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and 0.25 mM isopropyl-β-D-thiogalactoside (IPTG). As positive control, the leucine zipper from GCN4 was fused to the T18 and the T25 fragment. As a negative control, plasmids were co-transformed that only expressed the T18 or T25 fragment. All tested interactions were spotted on the same LB agar plates with positive control and all corresponding negative controls.

Western blot analysis

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Western blot analyses were performed as described (Sambrook and Russell, 2001) with rabbit polyclonal α-PomX (1:15000), α-PomY (1:15000), α-PomZ (1:10000) (Schumacher et al., 2017), α-PilC (1:3000) (Bulyha et al., 2009), or α-mCh (1:10000; Biovision) primary antibodies together with horseradish-conjugated goat α-rabbit immunoglobulin G (Sigma-Aldrich) as secondary antibody (1:25000). Blots were developed using Luminata Forte Western HRP Substrate (Millipore) and visualized using a LAS-4000 luminescent image analyzer (Fujifilm).

Protein purification

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His6-PomZ was purified from E. coli as described (Treuner-Lange et al., 2013). PomXWT-His6 and PomY-His6 were purified as described (Schumacher et al., 2017) using plasmids pEMR3 and pEMR1, respectively. PomXK13AR15A-His6 was purified as PomX-His6. Briefly, plasmid pSH58 was propagated in E. coli NiCo21(DE3) cells (NEB), grown in LB medium with 50 µg/ml kanamycin at 30°C to an OD600 of 0.6–0.7. Protein expression was induced with 0.4 mM IPTG for 16 hr at 18°C. Cells were harvested by centrifugation at 6000 g for 20 min at 4°C. Cells were washed with lysis buffer 1 (50mM NaH2PO4; 300 mM NaCl; 10 mM imidazole; pH 8.0 (adjusted with NaOH)) and lysed in 50 ml lysis buffer 2 (lysis buffer 1 with 0.1 mM EDTA; 1 mM β-mercaptoethanol; 100 mg/ml phenylmethylsulfonyl fluoride (PMSF); 1× complete protease inhibitor (Roche Diagnostics GmbH); 10 U/ml DNase 1) by sonication in three rounds of sonication for 5 min with a Branson Sonifier (Duty cycle 4; output control 40%) (Heinemann) on ice. Cell debris was removed by centrifugation at 4700 g for 45 min at 4°C. PomXK13AR15A-His6 was affinity purified with Protino Ni-NTA resin (Macherey-Nagel) from a batch, equilibrated in lysis buffer 1. PomXK13AR15A-His6 was eluted from the resin by washing 1× with 5 ml elution buffer 1 (lysis buffer 1 with 50 mM imidazole) and 3 x with 5 ml elution buffer 2 (lysis buffer 1 with 250 mM imidazole). Purified PomXK13AR15A-His6 was dialyzed 4× against 2 l dialysis buffer (50 mM Hepes/NaOH pH 7.2; 50 mM KCl; 0.1 mM EDTA; 1 mM β-mercaptoethanol; 10% (v/v) glycerol). Proteins were frozen in liquid nitrogen and stored at −80°C until used.

To purify PomXN-His6 plasmid pAH157 was propagated in E. coli NiCo21(DE3) cells, grown in 2×YT medium with 50 µg/ml kanamycin and 0.5% glucose at 30°C to an OD600 of 0.6–0.7. Protein expression was induced with 0.4 mM IPTG for 16 hr at 18°C. Cells were harvested and lysed as described for PomXK13AR15A. PomXN-His6 was affinity purified with Protino Ni-NTA resin (Macherey-Nagel) from a batch, equilibrated in lysis buffer 1. Contaminating proteins were eluted from the resin by washing 6× with 40 ml wash buffer 1 (lysis buffer 1 with 20 mM imidazole), 1× with 40 ml wash buffer 2 (lysis buffer 1 with 50 mM imidazole). PomXN-His6 was eluted from the resin by washing with 1 × 10 ml elution buffer 1 (lysis buffer 1 with 100 mM imidazole), 1× with 10 ml elution buffer 2 (lysis buffer 1 with 150 mM imidazole) and 1× with 10 ml elution buffer 3 (lysis buffer 1 with 200 mM imidazole). The elution fractions were pooled and loaded onto a HiLoad 16/600 Superdex 200 pg gel filtration column (GE Healthcare) that was equilibrated with dialysis buffer. Elution fractions were pooled. Proteins were frozen in liquid nitrogen and stored at −80°C until used.

For purification of PomXN_K13AR15A-His6 plasmid pAH165 was propagated in E. coli Rosetta2(DE3) cells, grown in 2×YT medium with 50 µg/ml kanamycin, 30 µg/ml chloramphenicol and 0.5% glucose at 30°C to an OD600 of 0.6–0.7. Protein expression was induced with 0.5 mM IPTG for 16 hr at 18°C. Cells were harvested and lysed as described for PomXK13AR15A. PomXN_K13AR15A-His6 was purified from cleared lysates with a 5 ml HiTrap Chelating HP column loaded with NiSO4 and equilibrated with lysis buffer 1. The column was washed with 20 column volumes (CVs) lysis buffer 1. The protein was eluted with elution buffer (50 mM NaH2PO4; 300 mM NaCl; 500 mM imidazole; pH 8.0 [adjusted with NaOH]) with a gradient of 20 CV. Fractions containing PomXN_K13AR15A-His6 were pooled and concentrated with an Amicon Ultra-15 centrifugation filter device with a cutoff of 3 kDa and loaded onto a HiLoad 16/600 Superdex 200 pg gel filtration column (GE Healthcare) that was equilibrated with dialysis buffer. Elution fractions were pooled. Proteins were frozen in liquid nitrogen and stored at −80°C until used.

To purify PomXC-His6 plasmid pAH152 was propagated in E. coli Rosetta2(DE3) cells, grown in 2×YT medium with 50 µg/ml kanamycin, 30 µg/ml chloramphenicol and 0.5% glucose at 37°C to an OD600 of 0.6–0.7. Protein expression was induced with 0.5 mM IPTG for 4 hr at 37°C. Cells were harvested and lysed as described for PomXK13AR15A. PomXC-His6 was affinity purified with Protino Ni-NTA resin (Macherey-Nagel) from a batch, equilibrated with lysis buffer 1. Contaminating proteins were eluted from the resin by washing 6× with 40 ml wash buffer 1 (lysis buffer 1 with 20 mM imidazole), 1× with 40 ml wash buffer 2 (lysis buffer 1 with 50 mM imidazole) and 2× with 40 ml wash buffer 3 (lysis buffer 1 with 100 mM imidazole) and 1× with 40 ml wash buffer 4 (lysis buffer 1 with 150 mM imidazole). PomXC-His6 was eluted from the resin with 3 × 10 ml elution buffer (lysis buffer 1 with 250 mM imidazole). The elution fractions were pooled and dialyzed against 4 × 2 l of dialysis buffer at 4°C. Proteins were frozen in liquid nitrogen and stored at −80°C until used.

For purification of PomXN-Strep, plasmid pDS232 was propagated in E. coli Rosetta2(DE3) cells, grown in LB medium with 50 µg/ml kanamycin, 30 µg/ml chloramphenicol and 0.5% glucose at 32°C to an OD600 of 0.6–0.7. Protein expression was induced with 0.5 mM IPTG for 2 hr at 32°C. Cells were harvested and lysed as described for PomXK13AR15A-His6 but in StrepTag lysis buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT)). PomXN-Strep was purified from cleared lysates with a 5 ml StrepTrap HP column equilibrated with StrepTag lysis buffer. The column was washed with 20 CV StrepTag lysis buffer. The protein was eluted with StrepTag elution buffer (StrepTag lysis buffer with 2.5 mM D-desthiobiotin). Fractions containing PomXN-Strep were pooled and dialyzed against 2 × 3 l of dialysis buffer at 4°C. Proteins were frozen in liquid nitrogen and stored at −80°C until used.

For purification of PomXC-Strep, plasmid pDS333 was propagated in E. coli Rosetta2(DE3) cells, grown in LB medium with 50 µg/ml kanamycin, 30 µg/ml chloramphenicol and 0.5% glucose at 32°C to an OD600 of 0.6–0.7. Protein expression was induced with 1 mM IPTG for 18 hr at 18°C. Cells were harvested and lysed as described for PomXN-Strep. PomXC-Strep was purified from a batch using 2 ml Strep-TactinXT 4Flow resin (iba), equilibrated with StrepTag lysis buffer. The resin was incubated with the cleared lysate for 1 hr at 4°C on a rotary shaker. Contaminating proteins were eluted from the resin by washing 5× with 10 ml StrepTag lysis buffer. The protein was eluted with 1× BXT buffer (100 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM EDTA, 50 mM biotin) (iba). Fractions containing PomXC-Strep were pooled and dialyzed against 2 × 5 l of dialysis buffer at 4°C. Proteins were frozen in liquid nitrogen and stored at −80°C until used.

Protein sedimentation assay

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Before sedimentation experiments, a clearing spin was performed for all proteins to be analyzed at 20,000 g for 10 min at 4°C. Proteins at a final concentration of 3 µM in a total volume of 50 µl were mixed and incubated for 1 hr at 32°C in a buffer (50 mM Hepes/NaOH, pH 7.2, 50 mM KCl, 1 mM β-mercaptoethanol, 10 mM MgCl2). Samples were separated into soluble and insoluble fractions by high-speed centrifugation (160,000 g, 60 min, 25°C). Insoluble and soluble fractions were separated, and volumes adjusted with 1× SDS sample buffer. Fractions were separated by SDS-PAGE and stained with Instant Blue (expedion) for 10 min.

In vitro pull-down experiments

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Ten µM protein alone or pre-mixed as indicated were incubated for 1 hr at 32°C in reaction buffer (50 mM HEPES/NaOH pH 7.2, 50 mM KCl, 10 mM MgCl2) in a total volume of 200 µl and applied to 20 µl 5% (v/v) MagStrepXT beads (iba) for 30 min. Magnetic beads were washed 10× with 200 µl reaction buffer. Proteins were eluted with 200 µl 1× BXT buffer (100 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM EDTA, 50 mM biotin) (iba). 10 µl per sample were separated by SDS-PAGE.

Negative stain transmission electron microscopy

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To fix and stain protein samples for negative stain TEM, 10 µl of a protein sample of interest (protein concentration before application onto the EM grid 3 µM) was applied onto an EM grid (Plano) and incubated for 1 min at 25°C. Residual liquid was blotted through the grid by applying the grid's unused side on Whatman paper. The grid was washed twice with double-distilled H2O. For staining, 10 µl of 1% uranyl acetate solution was applied onto the grid for 1 min and blotted through with a Whatman paper. If protein mixtures were applied to the EM grid, proteins of interest at a concentration of 3 µM were pre-mixed in a low-binding microtube (Sarstedt) and incubated for 10 min at 25°C before application onto the EM grid. Finished grids were stored in a grid holder for several months at room temperature. Electron microscopy was performed with a CM120 electron microscope (FEI) at 120kV.

ATPase assay

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ATP hydrolysis was determined using a 96-well NADH-coupled enzymatic assay (Kiianitsa et al., 2003) with modifications. Protein concentration was determined using Protein Assay Dye Reagent Concentrate (BioRad). Assays were performed in reaction buffer (50 mM HEPES/NaOH pH 7.2, 50 mM KCl, 10 mM MgCl2) with 0.5 mM nicotinamide adenine dinucleotide (NADH) and 2 mM phosphoenolpyruvate and 3 µl of a pyruvate kinase/lactate dehydrogenase mix (PYK/LDH; Sigma). PomXNPEP and PomXNPEP_K13AR15A peptides (MKKAFEQNVSRAKPRLRLGALT and MKKAFEQNVSRAAPALRLGALT) were purchased from Thermo Scientific. If appropriate, herring sperm DNA was added at a concentration of 60 µg/ml unless otherwise stated. Buffer was pre-mixed with proteins in low-binding microtubes (Sarstedt) on ice. To correct for glycerol in the assays, dialysis buffer was added if necessary. A total of 100 µl mixtures were transferred into transparent UV-STAR µCLEAR 96-well microplates (Greiner bio-one). The reaction was started by the addition of 1 mM ATP. Measurements were performed in an infinite M200PRO (Tecan) for 2 hr in 30 s intervals at 32°C shaking at 340 nm wavelength. To account for background by spontaneous ATP hydrolysis and UV-induced NADH decomposition, all assays were performed without the addition of His6-PomZ and measurements were subtracted. The light path was determined experimentally with known NADH concentrations to be 0.248 cm. The extinction coefficient of NADH ε340 = 6220 M−1cm−1 was used.

Analytical size-exclusion chromatography

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Experiments were carried out in dialysis buffer (50 mM Hepes/NaOH pH 7.2; 50 mM KCl; 0.1 mM EDTA; 1 mM β-mercaptoethanol; 10% (v/v) glycerol). PomXN-His6 and PomXN_K13AR15A-His6 were applied onto a Superdex 200 10/300 GL gel filtration column equilibrated with dialysis buffer. Blue dextran (2000 kDa), ferritin (440 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), RNAse A (13.7 kDa), and aprotinin (6.5 kDa) were used as standards with the same buffer conditions to calibrate the column.

Bioinformatics

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Gene and protein sequences of PomX, PomY, and PomZ were obtained from NCBI. PomX homologs were identified in a best-best hit reciprocal BlastP analysis from fully-sequenced genomes of Myxobacteria (Altschul et al., 1990). The similarity and identity of proteins were calculated from pairwise sequence alignments with EMBOSS Needle (Madeira et al., 2019). Domain analyses were performed with SMART (Letunic and Bork, 2018), PROSITE and Pfam (El-Gebali et al., 2019). Multiple sequence alignments were created with MUSCLE (Madeira et al., 2019) and further edited with Bioedit (https://bioedit.software.informer.com/7.2/). Consensus sequences of multiple sequence protein alignments were created with Weblogo 3 (Crooks et al., 2004). Proteins used: PomXMx (ABF89666; MXAN_0636), PomXMm (ATB45064; MYMAC_000648), PomXMh (AKQ69458; A176_006370), PomXMf (AKF79435; MFUL124B02_03625), PomXMs (AGC41991; MYSTI_00641), PomXCc (AFE03552; COCOR_00544), PomXMb (ATB30647; MEBOL_004108), PomXCf (ATB35470; CYFUS_000883), PomXAg (AKI99966; AA314_01593), PomXSa (ADO75429; STAUR_7674), PomXVi (AKU92216; AKJ08_2603), PomXAd (ABC83600; Adeh_3834). Spo0J Tt (AAS81946.1), Spo0J Bs (P26497.2) ParG TP228 Ec (WP_139578510.1), SopB F Ec (BAA97917.1), MinE Ng (AAK30127.1), MinE Ec (EFB7450413.1), TlpT Rs (ABA79218.1), McdB Se (ABB57864.1).

Statistics

The mean and standard deviation (STDEV) were calculated with Excel 2016. Localization patterns from fluorescence microscopy data were quantified based on the indicated n-value per strain. Boxplots were generated with SigmaPlot 14.0 (Systat). Statistical analysis was performed with SigmaPlot 14.0. All data sets were tested for normality using a Shapiro-Wilk test. For data with a non-normal distribution, a Mann-Whitney test was applied to test for significant differences.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (M. xanthus)pomXNCBImxan_0636
new locus tag
MXAN_RS03090
Gene (M. xanthus)pomYNCBImxan_0634
new locus tag
MXAN_RS03080
Gene (M. xanthus)pomZNCBImxan_0635
new locus tag
MXAN_RS03085
Strain, strain background (E. coli)Arctic Express DE3 RPAgilent TechnologiesE. 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 DE3NovagenF- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2 (CamR)Used for protein expression
Strain, strain background (E. coli)NiCo21(DE3)New England Biolabscan::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 ∆nin5Used for protein expression
Strain, strain background (E. coli)NEB TurboNew England BiolabsF' 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)DK1622DOI: 10.1073/pnas.76.11.5952Wildtype
Strain, strain background (M. xanthus)SA3108DOI: 10.1111/mmi.12094ΔpomZStrain with an in-frame deletion in pomZ
Strain, strain background (M. xanthus)SA3146DOI: 10.1111/mmi.12094ΔpomZ; attB::Pmxan0635 pomZD90A-mCh (pKA43)Strain expressing PomZD90A-mCh in a ΔpomZ background
Strain, strain background (M. xanthus)SA4223https://doi.org/10.1016/j.devcel.2017.04.011ΔpomXStrain with an in-frame deletion in pomX
Strain, strain background (M. xanthus)SA4252https://doi.org/10.1016/j.devcel.2017.04.011ΔpomX; attB::Pmxan0635 mCh-pomX (pAH53)Strain expressing mCh-PomX in a ΔpomX background
Strain, strain background (M. xanthus)SA4703https://doi.org/10.1016/j.devcel.2017.04.011ΔpomYStrain with an in-frame deletion in pomY
Strain, strain background (M. xanthus)SA4712https://doi.org/10.1016/j.devcel.2017.04.011ΔpomY; attB::PpilA pomY-mCh (pDS7)Strain expressing PomY-mCh in a ΔpomY background
Strain, strain background (M. xanthus)SA4797https://doi.org/10.1016/j.devcel.2017.04.011ΔmglA; ΔpomX; attB::Pmxan0635 mCh-pomX (pAH53)Strain expressing mCh-PomX in a non-motile ΔpomX background
Strain, strain background (M. xanthus)SA4297this studyWild-type; attB::Pmxan0635 mCh-pomX (pAH53)Strain expressing mCh-PomX in WT background
Strain, strain background (M. xanthus)SA6100this studypomX::pomXK13AR15AStrain with a replacement of pomX with the pomXK13AR15A allele
Strain, strain background (M. xanthus)SA7014this studyΔpomX; ΔpomZ; attB::Pmxan0635 pomZD90A-mCh (pKA43)Strain expressing PomZD90A-mCh in a pomX and pomZ deletion background
Strain, strain background (M. xanthus)SA7061this studyΔmglA; 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)SA7063this studyΔpomZ; ΔpomX; attB::Pmxan0635 mCh-pomX (pAH53)Strain expressing mCh-PomX in a pomX and pomZ deletion background
Strain, strain background (M. xanthus)SA8240this studypomX::pomXK13AR15A; ΔpomZ; attB::Pmxan0635 pomZD90A-mCh (pKA43)Strain expressing PomZD90A-mCh in a pomZ deletion background with pomXK13AR15A mutation.
Strain, strain background (M. xanthus)SA8250this studypomX::pomXK13AR15A; ΔpomY; attB::PpilA pomY-mCh (pDS7)Strain expressing PomZD90A-mCh in a pomXK13AR15A background
Strain, strain background (M. xanthus)SA8268this studypomX::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)SA9700this studypomX::pomXE6AStrain with a replacement of pomX with the pomXE6A allele
Strain, strain background (M. xanthus)SA9701this studypomX::pomXQ7AStrain with a replacement of pomX with the pomXQ7A allele
Strain, strain background (M. xanthus)SA9702this studypomX::pomXN8AStrain with a replacement of pomX with the pomXN8A allele
Strain, strain background (M. xanthus)SA9714this studypomX::pomXR11AStrain with a replacement of pomX with the pomXR11A allele
Strain, strain background (M. xanthus)SA9715this studypomX::pomXK3AStrain with a replacement of pomX with the pomXK3A allele
Strain, strain background (M. xanthus)SA9716this studypomX::pomXR17AStrain with a replacement of pomX with the pomXR17A allele
Strain, strain background (M. xanthus)SA9717this studypomX::pomXT22AStrain with a replacement of pomX with the pomXT22A allele
Strain, strain background (M. xanthus)SA9718this studypomX::pomXK2AStrain with a replacement of pomX with the pomXK2A allele
Strain, strain background (M. xanthus)SA9719this studypomX::pomXR15AStrain with a replacement of pomX with the pomXR15A allele
Strain, strain background (M. xanthus)SA9720this studyΔpomY; ΔpomZ; attB::Pmxan0635 pomZD90A-mCh (pKA43)Strain expressing PomZD90A-mCh in a pomZ and pomY deletion background
Strain, strain background (M. xanthus)SA9721this studyΔpomX; ΔpomY; attB::PpilA pomY-mCh (pDS7)Strain expressing PomY-mCh in a pomY and pomX deletion background
Strain, strain background (M. xanthus)SA9726this studyΔpomX; attB::Pmxan0635 mCh-pomXN (pDS252)Strain expressing mCh-PomXN in a pomX deletion background
Strain, strain background (M. xanthus)SA9727this studyWild-type; attB::Pmxan0635 mCh-pomXN (pDS252)Strain expressing mCh-PomXN in a WT background
Strain, strain background (M. xanthus)SA9731this studypomX::pomXK13AStrain with a replacement of pomX with the pomXK13A allele
Strain, strain background (M. xanthus)SA9732this studypomX::pomXS10AStrain with a replacement of pomX with the pomXS10A allele
Strain, strain background (M. xanthus)SA9739this studyΔpomX; attB::Pmxan0635 mCh-pomXQ7A (pDS317)Strain expressing mCh-PomXQ7A in a pomX deletion background
Strain, strain background (M. xanthus)SA9740this studyΔpomX; attB::Pmxan0635 mCh-pomXN8A (pDS318)Strain expressing mCh-PomXN8A in a pomX deletion background
Strain, strain background (M. xanthus)SA9741this studyΔpomX; attB::Pmxan0635 mCh-pomXR17A (pDS323)Strain expressing mCh-PomXR17A in a pomX deletion background
Strain, strain background (M. xanthus)SA9742this studyΔpomX; attB::Pmxan0635 mCh-pomXT22A (pDS324)Strain expressing mCh-PomXT22A in a pomX deletion background
Strain, strain background (M. xanthus)SA9743this studyΔpomX; attB::Pmxan0635 mCh-pomXS10A (pDS319)Strain expressing mCh-PomXS10A in a pomX deletion background
Strain, strain background (M. xanthus)SA9744this studyΔpomX; attB::Pmxan0635 mCh-pomXR11A (pDS320)Strain expressing mCh-PomXR11A in a pomX deletion background
Strain, strain background (M. xanthus)SA9747this studyΔpomX; attB::Pmxan0635 mCh-pomXK13A (pDS321)Strain expressing mCh-PomXK13A in a pomX deletion background
Strain, strain background (M. xanthus)SA9748this studyΔpomX; attB::Pmxan0635 mCh-pomXR15A (pDS322)Strain expressing mCh-PomXR15A in a pomX deletion background
Strain, strain background (M. xanthus)SA9749this studyΔpomX; attB::Pmxan0635 mCh-pomXK2A (pDS314)Strain expressing mCh-PomXK2A in a pomX deletion background
Strain, strain background (M. xanthus)SA9750this studyΔpomX; attB::Pmxan0635 mCh-pomXK3A (pDS315)Strain expressing mCh-PomXK3A in a pomX deletion background
Strain, strain background (M. xanthus)SA9751this studyΔpomX; attB::Pmxan0635 mCh-pomXE6A (pDS316)Strain expressing mCh-PomXE6A in a pomX deletion background
Strain, strain background (M. xanthus)SA9752this studyΔpomX; attB::Pmxan0635 mCh-pomXK13AR15A (pDS325)Strain expressing mCh-PomXK13AR15A in a pomX deletion background
Strain, strain background (M. xanthus)SA9753this studyΔmglA; ΔpomX; attB::Pmxan0635 mCh-pomXK13AR15A (pDS325)Strain expressing mCh-PomXK13AR15A in a non-motile pomX deletion background
Strain, strain background (M. xanthus)SA9754this studyΔmglA; 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)SA9755this studyWild-type; attB::Pmxan0635 mCh-pomXC (pDS329)Strain expressing mCh-PomXC in a WT background
Strain, strain background (M. xanthus)SA9756this studyΔpomY; attB::Pmxan0635 mCh-pomXC (pDS329)Strain expressing mCh-PomXC in a pomY deletion background
Strain, strain background (M. xanthus)SA9757this studyΔpomZ; attB::Pmxan0635 mCh-pomXC (pDS329)Strain expressing mCh-PomXC in a pomZ deletion background
Strain, strain background (M. xanthus)SA9762this studyΔpomX; attB::Pmxan0635 mCh-pomXC (pDS329)Strain expressing mCh-PomXC in a pomX deletion background
Recombinant DNA reagentpAH27
(plasmid)
https://doi.org/10.1016/j.devcel.2017.04.011Construct for in-frame deletion of pomX, KmR
Recombinant DNA reagentpAH53
(plasmid)
https://doi.org/10.1016/j.devcel.2017.04.011Pmxan0635 mCh-pomX, Mx8 attB, KmR
Recombinant DNA reagentpDS1
(plasmid)
https://doi.org/10.1016/j.devcel.2017.04.011Construct for in-frame deletion of pomY, KmR
Recombinant DNA reagentpDS7
(plasmid)
https://doi.org/10.1016/j.devcel.2017.04.011PpilA pomY-mCh, Mx8 attB, KmR
Recombinant DNA reagentpDS16
(plasmid)
https://doi.org/10.1016/j.devcel.2017.04.011Construct for in-frame deletion of pomY and pomZ, KmR
Recombinant DNA reagentpDS80
(plasmid)
https://doi.org/10.1016/j.devcel.2017.04.011Pmxan0635 pomZD90A, mxan_18–19 intergenic region, TcR
Recombinant DNA reagentpEMR3
(plasmid)
https://doi.org/10.1016/j.devcel.2017.04.011Overexpression of PomX-His6, KmR
Recombinant DNA reagentpKA1
(plasmid)
DOI: 10.1111/mmi.12094Construct for in-frame deletion of pomZ, KmR
Recombinant DNA reagentpKA3
(plasmid)
DOI: 10.1111/mmi.12094Overexpression of His6-PomZ, KmR
Recombinant DNA reagentpKA43
(plasmid)
DOI: 10.1111/mmi.12094Pmxan0635 pomZD90A-mCh, Mx8 attB, TcR
Recombinant DNA reagentpMAT12
(plasmid)
https://doi.org/10.1016/j.devcel.2017.04.011Construct for in-frame deletion of pomZ and pomX, KmR
Recombinant DNA reagentpAH152
(plasmid)
this studyOverexpression of PomXC-His6, KmR
Recombinant DNA reagentpSL16
(plasmid)
DOI: 10.1038/emboj.2011.291Construct for in-frame deletion of mglA, KmR
Recombinant DNA reagentpUT18
(plasmid)
https://doi.org/10.1073/pnas.95.10.5752BACTH plasmid
Recombinant DNA reagentpUT18C
(plasmid)
https://doi.org/10.1073/pnas.95.10.5752BACTH plasmid
Recombinant DNA reagentpKT25
(plasmid)
https://doi.org/10.1073/pnas.95.10.5752BACTH plasmid
Recombinant DNA reagentpKNT25
(plasmid)
https://doi.org/10.1073/pnas.95.10.5752BACTH plasmid
Recombinant DNA reagentpAH154
(plasmid)
this studyPmxan0635 mCh-pomXN, Mx8 attB, KmR
Recombinant DNA reagentpAH157
(plasmid)
this studyOverexpression of PomXN-His6, KmR
Recombinant DNA reagentpAH165
(plasmid)
this studyOverexpression of PomXN_K13AR15A-His6, KmR
Recombinant DNA reagentpDS100
(plasmid)
this studyBACTH plasmid for pomZ (pUT18C), AmpR
Recombinant DNA reagentpDS103
(plasmid)
this studyBACTH plasmid for pomX (pUT18C), AmpR
Recombinant DNA reagentpDS106
(plasmid)
this studyBACTH plasmid for pomX (pKT25), KmR
Recombinant DNA reagentpDS109
(plasmid)
this studyBACTH plasmid for pomZ (pUT18), AmpR
Recombinant DNA reagentpDS110
(plasmid)
this studyBACTH plasmid for pomX (pUT18), AmpR
Recombinant DNA reagentpDS114
(plasmid)
this studyBACTH plasmid for pomX (pKNT25) KmR
Recombinant DNA reagentpDS115
(plasmid)
this studyBACTH plasmid for pomZD90A (pUT18C), AmpR
Recombinant DNA reagentpDS117
(plasmid)
this studyBACTH plasmid for pomZD90A (pUT18), AmpR
Recombinant DNA reagentpDS120
(plasmid)
this studyBACTH plasmid for pomY (pUT18C), AmpR
Recombinant DNA reagentpDS122
(plasmid)
this studyBACTH plasmid for pomY (pUT18), AmpR
Recombinant DNA reagentpDS184
(plasmid)
this studyBACTH plasmid for pomXΔ2-21 (pUT18), AmpR
Recombinant DNA reagentpDS185
(plasmid)
this studyBACTH plasmid for pomXΔ2-21 (pUT18C), AmpR
Recombinant DNA reagentpDS186
(plasmid)
this studyBACTH plasmid for pomXΔ2-21 (pKT25), KmR
Recombinant DNA reagentpDS187
(plasmid)
this studyBACTH plasmid for pomXΔ2-21 (pKNT25) KmR
Recombinant DNA reagentpDS188
(plasmid)
this studyBACTH plasmid for pomXC (pUT18), AmpR
Recombinant DNA reagentpDS189
(plasmid)
this studyBACTH plasmid for pomXC (pUT18C), AmpR
Recombinant DNA reagentpDS190
(plasmid)
this studyBACTH plasmid for pomXC (pKT25), KmR
Recombinant DNA reagentpDS191
(plasmid)
this studyBACTH plasmid for pomXC (pKNT25) KmR
Recombinant DNA reagentpDS192
(plasmid)
this studyBACTH plasmid for pomXN (pUT18), AmpR
Recombinant DNA reagentpDS193
(plasmid)
this studyBACTH plasmid for pomXN (pUT18C), AmpR
Recombinant DNA reagentpDS194
(plasmid)
this studyBACTH plasmid for pomXN (pKT25), KmR
Recombinant DNA reagentpDS195
(plasmid)
this studyBACTH plasmid for pomXN (pKNT25) KmR
Recombinant DNA reagentpDS232
(plasmid)
this studyOverexpression of PomXN-Strep, KmR
Recombinant DNA reagentpDS252
(plasmid)
this studyPmxan0635 mCh-pomXN, Mx8 attB, KmR
Recombinant DNA reagentpDS253
(plasmid)
this studyBACTH plasmid for pomXN_K13AR15A (pUT18), AmpR
Recombinant DNA reagentpDS254
(plasmid)
this studyBACTH plasmid for pomXN_K13AR15A (pUT18C), AmpR
Recombinant DNA reagentpDS255
(plasmid)
this studyBACTH plasmid for pomXN_K13AR15A (pKT25), KmR
Recombinant DNA reagentpDS256
(plasmid)
this studyBACTH plasmid for pomXN_K13AR15A (pKNT25) KmR
Recombinant DNA reagentpDS257
(plasmid)
this studyBACTH plasmid for pomXN_Δ2-21 (pUT18), AmpR
Recombinant DNA reagentpDS258
(plasmid)
this studyBACTH plasmid for pomXN_Δ2-21 (pUT18C), AmpR
Recombinant DNA reagentpDS259
(plasmid)
this studyBACTH plasmid for pomXN_Δ2-21 (pKT25), KmR
Recombinant DNA reagentpDS260
(plasmid)
this studyBACTH plasmid for pomXN_Δ2-21 (pKNT25) KmR
Recombinant DNA reagentpDS303
(plasmid)
this studynat. site codon exchange for pomXK2A, KmR
Recombinant DNA reagentpDS304
(plasmid)
this studynat. site codon exchange for pomXK3A, KmR
Recombinant DNA reagentpDS305
(plasmid)
this studynat. site codon exchange for pomXE6A, KmR
Recombinant DNA reagentpDS306
(plasmid)
this studynat. site codon exchange for pomXQ7A, KmR
Recombinant DNA reagentpDS307
(plasmid)
this studynat. site codon exchange for pomXN8A, KmR
Recombinant DNA reagentpDS308
(plasmid)
this studynat. site codon exchange for pomXS10A, KmR
Recombinant DNA reagentpDS309
(plasmid)
this studynat. site codon exchange for pomXR11A, KmR
Recombinant DNA reagentpDS310
(plasmid)
this studynat. site codon exchange for pomXK13A, KmR
Recombinant DNA reagentpDS311
(plasmid)
this studynat. site codon exchange for pomXR15A, KmR
Recombinant DNA reagentpDS312
(plasmid)
this studynat. site codon exchange for pomXR17A, KmR
Recombinant DNA reagentpDS313
(plasmid)
this studynat. site codon exchange for pomXT22A, KmR
Recombinant DNA reagentpDS314
(plasmid)
this studyPmxan0635 mCh-pomXK2A, Mx8 attB, KmR
Recombinant DNA reagentpDS315
(plasmid)
this studyPmxan0635 mCh-pomXK3A, Mx8 attB, KmR
Recombinant DNA reagentpDS316
(plasmid)
this studyPmxan0635 mCh-pomXE6A, Mx8 attB, KmR
Recombinant DNA reagentpDS317
(plasmid)
this studyPmxan0635 mCh-pomXQ7A, Mx8 attB, KmR
Recombinant DNA reagentpDS318
(plasmid)
this studyPmxan0635 mCh-pomXN8A, Mx8 attB, KmR
Recombinant DNA reagentpDS319
(plasmid)
this studyPmxan0635 mCh-pomXS10A, Mx8 attB, KmR
Recombinant DNA reagentpDS320
(plasmid)
this studyPmxan0635 mCh-pomXR11A, Mx8 attB, KmR
Recombinant DNA reagentpDS321
(plasmid)
this studyPmxan0635 mCh-pomXK13A, Mx8 attB, KmR
Recombinant DNA reagentpDS322
(plasmid)
this studyPmxan0635 mCh-pomXR15A, Mx8 attB, KmR
Recombinant DNA reagentpDS323
(plasmid)
this studyPmxan0635 mCh-pomXR17A, Mx8 attB, KmR
Recombinant DNA reagentpDS324
(plasmid)
this studyPmxan0635 mCh-pomXT22A, Mx8 attB, KmR
Recombinant DNA reagentpDS325
(plasmid)
this studyPmxan0635 mCh-pomXK13AR15A, Mx8 attB, KmR
Recombinant DNA reagentpDS329
(plasmid)
this studyPmxan0635 mCh-pomXC, Mx8 attB, KmR
Recombinant DNA reagentpDS333
(plasmid)
this studyOverexpression of PomXC-Strep, KmR
Recombinant DNA reagentpEMR1
(plasmid)
this studyOverexpression of PomY-His6, KmR
Recombinant DNA reagentpSH1
(plasmid)
this studynat. site codon exchange for pomXK13AR15A, KmR
Recombinant DNA reagentpSH36
(plasmid)
this studyBACTH plasmid for pomXK13AR15A (pKNT25) KmR
Recombinant DNA reagentpSH37
(plasmid)
this studyBACTH plasmid for pomXK13AR15A (pKT25), KmR
Recombinant DNA reagentpSH38
(plasmid)
this studyBACTH plasmid for pomXK13AR15A (pUT18), AmpR
Recombinant DNA reagentpSH39
(plasmid)
this studyBACTH plasmid for pomXK13AR15A (pUT18C), AmpR
Recombinant DNA reagentpSH58
(plasmid)
this studyOverexpression of PomXK13AR15A-His6, KmR
Sequence-based reagentpomX BTH fwd XbaIthis studyPCR primer5’-GCGTCTAGAGATGAAGAAAGCCTTTGAAC-3’
Sequence-based reagentpomX BTH rev KpnIthis studyPCR primer5’-GCGGGTACCCGGCGCACCGTGGCCTGAC-3’
Sequence-based reagentpomY BTH fwd XbaIthis studyPCR primer5’-GCGTCTAGAGGTGAGCGACGAGCGTCCG-3’
Sequence-based reagentpomY BTH rev KpnIthis studyPCR primer5’-GCGGGTACCCGAGCGGCGAAGTATTTGTG-3’
Sequence-based reagentpomZ BTH fwd XbaIthis studyPCR primer5’-GCGTCTAGAGATGGAAGCGCCGACGTAC-3’
Sequence-based reagentpomZ BTH rev KpnIthis studyPCR primer5’-GCGGGTACCCGGCCGGCCTGCTGGGTGCC-3’
Sequence-based reagentpomXΔ2–21 BTH fwd XbaIthis studyPCR primer5’-GCGTCTAGAGATGACGGGCCTCGTCGACCCC-3’
Sequence-based reagentpomXC BTH fwd XbaIthis studyPCR primer5’-GCGTCTAGAGATGGCCACCGTGGCGGAGGCG-3’
Sequence-based reagentpomXN BTH rev KpnIthis studyPCR primer5’-GCGGGTACCCGGGGCAGCGGCTCCGGGCG-3’
Sequence-based reagent0636 up fwdthis studyPCR primer5’-GCGGGATCCGTCACCCCAAGCCATTC-3’
Sequence-based reagentPomX K2A rev nativethis studyPCR primer5’-CAAAGGCTTTCGCCATGGTTCTCAG-3’
Sequence-based reagentPomX K2A fwd nativethis studyPCR primer5’-CTGAGAACCATGGCGAAAGCCTTTG-3’
Sequence-based reagent0636 HindIII rev stopthis studyPCR primer5’-GCGAAGCTTTCAGCGCACCGTGGCCTGAC-3’
Sequence-based reagentPomX K3A rev nativethis studyPCR primer5’-CTGTTCAAAGGCCGCCTTCATGGTTC-3’
Sequence-based reagentPomX K3A fwd nativethis studyPCR primer5’-GAACCATGAAGGCGGCCTTTGAACAG-3’
Sequence-based reagentPomX E6A revthis studyPCR primer5’-GGACACGTTCTGCGCAAAGGCTTTCTT-3’
Sequence-based reagentPomX E6A fwdthis studyPCR primer5’-AAGAAAGCCTTTGCGCAGAACGTGTCC-3’
Sequence-based reagentPomX Q7A revthis studyPCR primer5’-GCGGGACACGTTCGCTTCAAAGGCTTT-3’
Sequence-based reagentPomX Q7A fwdthis studyPCR primer5’-AAAGCCTTTGAAGCGAACGTGTCCCGC-3’
Sequence-based reagentPomX N8A revthis studyPCR primer5’-GGCGCGGGACACCGCCTGTTCAAAGGC-3’
Sequence-based reagentPomX N8A fwdthis studyPCR primer5’-GCCTTTGAACAGGCGGTGTCCCGCGCC-3’
Sequence-based reagentPomX S10A revthis studyPCR primer5’-CGGCTTGGCGCGCGCCACGTTCTGTTC-3’
Sequence-based reagentPomX S10A fwdthis studyPCR primer5’-GAACAGAACGTGGCGCGCGCCAAGCCG-3’
Sequence-based reagentPomX R11A revthis studyPCR primer5’-GCGCGGCTTGGCCGCGGACACGTTCTG-3’
Sequence-based reagentPomX R11A fwdthis studyPCR primer5’-CAGAACGTGTCCGCGGCCAAGCCGCGC-3’
Sequence-based reagentPomX K13A revthis studyPCR primer5’-GCGGAGGCGCGGCGCGGCGCGGGACAC-3’
Sequence-based reagentPomX K13A fwdthis studyPCR primer5’-GTGTCCCGCGCCGCGCCGCGCCTCCGC-3’
Sequence-based reagentPomX R15A revthis studyPCR primer5’-GCCCAGGCGGAGCGCCGGCTTGGCGCG-3’
Sequence-based reagentPomX R15A fwdthis studyPCR primer5’-CGCGCCAAGCCGGCGCTCCGCCTGGGC-3’
Sequence-based reagentPomX R17A revthis studyPCR primer5’-CAGCGCGCCCAGCGCGAGGCGCGGCTT-3’
Sequence-based reagentPomX R17A fwdthis studyPCR primer5’-AAGCCGCGCCTCGCGCTGGGCGCGCTG-3’
Sequence-based reagentPomX T22A revthis studyPCR primer5’-GTCGACGAGGCCCGCCAGCGCGCCCAG-3’
Sequence-based reagentPomX T22A fwdthis studyPCR primer5’-CTGGGCGCGCTGGCGGGCCTCGTCGAC-3’
Sequence-based reagentPomX K13AR15A revthis studyPCR primer5’-CAGAACGTGTCCCGCGCCGCGCCGGCCCTCCGCCTGGGCGCGCTG-3’
Sequence-based reagentPomX K13AR15A fwdthis studyPCR primer5’-CAGCGCGCCCAGGCGGAGGGCCGGCGCGGCGCGGGACACCTTCTG-3’
Sequence-based reagentmCherry XbaI fwdthis studyPCR primer5’-GCGTCTAGAGTGAGCAAGGGCGAGGAG-3’
Sequence-based reagentPomX K2A revthis studyPCR primer5’-TTCAAAGGCTTTCGCCATGGCTCCGCC-3’
Sequence-based reagentPomX K2A fwdthis studyPCR primer5’-GGCGGAGCCATGGCGAAAGCCTTTGAA-3’
Sequence-based reagentKA348this studyPCR primer5’-GCCAAGCTTTCAGCGCACCGTGGCCTG-3’
Sequence-based reagentPomX K3A fwdthis studyPCR primer5’-GGAGCCATGAAGGCGGCCTTTGAACAG-3’
Sequence-based reagentPomX K3A revthis studyPCR primer5’-CTGTTCAAAGGCCGCCTTCATGGCTCC-3’
Sequence-based reagentAH142this studyPCR primer5’-GGAATTCCATATGGCCACCGTGGCGGAGGCG-3’
Sequence-based reagentKA346this studyPCR primer5’-GCCAAGCTTGCGCACCGTGGCCTGACTC-3’
Sequence-based reagentAH143this studyPCR primer5’-CCCAAGCTTGGGCAGCGGCTCCGGGCG-3’
Sequence-based reagentNdeI PomX fwdthis studyPCR primer5’-GGAATTCCATATGAAGAAAGCCTTTGAACAG-3’
Sequence-based reagentAH144this studyPCR primer5’-GCCAAGCTTTCAGGGCAGCGGCTCCGGGCG-3’
Sequence-based reagentKA384this studyPCR primer5’-GCGGGATCCGGCGGAGCCATGAAGAAAGCCTTTGAACAG-3’
Sequence-based reagentDS276this studyPCR primer5’-GCGAAGCTTACTTCTCGAACTGTGGGTGACTCCAGCGCACCGTGGCCTGAC-3’
Sequence-based reagentDS277this studyPCR primer5’-GCGCCATGGCCACCGTGGCGGAGGCG-3’
Sequence-based reagentPomX BspHI fwdthis studyPCR primer5’-GCGTCATGAAGAAAGCCTTTGAACAGAACG-3’
Sequence-based reagentPomXN rev strep-tagthis studyPCR primer5’-GCGAAGCTTACTTCTCGAACTGTGGGTGACTCCAGGGCAGCGGCTCCGGGCG-3’
Sequence-based reagentNdeI-PomY fwdthis studyPCR primer5’-GGAATTCCATATGAGCGACGAGCGTCCGGAC-3’
Sequence-based reagentPomY C-term his revthis studyPCR primer5’-CGGAAGCTTAGCGGCGAAGTATTTGTGC-3’
Sequence-based reagentAH141this studyPCR primer5’-GCGGGATCCGGCGGAGCCGCCACCGTGGCGGAGGCG-3’
Antibodyα-PomX (rabbit, polyclonal)https://doi.org/10.1016/j.devcel.2017.04.011Western Blot (1:15000)
Antibodyα-PomY (rabbit, polyclonal)https://doi.org/10.1016/j.devcel.2017.04.011Western Blot (1:15000)
Antibodyα-PomZ (rabbit, polyclonal)DOI: 10.1111/mmi.12094Western Blot (1:10000)
Antibodyα-PilC (rabbit, polyclonal)DOI:10.1111/j.1365–2958.2009.06891.xWestern Blot (1:3000)
Antibodyα-mCherry (rabbit, polyclonal)BiovisionCat# 5993Western Blot (1:10000)
Antibodyhorseradish-conjugated
α-rabbit immunoglobulin G
(goat,polyclonal)
Sigma-AldrichCat# A0545-1MLWestern Blot (1:25000)
Peptide, recombinant proteinPomXNPEPThermo ScientificMKKAFEQNVSRAKPRLRLGALT
Peptide, recombinant proteinPomXNPEPK13AR15AThermo ScientificMKKAFEQNVSRAAPALRLGALT
Software, algorithmMetamorph_v 7.5Molecular Devices
Software, algorithmOuftiDOI: 10.1111/mmi.13264http://www.oufti.org/
Software, algorithmMatlab R2018aMathWorks
Commercial assay or kitLuminata ForteFisher scientificCat# 10394675

Data availability

All data supporting this study are available within the article and supporting files. Source data files have been provided for Figures 1, 2, 3, 4, 5 and 6.

References

  1. Book
    1. Sambrook J
    2. Russell DW
    (2001)
    Molecular Cloning: A Laboratory Manual
    Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Decision letter

  1. Gisela Storz
    Senior and Reviewing Editor; National Institute of Child Health and Human Development, United States
  2. Joe Lutkenhaus
    Reviewer; University of Kansas Medical Center, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study examines a bacterial system that positions the cell division septum in Myxococcus xanthus, called PomXYZ, a tripartite protein-protein interaction system. It will be of broad interest to readers who are interested in how macromolecular complexes and DNA are dynamically positioned inside cells.

Decision letter after peer review:

Thank you for submitting your article "PomX, a ParA/MinD ATPase activating protein, is a triple regulator of cell division in Myxococcus xanthus" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Gisela Storz serving as the Reviewing and Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Joe Lutkenhaus (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential Revisions:

The reviewers, who were all enthusiastic about this study, have several comments for improving this manuscript.

1) Given that PomX polymerizes so readily, what is the form of this protein in the assays? Polymerized or not and how does that affect the results.

2) Related to the first point, how is PomX polymerization is regulated? Is it just by buffer conditions?

3) In the text it is stated that PomY also stimulates the ATPase activity of PomZ. Is that really the case? Does it have an N-terminal are like PomX?

4) The K3A mutation (which behaves as a null mutation) lowers the intracellular level of PomX below that detectable, which is understandable. However this ignores a potentially critical residue. The role of the residue could be tested using peptides (as was done with K13AR15A). ATPase assays with a K3A peptide would be useful information and might uncover a critical residue.

5) Some of the strong conclusions about ATP hydrolysis stimulation need to be tempered.

Reviewer #1 (Recommendations for the authors):

I have some suggestions that should be straightforward for the authors to incorporate if they wish.

1) Subsection “PomX AAP activity resides in PomXN”. The authors comment on the cooperativity of the ATPase activity of PomZ and make a statement on the speed of the reaction relative to other ATPases. It appears that the reactions in Figure 2E-H were not performed in a condition where one substrate was limiting (thus, the reactions were unable to reach a plateau, as stated in the aforementioned subsection). As such, the reaction did not reach saturation, so it is difficult to draw conclusions regarding the shape of the curve (i.e., first order, second order, etc) and therefore whether the hydrolysis is cooperative. Since the reaction did not produce a classical Michaelis-Menten curve, it is also not possible to calculate a kcat value (turnover) for the reaction (mol ATP hydrolyzed/min/mol protein), which would allow a conclusion about how “slow” the enzyme is. One way to achieve this is to hold the protein concentration constant (low) and vary the nucleotide level. In lieu of repeating these experiments, since these conclusions are not central to the authors' overall story, the authors may consider omitting the specific conclusions regarding enzyme kinetics.

Reviewer #2 (Recommendations for the authors):

1) The authors show that PomX, as in other AAPs, stimulates ATP hydrolysis via a short N-terminal segment that can be replaced by peptides in vitro. They conclude that K13 and R15 are important residues for the PomZ interaction, but admit that they cannot be the only ones since the K13AR15A mutant still retains partial function. But they do not discuss K3 because the K3A mutation (which behaves as a null mutation) lowers the intracellular level of PomX below that detectable, which is understandable. However this ignores a potentially critical residue and they could test the role of the residue using peptides (as they did with K13AR15A). ATPase assays with a K3A peptide would be useful information and might uncover a critical residue.

2) The ParA ATPase has also been implicated in splitting ParB complexes/foci (see Ah-Seng et al., 2013), which extends similarities with ParA-like systems and could be mentioned.

3) I found Figure 3D, right panel, confusing and found it difficult to discern what data points were plotted and what they mean. For example, what does the % mean where labelled directly on the graph? More explanation in the legend could be included, or the data plotted in a more simplified representation.

Reviewer #3 (Recommendations for the authors):

The authors hone in on the N-ter peptide of PomX and find that it is necessary for interaction with PomZ as well as ability to stimulate the ATPase activity of PomZ.

Abstract: I might reword the fifth sentence. The C-terminal domain interacts with PomY and forms polymers which serve as a scaffold for PomXYZ complex formation.

The focus in this paper is on PomX but it is not clear why since PomY has also been shown to stimulate the ATPase activity of PomZ to a similar degree (Introduction). Some rationale should be provided for focusing on PomX. Is the stimulation by PomY reproducible? Does it have a role? Seems incomplete without this.

Introduction – triple regulator – why not say PomX displays three activities required to regulate cell division – AAP, scaffold and along with PomZ is required for fission of the complex.

Figure 1—figure supplement 1B. What are the extra bands in the rightmost Western panel when a-mCh antibody is used? The bands at 72kDa and ~45kDa do not show up (or are less intense) in the left and center panels with the same antibody.

Results. ∆pomX cells have only 2X the average length but produce minicells. Does that mean the frequency of cell division is not affected? Or are not many minicells produced?

Results. From Figure 2A it appears that the BATCH for the interaction between PomZ and PomXN only works well in one orientation. Does T18 attached to the N-ter of PomX interfere with the interaction? T25 does not seem to interfere. A comment about this should appear somewhere.

Results. Spontaneous filament formation by PomX and PomXC. How is assembly regulated in vitro. What is added to the solution to induce assembly? Is it induced by changing the salt concentration? When PomX or PomXC are isolated are filaments already present? Perhaps this has been addressed in an earlier publication but I did not see it.

In the ATPase assays what state is PomX in? Is it polymerized? Does it make a difference whether it is or not.

Figure 5A. Any reason why PomXN was not included as a control?

"all four PomXNPEP mutants". Aren't there only two mutants? Each containing two changes.

Is the NPEP predicted to be an α helix. The N-terminus of MinE bound to MInD is an α helix so I would expect this to be the same although it does not have to be.

Discussion. It seems in an earlier publication there was stimulation of the ATPase activity by both PomX and PomY in the absence of DNA. Please clarify.

Subsection “The PomX/PomZ interaction is important for PomX/Y/Z cluster fission during division”. What is the consequence of not splitting the cluster?

Discussion. Although the precise mechanism is unknown, a mechanism has been proposed for MinD-MInE as well as a comparison with RAS in Park et al., 2012.

Can the authors comment on parallels to ParA/ParB where ParB is bound to DNA to presumably have a large cluster of ParB (the AAP) whereas in the system studied here PomX polymerizes to form large clusters. This is presumably needed to migrate on the DNA.

https://doi.org/10.7554/eLife.66160.sa1

Author response

Essential Revisions:

The reviewers, who were all enthusiastic about this study, have several comments for improving this manuscript.

1) Given that PomX polymerizes so readily, what is the form of this protein in the assays? Polymerized or not and how does that affect the results.

Thank you for drawing our attention to these questions! Purified PomX-His6, as mentioned in the comment below, forms filaments/polymerizes under all conditions tested in vitro. The buffer conditions for the ATPase assay, sedimentation assays, and the EM analyses are identical. Moreover, the concentration range of PomX used in these different assays is the same. This also means that the protein variants of PomX and PomXC that are added to the ATPase assays are polymers. We would like to add that in all the assays, we very carefully monitor the amount of PomX added to be able to make clear statements about the concentration of PomX. Importantly, PomXN, which does not form polymers/filaments, stimulates PomZ ATPase activity as efficiently as PomX full-length protein. These observations support that the PomXN domains in a PomX polymer act independently of each other to stimulate PomZ ATPase activity. In the revised manuscript, we have modified the text to emphasize this. We return to this point in the Discussion.

2) Related to the first point, how is PomX polymerization is regulated? Is it just by buffer conditions?

Upon (over)production of PomX in E. coli, it spontaneously forms filaments under all conditions tested in vitro. We have used different tags, attached the tag to the N-terminus or C-terminus, tried different expression levels, used different temperatures during expression, and used different buffer conditions for purifying the protein. Under all these different conditions, PomX always purifies as a polymer. Even upon expression of native PomX in E. coli it purifies as a polymer. Also, upon expression of mCh-tagged PomX in M. xanthus, the protein forms a filamentous structure. Therefore, we have not been able to identify any physiological condition where PomX does not form a polymer/filament. In other words, polymerization is spontaneous and independent of any cofactor (that we know of). Therefore, we have as a working model that PomX upon synthesis spontaneously polymerizes and that this polymerization is not regulated. The buffer conditions for the ATPase assay, sedimentation assays, and the EM analyses are identical. Moreover, the concentration range of PomX used in these different assays is the same. This also means that the protein variants of PomX and PomXC that are added to the ATPase assays are polymers. We would like to add that in all the assays, we very carefully monitor the amount of PomX added to be able to make clear statements about the concentration of PomX. To make clear that PomX spontaneously polymerizes in a cofactor-independent manner, we have modified the text.

3) In the text it is stated that PomY also stimulates the ATPase activity of PomZ. Is that really the case? Does it have an N-terminal are like PomX?

In the revised manuscript, we have now included a brief description of the rationale for focusing our analysis on PomX in this manuscript. To understand the mechanism of the PomX/Y/Z system we are of course also working on PomY (which we can assure and confirm has AAP activity as previously published); but that work has not been finished yet. We have also added that “Interestingly, PomY, the second AAP of PomZ (Schumacher et al., 2017), does not have an N-terminus enriched in positively charged residues suggesting that its mode of action could be different from that of previously described AAPs”.

4) The K3A mutation (which behaves as a null mutation) lowers the intracellular level of PomX below that detectable, which is understandable. However this ignores a potentially critical residue. The role of the residue could be tested using peptides (as was done with K13AR15A). ATPase assays with a K3A peptide would be useful information and might uncover a critical residue.

We apologize for this confusion. The untagged PomXK3A variant does not accumulate in M. xanthus (and therefore does not complement the ΔpomX mutant). However, the mCh-tagged PomXK3A variant accumulates in M. xanthus at a level similar to that of the WT protein and complements the cell length and division defects of the ΔpomX mutant (Shown in Figure 4—figure supplement 1A, B). Therefore, we conclude that the Lys3 residue is not important for PomX function. To make this conclusion more clear, we have modified the text. Moreover, we included the cell length of the ΔpomX mutant expressing mCh-PomXK3A. Because the mCh-PomXK3A variant is fully active, we have not done any experiments to specifically explore the function of Lys3 in the activation of PomZ ATPase activity.

5) Some of the strong conclusions about ATP hydrolysis stimulation need to be tempered.

Thanks for pointing this out to us. Throughout the manuscript, we have deleted comments and conclusions about enzyme kinetics.

Reviewer #1 (Recommendations for the authors):

I have some suggestions that should be straightforward for the authors to incorporate if they wish.

1) Subsection “PomX AAP activity resides in PomXN”. The authors comment on the cooperativity of the ATPase activity of PomZ and make a statement on the speed of the reaction relative to other ATPases. It appears that the reactions in Figure 2E-H were not performed in a condition where one substrate was limiting (thus, the reactions were unable to reach a plateau, as stated in the aforementioned subsection). As such, the reaction did not reach saturation, so it is difficult to draw conclusions regarding the shape of the curve (i.e., first order, second order, etc) and therefore whether the hydrolysis is cooperative. Since the reaction did not produce a classical Michaelis-Menten curve, it is also not possible to calculate a kcat value (turnover) for the reaction (mol ATP hydrolyzed/min/mol protein), which would allow a conclusion about how “slow” the enzyme is. One way to achieve this is to hold the protein concentration constant (low) and vary the nucleotide level. In lieu of repeating these experiments, since these conclusions are not central to the authors' overall story, the authors may consider omitting the specific conclusions regarding enzyme kinetics.

Thanks for pointing this out to us. Throughout the manuscript we have deleted comments and conclusions about enzyme kinetics.

Reviewer #2 (Recommendations for the authors):

1) The authors show that PomX, as in other AAPs, stimulates ATP hydrolysis via a short N-terminal segment that can be replaced by peptides in vitro. They conclude that K13 and R15 are important residues for the PomZ interaction, but admit that they cannot be the only ones since the K13AR15A mutant still retains partial function. But they do not discuss K3 because the K3A mutation (which behaves as a null mutation) lowers the intracellular level of PomX below that detectable, which is understandable. However this ignores a potentially critical residue and they could test the role of the residue using peptides (as they did with K13AR15A). ATPase assays with a K3A peptide would be useful information and might uncover a critical residue.

We apologize for this confusion. The untagged PomXK3A variant does not accumulate in M. xanthus (and therefore does not complement the ΔpomX mutant). However, the mCh-tagged PomXK3A variant accumulates in M. xanthus at a level similar to that of the WT protein and complements the cell length and division defects of the ΔpomX mutant (Shown in Figure 4—figure supplement 1A, B). Therefore, we conclude that the Lys3 residue is not important for PomX function. To make this conclusion more clear, we have modified the text. Moreover, we included the cell length of the ΔpomX mutant expressing mCh-PomXK3A. Because the mCh-PomXK3A variant is fully active, we have not done any experiments to specifically explore the function of Lys3 in the activation of PomZ ATPase activity.

2) The ParA ATPase has also been implicated in splitting ParB complexes/foci (see Ah-Seng et al., 2013), which extends similarities with ParA-like systems and could be mentioned.

Thank you for pointing this out to us. We have now included a brief comparison to the SopA/B system in the Discussion.

3) I found Figure 3D, right panel, confusing and found it difficult to discern what data points were plotted and what they mean. For example, what does the % mean where labelled directly on the graph? More explanation in the legend could be included, or the data plotted in a more simplified representation.

We apologize for this confusion. We use Figure 3D right panel to compare cell length, position of cell division constrictions and the constriction frequency. To make the figure more accessible, we now precisely refer to the left and right panels of this figure in the main text. In addition, we changed the figure legend to explain what we are plotting more carefully.

Reviewer #3 (Recommendations for the authors):

The authors hone in on the N-ter peptide of PomX and find that it is necessary for interaction with PomZ as well as ability to stimulate the ATPase activity of PomZ.

Abstract: I might reword the fifth sentence. The C-terminal domain interacts with PomY and forms polymers which serve as a scaffold for PomXYZ complex formation.

Thanks and changed as suggested.

The focus in this paper is on PomX but it is not clear why since PomY has also been shown to stimulate the ATPase activity of PomZ to a similar degree (Introduction). Some rationale should be provided for focusing on PomX. Is the stimulation by PomY reproducible? Does it have a role? Seems incomplete without this.

In the revised manuscript, we have now included a brief description of the rationale for focusing our analysis on PomX in this manuscript. To understand the mechanism of the PomX/Y/Z system we are of course also working on PomY (which we can assure and confirm has AAP activity as previously published); but that work has not been finished yet. We also think that the manuscript would be far too long to include data on how PomY activates PomZ ATPase activity. We have added that “Interestingly, PomY, the second AAP of PomZ (Schumacher et al., 2017), does not have an N-terminus enriched in positively charged residues suggesting that its mode of action could be different from that of previously described AAPs”.

Introduction – triple regulator – why not say PomX displays three activities required to regulate cell division – AAP, scaffold and along with PomZ is required for fission of the complex.

Changed essentially as suggested (Introduction).

Figure 1—figure supplement 1B. What are the extra bands in the rightmost Western panel when a-mCh antibody is used? The bands at 72kDa and ~45kDa do not show up (or are less intense) in the left and center panels with the same antibody.

The bands at approximately 72kDa and 45kDa are unspecific bands that sometimes and sometimes not appear in Western blots with α-mCherry antibodies. Please note that these bands are also present in the cell extract of the WT and ΔpomX strains, none of which express an mCherry protein. We are struggling to find out why these bands sometimes appear. We would like to add that the presence of these bands does not affect any of our conclusions. In the revised manuscript, we have clearly marked these bands in Figure 1—figure supplement 1B and explained in the legend that “Note that the three bands labeled * in the right and left α-mCh Western blot of B are unspecific bands that sometimes appear in the Western blots with α-mCh antibodies”.

Results. ∆pomX cells have only 2X the average length but produce minicells. Does that mean the frequency of cell division is not affected? Or are not many minicells produced?

The constriction frequency is reduced approximately 5-fold in the ΔpomX mutant compared to the WT. Moreover, the constrictions are not only at midcell. Altogether, this results in the formation of filamentous cells and minicells. Importantly, the variance in cell length of the filamentous cells in the ΔpomX mutant (see Figure 1D) is much large compared to WT and some cells have a length >40 µm as would be expected with a 5-fold reduction of the constriction frequency.

Results. From Figure 2A it appears that the BATCH for the interaction between PomZ and PomXN only works well in one orientation. Does T18 attached to the N-ter of PomX interfere with the interaction? T25 does not seem to interfere. A comment about this should appear somewhere.

As shown in Figure 2A, the interaction between PomZ and PomX depends on where the tag (T18) is added to PomZ but is independent of where the tag (T25) is added to PomX and PomXN. We have not tested whether T18 attached to the N-ter of PomX interferes with the interaction to PomZ; but we would expect that this is not the case because T25 added to the N-terminus of PomX does not interfere with its interaction with PomZ. It is quite common that interactions observed in BACTH assays depend on where the tag is added. So, the important take-home message from the BACTH is that PomX and PomXN interact with PomZ. Therefore, we decided – and we hope that you agree with this decision – not to include a comment about the interactions that we did not see. Instead, we went on to check for interactions in vitro using purified proteins.

Results. Spontaneous filament formation by PomX and PomXC. How is assembly regulated in vitro. What is added to the solution to induce assembly? Is it induced by changing the salt concentration? When PomX or PomXC are isolated are filaments already present? Perhaps this has been addressed in an earlier publication but I did not see it.

Upon (over)production of PomX in E. coli, it spontaneously forms filaments under all conditions tested in vitro. We have used different tags, attached the tag to the N-terminus or C-terminus, tried different expression levels, used different temperatures during expression, and used different buffer conditions for purifying the protein. Under all these different conditions, PomX always purifies as a polymer. Even upon expression of native PomX in E. coli it purifies as a polymer. Also, upon expression of mCh-tagged PomX in M. xanthus, the protein forms a filamentous structure. Therefore, we have not been able to identify any physiological condition where PomX does not form a polymer/filament. In other words, polymerization is spontaneous and independent of any cofactor (that we know of). Therefore, we have as a working model that PomX upon synthesis spontaneously polymerizes and that this polymerization is not regulated. The buffer conditions for the ATPase assay, sedimentation assays, and the EM analyses are identical. Moreover, the concentration range of PomX used in these different assays is the same. This also means that the protein variants of PomX and PomXC that are added to the ATPase assays are polymers. We would like to add that in all the assays we very carefully monitor the amount of PomX added to be able to make clear statements about the concentration of PomX. To make clear that PomX spontaneously polymerizes in a cofactor-independent manner, we have modified the text.

In the ATPase assays what state is PomX in? Is it polymerized? Does it make a difference whether it is or not.

Thank you for drawing our attention to these questions! Purified PomX-His6, as mentioned in the comment above, forms filaments/polymerizes under all conditions tested in vitro. The buffer conditions for the ATPase assay, sedimentation assays, and the EM analyses are identical. Moreover, the concentration range of PomX used in these different assays is the same. This also means that the protein variants of PomX and PomXC that are added to the ATPase assays are polymers. We would like to add that in all the assays, we very carefully monitor the amount of PomX added to be able to make clear statements about the concentration of PomX. Importantly, PomXN, which does not form polymers/filaments, stimulates PomZ ATPase activity as efficiently as PomX full-length protein. These observations support that the PomXN domains in a PomX polymer act independently of each other to stimulate PomZ ATPase activity. In the revised manuscript, we have modified the text to emphasize this. We return to this point in the Discussion.

Figure 5A. Any reason why PomXN was not included as a control?

We decided not to include PomXN as a control because (1) it is already included in Figure 2A, and (2) to keep the figure simple. We have changed the legend to Figure 5A to make clear that all these experiments were done in parallel and can be compared directly.

"all four PomXNPEP mutants". Aren't there only two mutants? Each containing two changes.

We apologize for the confusion. It actually is four variants and we have clarified that in the text.

Is the NPEP predicted to be an α helix. The N-terminus of MinE bound to MInD is an α helix so I would expect this to be the same although it does not have to be.

Predictions programs (Psipred, Predictprotein and JPred4) that identify an α-helix in the N-terminus of MinE do not predict an α-helix in PomXNPEP.

Discussion. It seems in an earlier publication there was stimulation of the ATPase activity by both PomX and PomY in the absence of DNA. Please clarify.

Using PomX-His6 and PomY-His6 and a MalE-PomZ protein in a malachite-green colorimetric endpoint measurement, we previously reported that PomX-His6 and PomY-His6 weakly but significantly stimulate PomZ ATPase activity in the absence of DNA (Schumacher et al., 2017). Moreover, we reported that PomX-His6 and PomY-His6 stimulated PomZ ATPase activity strongly in the presence of DNA. Thus, overall our previous results and those reported in the current manuscript are in agreement. In the current manuscript, we purified PomZ-His6 (instead of MalE-PomZ) to improve and increase protein yield and purity. Along the same lines, we used an NADH-coupled enzymatic assay that allows us to measure ADP release in real-time and which is not susceptible to perturbation by free phosphate in the buffers used. We believe that these changes explain the slight difference between our previous results and the ones that we report here.

Subsection “The PomX/PomZ interaction is important for PomX/Y/Z cluster fission during division”. What is the consequence of not splitting the cluster?

Thanks for pointing out that we had not addressed this in the original version of the manuscript. We have now included that “We speculate that the reduced frequency of cluster fission events contributes to the cell division defect in the PomX AAP mutants”.

Discussion. Although the precise mechanism is unknown, a mechanism has been proposed for MinD-MInE as well as a comparison with RAS in Park et al., 2012.

Thanks for pointing this out to us. We have included mentioning of this proposed mechanism in the Discussion.

Can the authors comment on parallels to ParA/ParB where ParB is bound to DNA to presumably have a large cluster of ParB (the AAP) whereas in the system studied here PomX polymerizes to form large clusters. This is presumably needed to migrate on the DNA.

Thanks for this suggestion. We have now included a comparison between the PomX/Y complex, ParB-parS complexes, cytoplasmic chemoreceptor clusters and carboxysomes to point out that they may not only be similar in terms of the mechanism of the AAP but also in terms of translocation on the nucleoid (Discussion).

https://doi.org/10.7554/eLife.66160.sa2

Article and author information

Author details

  1. Dominik Schumacher

    Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch, Marburg, Germany
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Andrea Harms

    Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch, Marburg, Germany
    Contribution
    Data curation, Validation, Investigation
    Competing interests
    No competing interests declared
  3. Silke Bergeler

    Arnold Sommerfeld Center for Theoretical Physics and Center for NanoScience, Department of Physics, Ludwig-Maximilians-Universität München, München, Germany
    Contribution
    Software, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Erwin Frey

    Arnold Sommerfeld Center for Theoretical Physics and Center for NanoScience, Department of Physics, Ludwig-Maximilians-Universität München, München, Germany
    Contribution
    Supervision, Funding acquisition, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8792-3358
  5. Lotte Søgaard-Andersen

    Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch, Marburg, Germany
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    sogaard@mpi-marburg.mpg.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0674-0013

Funding

Deutsche Forschungsgemeinschaft (TRR 174)

  • Erwin Frey
  • Lotte Søgaard-Andersen

Max Planck Society

  • Lotte Søgaard-Andersen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Sabrina Huneke-Vogt for assistance with plasmid constructions, Manon Wigbers for help with the image analysis script, and Anke Treuner-Lange for many helpful discussions.

Senior and Reviewing Editor

  1. Gisela Storz, National Institute of Child Health and Human Development, United States

Reviewer

  1. Joe Lutkenhaus, University of Kansas Medical Center, United States

Publication history

  1. Received: December 30, 2020
  2. Accepted: March 17, 2021
  3. Accepted Manuscript published: March 18, 2021 (version 1)
  4. Version of Record published: March 24, 2021 (version 2)

Copyright

© 2021, Schumacher et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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