A deterministic, c-di-GMP-dependent genetic program ensures the generation of phenotypically similar, symmetric daughter cells during cytokinesis

Phenotypic heterogeneity in bacteria results from stochastic processes or deterministic genetic programs. These deterministic programs often incorporate the versatile second messenger c-di-GMP, and by deploying c-di-GMP metabolizing enzyme(s) asymmetrically during cell division give rise to daughter cells with different c-di-GMP levels. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum. Here, we identify a deterministic c-di-GMP-dependent genetic program that is hardwired into the cell cycle of Myxococcus xanthus to minimize phenotypic heterogeneity and guarantee the formation of phenotypically similar daughter cells during division. Cells lacking the diguanylate cyclase DmxA have an aberrant motility behaviour. DmxA is recruited to the cell division site and its activity switched on during cytokinesis, resulting in a dramatic but transient increase in the c-di-GMP concentration. During cytokinesis, this c-di-GMP burst ensures the symmetric incorporation and allocation of structural motility proteins and motility regulators at the new cell poles of the two daughters, thereby generating mirror-symmetric, phenotypically similar daughters with correct motility behaviours. These findings suggest a general c-di-GMP-dependent mechanism for minimizing phenotypic heterogeneity, and demonstrate that bacteria by deploying c-di-GMP metabolizing enzymes to distinct subcellular locations ensure the formation of dissimilar or similar daughter cells.


Introduction
In bacteria, motility and its regulation contribute to colonization of hosts and other habitats, biofilm formation, virulence and predation 1 .The ubiquitous second messenger cyclic di-GMP (c-di-GMP) is a key regulator of bacterial motility [2][3][4] .Generally, high c-di-GMP levels inhibit flagella-based swimming motility and stimulate surface adhesion and type IV pili (T4P)-dependent surface motility, thereby promoting surface colonization, biofilm formation and virulence [2][3][4] .Several bacterial species that alternate between planktonic and surfaceadhered lifestyles harness this duality of c-di-GMP -inhibition of flagella-based motility and stimulation of surface adhesion and T4P-based motility -to establish deterministic genetic programs that are hardwired into the cell cycle to produce phenotypically distinct daughter cells during cell division, thereby generating phenotypic heterogeneity within a population of genetically identical cells 4,5 .Here, we report that a c-di-GMP-based, deterministic genetic program is hardwired into the cell cycle of Myxococcus xanthus to minimize phenotypic heterogeneity and guarantee the formation of phenotypically similar daughter cells during division.
M. xanthus does not have flagella and only translocates on surfaces.For this, the rodshaped cells use two motility systems, one for T4P-dependent motility and one for gliding 6 .
Motility and its regulation are important for the social behaviours of M. xanthus, including the formation of spreading colonies in which cells prey on other microbes and spore-filled fruiting bodies in the absence of nutrients 7 .Both motility systems are highly polarized, i.e. the core T4P machine (T4PM) is present at both cell poles [8][9][10][11] but only active at one pole at a time ensuring unipolar T4P assembly 12 , and the Agl/Glt machine for gliding only assembles at one pole at a time [13][14][15][16][17][18] .Because the two machines are active at the same pole, cells translocate unidirectionally across surfaces with a piliated leading and non-piliated lagging cell pole [19][20][21] .To regulate their social behaviours, M. xanthus cells occasionally reverse their direction of movement 22 .The Frz chemosensory system induces reversals 22 , which at the cellular level entails the inversion of the polarity of the two motility machines 6 .
The so-called polarity module establishes the unipolar assembly of the active motility machines.The output of this module is generated by the small GTPase MglA, a nucleotidedependent molecular switch that is inactive in its GDP-bound and active in its GTP-bound state 23,24 .GTP-bound MglA localizes to the leading pole 23,24 where it interacts with effectors to stimulate T4P formation and assembly of the Agl/Glt machine 18,20,21,25 .The bipartite RomR/RomX and MglB/RomY complexes of the polarity module regulate the nucleotide-bound state and bring MglA-GTP to the leading pole by functioning as a guanine nucleotide exchange factor (GEF) 26 and a GTPase activating protein (GAP) 23,24,27 , respectively.These four proteins and the MglC adaptor protein also localize asymmetrically to the poles 23,24,[26][27][28][29] .During reversals, the Frz system acts on the polarity module to induce an inversion of its polarity 30,31 , laying the foundation for activating the motility machineries at the new leading pole.The asymmetric polar localization of the polarity proteins relies on an intricate set of positive and negative feedback loops between the six proteins 23,24,29,32 .
Consequently, mutants lacking one or more polarity proteins have an abnormal localization of the remaining proteins, and such mutants are either non-motile or have an abnormal motility behaviour, i.e. hyper-reverse independently of the Frz system or hypo-reverse and are non-responsive to Frz signalling 18,23,24,26,27,29,33 .
The c-di-GMP level is determined by the opposing activities of diguanylate cyclases (DGCs), which contain the catalytic GGDEF domain and synthesize c-di-GMP, and phosphodiesterases (PDEs), which degrade c-di-GMP 2,3 .M. xanthus encodes 11 GGDEF domain proteins predicted to have DGC activity 34 .The systematic inactivation of the 11 corresponding genes identified DmxA as the only DGC implicated in motility during growth 34   , while the DGC DmxB is specifically important for fruiting body formation 35 .The functions of the remaining nine DGCs are not known.
Here, we addressed the mechanism of DmxA.We report that DmxA is recruited to the cell division site, and its DGC activity switched on late during cytokinesis, resulting in a dramatic but transient increase in the c-di-GMP concentration.The burst in c-di-GMP ensures the equal incorporation and allocation of structural motility proteins and polarity proteins at the new cell poles of the two daughters, thereby generating mirror-symmetric, phenotypically similar daughters with correct motility behaviour.Thus, for the first time, evidence is provided that, during cell division, c-di-GMP guarantees the generation of phenotypically similar offspring.

DmxA is a dimeric DGC with a low-affinity I-site
Based on sequence analysis, DmxA has an N-terminal transmembrane domain (TMD) with six α-helices, followed by two GAF domains, and the catalytic GGDEF domain with the active (A)-site and a c-di-GMP-binding inhibitory (I)-site (Fig. 1a), which is involved in allosteric feedback inhibition of activity in other DGCs 36 .A His6-DmxA variant comprising the two GAF domains and the GGDEF domain is enzymatically active and binds c-di-GMP in vitro 34 .To understand how the different domains contribute to catalytic activity, we purified five soluble MalE-tagged DmxA variants (Fig. 1b; S1a-b), i.e. variants containing the two GAF domains and the GGDEF domain (MalE-DmxA WT ), only the two GAF domains (MalE-DmxA GAF×2 ), only the GGDEF domain (MalE-DmxA GGDEF ), and MalE-DmxA WT variants with substitutions in either the catalytic site (MalE-DmxA E626A ) or the I-site (MalE-DmxA R615A ).In size exclusion chromatography (SEC), all variants except MalE-DmxA GGDEF eluted at sizes corresponding to dimers, while MalE-DmxA GGDEF eluted at a size corresponding to a monomer (Fig. 1b; S1a), indicating that the GAF domain-containing region is important for dimer formation.Indeed, a high-confidence Alphafold-Multimer structural model supports that DmxA forms a symmetric dimer in which the protomers interact extensively via two αhelices connecting GAF1 to GAF2, and GAF2 to the GGDEF domain and in which the two Asites are in close proximity and facing each other (Fig. 1c; S1c).Consistently, active DGCs are dimeric, dimerization is mediated by domain(s) outside of the GGDEF domain 37,38 , and in solved structures of DGCs the two A-sites are in close proximity [37][38][39][40] .
We conclude that DmxA has DGC activity and a low-affinity I-site.Moreover, the GAF domain-containing region is important for dimerization, as described for eukaryotic PDEs with an analogous domain architecture 42 .
The ΔdmxA mutant has an aberrant motility behaviour with aberrant reversals and a cell polarity defect A disruption of dmxA by a plasmid insertion (ΩdmxA) caused reduced T4P-dependent motility 34 .We generated an in-frame deletion mutation of dmxA (ΔdmxA) to understand the underlying mechanism.In population-based motility assays on 0.5% agar, which is most favourable for T4P-dependent motility, wild-type (WT) generated expanding colonies with the characteristic flares, while the ΔpilA negative control, which lacks the major pilin of T4P 43 , did not (Fig. 2a).The ΔdmxA mutant displayed significantly reduced colony expansion, which was restored upon complementation (Fig. 2a).A ΔdmxAΔgltB double mutant, which lacks a component of the Agl/Glt machine 13 , also had significantly reduced colony expansion compared to the ΔgltB mutant (Fig. 2a).Thus, ΔdmxA causes a T4P-dependent motility defect.On 1.5% agar, which is most favourable for gliding, WT displayed the characteristic single cells at the colony edge, while the ΔgltB negative control did not (Fig. 2a).The ΔdmxA mutant had single cells at the colony edge but significantly reduced colony expansion, which was restored upon complementation (Fig. 2a).A ΔdmxAΔpilA double mutant had single cells at the colony edge but a more significant expansion defect than the ΔpilA mutant (Fig. 2a).Thus, the ΔdmxA mutation also causes a gliding defect.We previously reported that the ΩdmxA mutant only had a defect in T4P-dependent motility 34 .
However, in those experiments, we exclusively focused on the presence or absence of single cells at the colony edge and not overall colony expansion.
We assayed single-cell motility to uncover how the ΔdmxA mutation causes motility defects (Fig. 2b-c).ΔdmxA cells had the same speed as WT but reversed aberrantly and had a much broader distribution of reversal frequencies than WT, ranging from cells that did not reverse to cells that reversed up to eight times during the experiment.The reversal defect was corrected upon complementation.Moreover, while ΔfrzE cells, which lack the FrzE kinase essential for Frz-induced reversals 44 , did not reverse, many ΔdmxAΔfrzE cells still reversed.Mutants with Frz-independent reversals generally lack polar MglB/RomY GAP activity, causing MglA-GTP to localize to both cell poles 18,23,24,27 .Interestingly, the ΔdmxA mutant also had a much broader distribution of reversal frequencies than the ΔmglB mutant, which lacks MglB/RomY GAP activity, causing essentially all cells to reverse or hyperreverse.
The structural proteins of the two motility machines, the Frz proteins, and the polarity proteins accumulated at the same level in WT and the ΔdmxA mutant (Fig. S2b-g), supporting that changes in protein accumulation are not responsible for the aberrant motility behaviour.
We conclude that cells lacking DmxA have motility defects caused by aberrant reversals and have an unusually broad distribution of reversal frequencies.Moreover, the ΔdmxA mutant has a cell polarity defect, and the underlying mechanism differs from that of a mutant lacking MglB/RomY GAP activity.

DmxA is recruited to the division site by the divisome late during cytokinesis
To understand how DmxA impacts motility behaviour and cell polarity, we determined the localization of an active (see below) DmxA-mVenus fusion expressed from the native site.
Remarkably, DmxA-mVenus localized at mid-cell in ~5% of cells, while the remaining cells had a speckle-like pattern along the cell body (Fig. 3a).In all cells with a mid-cell cluster, the cluster co-localized with a cell division constriction.Still, not all constricting cells had a DmxA-mVenus cluster, suggesting that DmxA-mVenus is recruited to the division site late during cytokinesis.Indeed, we observed by time-lapse microscopy (Fig. 3b) that (i) DmxA-mVenus cluster formation at the division site was visible in each division event, (ii) the cluster appeared at the division site 20±15 min before completion of cytokinesis, and (iii) disintegrated upon completion of cytokinesis.Consistently, the mean cluster lifetime was 20±16 min (Fig. 3b).The 5 h doubling time of M. xanthus and this cluster lifetime agree well with the percentage of cells with a mid-cell cluster in an unsynchronized cell population.
DmxA is encoded in an operon with the FtsB divisome protein, and this genetic organization is conserved in related species (Fig. S3a-d).However, ΔdmxA cells had neither a growth (Fig. S3e) nor a cell length defect (Fig. S3f).Therefore, to test whether DmxA is recruited to the division site by the divisome, we first treated cells with cephalexin, which blocks cytokinesis after initiation of constriction in M. xanthus 45 .In cells treated with cephalexin for one generation, the frequency of cells with a constriction and a DmxA-mVenus cluster at mid-cell had significantly increased (Fig. 3a).DmxA-mVenus accumulated at the same level in cephalexin-treated and untreated cells (Fig. S4a), hinting that DmxA-mVenus synthesis is not cell cycle-regulated.Next, we depleted dmxA-mVenus expressing cells of the essential cell division protein FtsZ 45 using a strain in which the only copy of ftsZ was expressed from a vanillate-inducible promoter (Pvan).In the presence of vanillate, DmxA-mVenus localized at constrictions in 3% of cells (Fig. 3c).At 10 h of depletion, FtsZ was undetectable by immunoblotting (Fig. S4b), and cells had neither constrictions nor DmxA-mVenus clusters at mid-cell (Fig. 3c) despite the protein accumulating at a slightly higher level than in untreated cells (Fig. S4b).Finally, we observed in proximity labelling experiments using strains expressing either DmxA or sfGFP fused to the promiscuous biotin ligase miniTurbo (Fig. S4c) that the cell division protein FtsK, which localizes to the division site in M. xanthus 46 , was significantly enriched in the DmxA-miniTurbo samples (Fig. 3d; Table S1).Equally, in the reciprocal experiment using an FtsK-miniTurbo construct (Fig. S4c; Table S2), DmxA was significantly enriched (Fig. 3e).
Altogether, these observations support that DmxA accumulation is not cell cycle-regulated and that DmxA interacts with protein(s) of the divisome, and is thereby recruited to the division site late during cytokinesis.
DmxA function depends on DGC activity and is recruited to the division site by the TMD To determine whether DGC activity and which domains contribute to DmxA function and localization, we replaced dmxA with mVenus-fused versions of full-length dmxA WT , dmxA E626A and dmxA R615A as well as with the truncated variants dmxA ΔGAF×2 , dmxA ΔTMD and dmxA TMD (Fig. 3f).By immunoblot analysis, all variants accumulated at similar levels except DmxA ΔGAF×2 -mVenus and dmxA TMD -mVenus, which accumulated at slightly lower levels (Fig. S5a).Among these variants, only DmxA WT -mVenus and DmxA R615A -mVenus supported WT motility (Fig. 3g; S5b).DmxA-mVenus localization to the division site was independent of DGC activity, the I-site and the two GAF domains (Fig. 3g).By contrast, the TMD was not only essential but also sufficient for DmxA-mVenus localization to the division site (Fig. 3g).
Thus, DmxA function depends on DGC activity but not on c-di-GMP binding to the I-site, and DmxA is recruited to the division site by the TMD.

DmxA DGC activity is activated upon recruitment to the division site
Based on the DmxA localization pattern, we hypothesized that DmxA activity is cell cycleregulated and explicitly switched on late during cytokinesis.To test this idea, we used the genetically-encoded c-di-GMP biosensor cdGreen2 47 , for which binding of c-di-GMP results in conformational changes leading to increased green fluorescence, thus allowing real-time measurements of c-di-GMP levels at single-cell resolution over the entire cell cycle.For normalization of the cdGreen2 signal, cdGreen2 was co-expressed with a gene encoding the fluorescent protein mScarlet-I 47 .
For WT, we observed significant cell-to-cell variability of the cdGreen2 signal, while the mScarlet-I signal varied much less (Fig. 4a; S6a-b).Intriguingly, this cell-to-cell variability was clearly bimodal and only long cells with a constriction and some very short cells had a very high cdGreen2 signal (Fig. 4a; S6a).To focus on DmxA, we generated a mutant lacking 10 of the 11 predicted DGCs in M. xanthus, leaving DmxA as the only DGC (henceforth the Δ10 mutant).In the Δ10 mutant, DmxA-mVenus localized to the division site as in WT (Fig. S6c).Remarkably, the cdGreen2 signal was even more clearly bimodal in this strain, i.e. high in long cells with a constriction and in some very short cells (Fig. 4a; S6b).By contrast, all ΔdmxA cells, i.e. cells only lacking DmxA but retaining the remaining 10 DGCs, had the same low cdGreen2 background signal (Fig. 4a; S6b).Time-lapse microscopy of Δ10 and WT cells revealed that the cdGreen2 signal increased dramatically shortly before completion of cytokinesis and then decreased rapidly in the two daughters, which had equal levels of cdGreen2 fluorescence (Fig. 4b; S6d).By contrast, ΔdmxA cells completely lacked the transient increase in c-di-GMP (Fig. 4c).The increase in c-di-GMP initiated 22±12 min before completion of cytokinesis, and remained high for 19±9 min (Fig. 4b).Remarkably, DmxA localizes to the division site with a similar timing (Fig. 3b).
Moreover, in cephalexin-treated cells, the DmxA-mVenus clusters persisted longer at the division site and then eventually disintegrated (Fig. S6e).Notably, the high cdGreen2 signal also persisted longer in cephalexin-treated cells and eventually also vanished (Fig. S6f).We conclude that DmxA is sufficient and required for the transient c-di-GMP burst during cytokinesis.
The fully active DmxA R615A I-site variant also supported the burst in c-di-GMP, while the noncomplementing DmxA ΔTMD and DmxA ΔGAF×2 variants did not (Fig. 4a; S6b).Importantly, DmxA ΔTMD -mVenus is similar to MalE-DmxA WT , which has DGC activity in vitro (Fig. 1d), but DmxA ΔTMD -mVenus does not localize to the division site (Fig. 4a), arguing that DmxA DGC activity in vivo is explicitly activated upon its recruitment to the division site.Furthermore, DmxA ΔGAF×2 localizes to mid-cell but lacks the region for DmxA dimerization, arguing that this region also in vivo mediates dimerization and, thus, DGC activity.Finally, we observed no significant difference in the c-di-GMP levels in unsynchronized populations of WT and ΔdmxA cells (Fig. S6g), corroborating that DmxA only displays DGC activity for a brief period (~20 min) of the 5 h cell cycle.
To resolve whether the increase in c-di-GMP initiates before or after the separation of the cytoplasm of the two daughters, we performed fluorescence recovery after photobleaching (FRAP) experiments in which we bleached the mScarlet-I signal in one half of pre-divisional cells with a high cdGreen2 signal.The bleaching event caused a decrease in the fluorescence signal in both cell halves in 50% of cells and only affected the signal in the bleached half in the remaining 50% (Fig. 4d).
We conclude that DmxA causes the transient c-di-GMP burst during cytokinesis and that DGC activity is explicitly activated upon recruitment to the division site and before the cytoplasm of the daughters is separated.By coupling DmxA activity and cytokinesis, two daughters with equal levels of c-di-GMP are generated.
DmxA is essential for the symmetric incorporation and allocation of the core T4PM at the nascent and new cell poles We next sought to establish the link between DmxA DGC activity during cytokinesis, motility and cell polarity.Because the T4PM core is present at both cell poles, is symmetrically incorporated at the nascent and new poles during and immediately after completion of cytokinesis 10,48 , and DmxA is active during cytokinesis, we initially focused on the polar assembly of the core T4PM.This assembly starts with forming the PilQ secretin in the outer membrane (OM), progresses inward, and culminates with the incorporation of cytoplasmic PilM 10 (Fig. S2b).The PilB and PilT ATPases bind to the cytoplasmic base of the core T4PM at the leading pole in a mutually exclusive manner to stimulate T4P extension and retraction, respectively 9,49,50 (Fig. S2b).Note that PilT localizes bipolarly, while PilB almost exclusively localizes unipolarly to the leading pole 9,21 .
First, we performed time-lapse microscopy using PilQ-sfGFP as a readout for assembly of the core T4PM.As reported, in WT, PilQ-sfGFP incorporation was initiated during cytokinesis and, upon completion of cytokinesis, continued symmetrically at the new poles in the two daughters (Fig. 5a; S7), giving rise to mirror-symmetric daughters.Strikingly, in the absence of DmxA, PilQ-sfGFP incorporation was not only significantly delayed but also asymmetric in the two daughters, resulting in asymmetric daughter pairs and with many cells having PilQ-sfGFP clusters of very different intensities or even only a single, unipolar PilQ-sfGFP cluster at the old pole (Fig. 5a; S7).Moreover, this defect in PilQ-sfGFP polarity was typically not fully corrected before the subsequent cell division (Fig. 5a; S7), resulting in propagation of the polarity defect.
Consistent with the faulty polar PilQ incorporation, we observed in quantitative analyses of snapshot microscopy images that PilQ-sfGFP, mCherry-PilM and mCherry-PilT localization in the ΔdmxA mutant was significantly shifted toward unipolar and, thus less symmetric than in WT (Fig. 5b).Similarly, PilB-mCherry was significantly more unipolar in ΔdmxA cells.The shifts toward asymmetry were primarily caused by many ΔdmxA cells having no or a strongly decreased fluorescence signal at the pole with the lowest fluorescence (Fig. 5b).Thus, in the absence of DmxA, all tested T4PM proteins localize significantly more asymmetrically than in WT.
We conclude that DmxA and the burst in c-di-GMP are essential for the symmetric incorporation and allocation of PilQ during and after cytokinesis, thereby generating mirrorsymmetric daughters.Moreover, we infer that the faulty polar PilQ incorporation and allocation contribute to the more asymmetric localization of the other tested T4PM proteins in the ΔdmxA mutant.

DmxA is essential for the symmetric allocation of the polarity proteins to the daughters during cytokinesis
We predicted that the proteins of the polarity module would also be symmetrically allocated during cytokinesis to generate mirror-symmetry of these proteins in the daughters.Briefly, RomR alone localizes polarly in the absence of the remaining five polarity proteins and brings about polar localization of these proteins 29,32 .Moreover, RomX and MglC localization follows that of RomR 26,29 and RomY the highest concentration of MglB 27 (Fig. S2f).
Therefore, we used RomR and MglB as well as MglA, which generates the output of the polarity module, as readouts for the localization of the proteins of the polarity module.
First, to test our prediction, we performed time-lapse microscopy of WT expressing RomR-mCherry.These analyses revealed a precise order of events in which, late during cytokinesis, RomR-mCherry was released from the old poles, then localized to the division site, and, upon completion of cytokinesis, was symmetrically allocated to the two daughters (Fig. 6a), giving rise to mirror-symmetric daughters.Remarkably, in the absence of DmxA, we observed very different patterns.During most division events, RomR-mCherry was released from the old poles, however, it was either not recruited at the division site but instead switched to the opposite pole (Fig. 6a, upper), or if it localized to the division site, it was asymmetrically allocated to the daughters (Fig. 6a, lower).Consequently, the daughters of a division event contained different amounts of RomR-mCherry, were not mirrorsymmetric, and had polar clusters of very different intensities or even only a single RomR-mCherry cluster at the old pole.The defects in RomR-mCherry polarity were typically also not fully corrected before the subsequent cell division (Fig. 6a).
Consistent with the faulty polar RomR allocation to the daughters in the absence of DmxA, we observed in quantitative analyses of snapshot microscopy images that RomR-mCherry, MglB-mVenus and MglA-mVenus in the ΔdmxA mutant localized in highly aberrant patterns and had largely lost their defined polar asymmetry in individual cells, displaying much broader variations in asymmetry values compared to WT (Fig. 6b).The aberrant RomR-mCherry and MglB-mVenus asymmetry resulted from the much broader variations in the fluorescence signals at both poles (Fig. 6b); accordingly, MglA polar localization was significantly reduced in many cells (Fig. 6b).
We conclude that RomR polarity is reset during cytokinesis in WT and that DmxA together with the burst in c-di-GMP are essential for this reset and the symmetric allocation of RomR to the two daughters.Moreover, we infer that the highly aberrant localization of MglB and MglA in the ΔdmxA mutant results from the faulty RomR allocation during cytokinesis.
Finally, we aimed to establish the link between the aberrant localization of the T4PM and the polarity proteins to the aberrant T4P-dependent motility behaviour of the ΔdmxA cells.
Because the polar signals of PilB-mCherry and MglA-mVenus are low, it is technically difficult to follow these fusions in time-lapse recordings.Therefore, we followed the localization of the MglA-GTP effector SgmX-mVenus, which is recruited to the leading pole by MglA, and then recruits the PilB ATPase to stimulate T4P extension 20,21 .In WT, SgmX-mVenus localized with a large cluster at the leading pole in 100% of cells and switched polarity during reversals (n=48) (Fig. 6c).In the ΔdmxA mutant, SgmX-mVenus localized as in WT in ~75% of cells (n=84) but in the remaining ~25%, SgmX-mVenus localized aberrantly with either a bipolar pattern and/or more unstably at the leading pole, i.e. the intensity at the leading pole would shortly decrease, and this was occasionally accompanied by a brief increase in fluorescence at the opposite pole (Fig. 6c).Importantly, many of these ~25% of cells hyper-reversed (Fig. 6c).
Altogether, these observations support that the aberrant localization of the T4PM and the polarity proteins caused by lack of DmxA results in motility defects with aberrant reversals.

Discussion
Here, we describe a c-di-GMP-dependent genetic program that is hardwired into the M. xanthus cell cycle and guarantees the formation of mirror-symmetric, phenotypically similar daughter cells.Specifically, the DGC DmxA is explicitly recruited to the division site late during cytokinesis, and released upon completion of cytokinesis.During this brief period of the cell cycle, its DGC activity is switched on resulting in a dramatic but transient increase in the c-di-GMP concentration.This c-di-GMP burst, in turn, ensures the equal and symmetric allocation of core T4PM proteins and polarity proteins to the two daughters.In the absence of DmxA, the daughters inherit unequal amounts of these proteins causing aberrant T4PM localization and cell polarity and, consequently, aberrant motility behaviour.Thus, M.
xanthus harnesses DmxA and c-di-GMP to ensure the generation of mirror-symmetric, phenotypically similar daughters in each cell division event.
DmxA recruitment to the division site late during cytokinesis depends on the TMD of DmxA and the divisome, suggesting that the TMD interacts with the divisome.Indeed, using proximity labelling, we identified the transmembrane divisome protein FtsK as a potential direct interaction partner of DmxA.Several lines of evidence support that DmxA is activated upon recruitment to the division site.First, the timing of DmxA localization to the division site and the burst in c-di-GMP perfectly correlate.Second, DmxA is required and sufficient for the burst in c-di-GMP.Third, DmxA ΔTMD does not localize to the division site and is not active in vivo; however, the protein has DGC activity in vitro.Although we cannot rule out that DmxA ΔTMD is less active than full-length DmxA, these observations jointly support that DmxA is activated at the division site.Finally, in cephalexin-treated cells and in cells depleted of FtsZ, DmxA accumulates at the same level as in untreated cells, strongly indicating that DmxA accumulation is not cell cycle-regulated and that DmxA activity is not regulated by the total cellular concentration.Based on these observations, we suggest that DmxA DGC activity, upon recruitment to the division site, is switched on either by interacting with protein(s) of the divisome or, alternatively, the high local DmxA concentration stimulates formation of the enzymatically active dimer.We speculate that the low-affinity I-site allows DmxA to synthesize high concentrations of c-di-GMP, and may solely be relevant at very high concentrations to avoid excessive overproduction of c-di-GMP.Upon completion of cytokinesis, the DmxA cluster disintegrates, likely subsequent to the disassembly of the divisome.As a consequence, c-di-GMP synthesis ceases and its level decreases rapidly.
Interestingly, none of the six predicted PDEs of M. xanthus have been implicated in motility 34   .In the future, it will be interesting to determine which PDE(s) are involved in the rapid decrease in the c-di-GMP concentration.
Despite only being active during a brief period of the cell cycle, DmxA is essential for WT motility behaviour.During this brief period, DmxA guarantees the symmetric incorporation and allocation at the nascent and new poles of PilQ of the T4PM and RomR of the polarity module.Because PilQ and RomR are at the base of the assembly of the core T4PM and the polar localization of the proteins of the polarity module, respectively, we suggest that the defects in PilQ and RomR polar localization during cytokinesis in the absence of DmxA causes the observed misincorporation of the core T4PM and the remaining polarity regulators, respectively.Because the polarity defects that arise during one division in cells lacking DmxA are not fully corrected until the next division, and mutants with aberrant localization of the polarity proteins have aberrant cell behaviours with altered reversal frequencies 18,23,24,26,27,29,33 , we suggest that the aberrant T4P-dependent motility behaviour in the absence of DmxA is the result of dual defects, i.e. the defects in the polar incorporation of the core T4PM and in the localization of polarity proteins.We also speculate, but have not shown, that the aberrant gliding behaviour is not only a consequence of the mislocalized polarity proteins but also involves defects in the polar incorporation of structural proteins of the Agl/Glt machine.
How, then, does the burst in c-di-GMP ensure the correct incorporation and allocation of polarly localized motility proteins and regulators?The effects of changing c-di-GMP levels are implemented by the binding of c-di-GMP to downstream effectors 2,3 .Because polar PilQ incorporation depends on its peptidoglycan-binding AMIN domains 51 , we suggest that the cdi-GMP burst brings about the localization of a landmark protein, which possibly binds peptidoglycan, at the nascent and new poles that assist polar recruitment of PilQ.The cytoplasmic RomR protein by an unknown mechanism localizes polarly in the absence of the other polarity proteins 32 .The symmetric allocation of RomR to the daughters involves a polarity reset involving three steps, i.e.RomR release from the old poles, its recruitment to the division site, and its symmetric allocation to the two daughters.In the absence of DmxA, RomR was still released from the old poles in most cells, suggesting that this step is independent of DmxA and c-di-GMP.However, RomR was either not recruited to the division site or, if it was recruited, then it was not symmetrically allocated to the two daughters.
Therefore, we suggest that c-di-GMP brings about the localization of a landmark protein at the division site that is recognized by RomR.Because the defects in PilQ and RomR polar localization are not fully corrected between division events, we also suggest that these landmarks may only be transiently active.In future experiments, it will be important to identify the effector(s) involved in the response to DmxA-generated c-di-GMP and to address whether these effector(s) serve as landmark(s) or to recruit landmark(s).Nonetheless, we speculate that an advantage of engaging a DGC in setting up correct cell polarity during cytokinesis could be that c-di-GMP would allow the transient function of effector(s)/polar landmark(s).
Consequently, one daughter has low c-di-GMP and becomes the flagellated swimming daughter, while the other daughter has high c-di-GMP and becomes the surface-associated daughter.By contrast, M. xanthus places the DGC DmxA at the division site, thereby enabling the formation of mirror-symmetric and phenotypically similar daughters.Thus, by deploying c-di-GMP synthesizing and degrading enzymes to distinct subcellular locations, bacteria harness c-di-GMP to establish deterministic genetic programs that are hardwired into the cell cycle to generate or, as shown here, minimize phenotypic heterogeneity (Fig. 7).
Similar to stochastically generated phenotypic heterogeneity [60][61][62] , the deterministic generation of phenotypic heterogeneity has been suggested to be part of a bet-hedging strategy in which the diversification of phenotypes optimizes the survival of the population and/or a division of labour strategy by enabling the colonization of multiple habitats in parallel 55,58,59,63 .Because M. xanthus translocates on surfaces in large cooperative swarms in which the motility of individual cells is highly coordinated, we speculate that reducing phenotypic heterogeneity during cell division optimizes its ability to perform its motilitydependent social behaviours.
Oxytetracyline and kanamycin at concentrations of 10 μg ml -1 and 50 µg ml -1 , respectively were added when needed.Cephalexin was added to a concentration of 35 µg ml -1 in liquid medium and 20 µg ml -1 on agarose.All M. xanthus strains are derivatives of the WT strain DK1622 12 .M. xanthus strains, plasmids and oligonucleotides used in this work are listed in Tables S3-S5, respectively.In-frame deletions or gene replacements were generated as described 65 , plasmids were integrated in a single copy by site-specific recombination at the Mx8 attB site or by homologous recombination at the MXAN_18-19 site or at the endogenous locus.All in-frame deletions and plasmid integrations were verified by PCR.
Motility assays.Population-based motility assays were performed as described 66 .Briefly, exponentially growing suspension cultures were harvested (3 min, 8,000 g, RT) and resuspended in 1% CTT to a calculated density of 7×10 9 cells ml -1 .5 µl aliquots of cell suspensions were spotted on 0.5% agar (Invitrogen) and 1.5% agar (Gibco) supplemented with 0.5% CTT and incubated at 32°C.Cells were imaged at 24 h using a M205FA Stereomicroscope (Leica) and a DMi8 inverted microscope (Leica) equipped with a Hamamatsu ORCA-Flash4.0V2 digital CMOS C11440 camera (Hamamatsu Photonics) and DFC9000 GT camera (Leica), respectively.To visualise single cells moving by T4Pdependent motility, 5 µl exponentially growing cells in suspension were placed in a 24-well polystyrene plate (Falcon).After 10 min incubation in the dark at RT, 200 µl of 1% methylcellulose in MMC buffer (10 mM MOPS, 4 mM MgSO4, 2 mM CaCl2, pH 7.6) were added, and cells incubated for 30 min in the dark at RT. Cells were imaged for 15 min with 30 s intervals.To visualize individual cells moving by gliding, exponentially growing cells in suspension were diluted to 3×10 8 and 5 µl spotted on 1.5% agar (Gibco) supplemented with 0.5% CTT and immediately covered with a coverslide.Cells were incubated 2 h at 32°C and then visualized for 15 min with 30 s intervals at RT. Cells were imaged using a DMi8 Inverted microscope and DFC9000 GT camera.Images were analysed using ImageJ 67 .
Negative stain transmission electron microscopy.10 µl of M. xanthus cells exponentially grown in suspension were placed on one side of the electron microscopy grid (Plano) and incubated at RT for 40 min.To avoid evaporation during this step, the grid was incubated in humid air.The liquid was blotted through the grid by capillarity by applying the side of the grid on Whatman paper.Cells were washed three times with 10 µl of double-distilled water and stained with UA-Zero EM Stain (Plano) (diluted to 0.25% (v/v) in double distilled water).
Operon mapping.Mapping of the ftsB-dmxA operon was performed as described 51 .Briefly, 1×10 9 WT cells from an exponentially growing suspension culture were harvested (3 min, 8,000 g, room temperature (RT)) and resuspended in 200 μl lysis-buffer (100 mM Tris-HCl pH 7.6, 1 mg ml -1 lysozyme).After incubation at 25°C for 5 min, cells were lysed and RNA purified using the Monarch Total RNA Miniprep Kit (NEB) according to the manufacturer's instructions except that the on-column DNase treatment was omitted.RNA was eluted in RNase-free water, treated with Turbo DNase (Invitrogen) and purified using the Monarch RNA Cleanup Kit (50 μg) (NEB) and eluted in RNase-free water. 1 μg of RNA was used for cDNA synthesis using the LunaScript RT SuperMix Kit (NEB) with and without reversetranscriptase.cDNA, the mock reaction without reverse-transcriptase, or genomic DNA were used as template for PCR using the primers listed in Table S5.
Cell length determination.5-µl aliquots of exponentially growing suspension cultures were spotted on 1% agarose supplemented with 0.2% CTT.Cells were immediately covered with a coverslide, and imaged using a DMi8 Inverted microscope and DFC9000 GT camera.To assess cell length, cells were segmented using Omnipose 70 , segmentation was manually curated using Oufti 71 , analysed using Matlab R2020a (The MathWorks) and plotted using GraphPad Prism (GraphPad Software, LLC).Fluorescence microscopy.In all time-lapse microscopy experiments except for those involving SgmX-mVenus, cells were visualized as in 72 with slight modifications.Briefly, 5 µl exponentially growing cells in suspension were placed on a glass coverslide attached to a plastic frame.Cells were covered with a thick 1% agarose pad supplemented with 0.2% CTT, the pad sealed with parafilm to reduce evaporation, and cells imaged after 180 min.To avoid that cells would move out of the field of view, strains contained the ΔgltB mutation.To clearly distinguish leading and lagging cell poles of cells moving by T4P-dependent motility, time-lapse microscopy experiments involving SgmX-mVenus, were done on Chitosan-coated µ-Dishes (Ibidi) as described 73 .Briefly, a 100 µl-aliquot of exponentially growing cells was diluted in 900 µl MC7 buffer (10 mM MOPS pH 7.0, 1 mM CaCl2), spotted on the chitosancoated µ-Dish, and imaged after 30 min.Snapshot microscopy images were captured from cells on 1% agarose pad supplemented with 0.2% CTT (biosensor and DmxA-mVenus) or on Chitosan-coated µ-Dishes (all other fluorescent proteins).Cells were imaged using a DMi8 inverted microscope and a Hamamatsu ORCA-Flash4.0V2 Digital CMOS C11440 or a DFC9000 GT camera.Data was analysed using Oufti 71  were binned from the ω values as follows: unipolar (ω>0.9),bipolar asymmetric (0.9≥ω>0.2) and bipolar symmetric (0.2≥ω).Diffuse localization was determined when no polar signal was detected.Polar fluorescence of moving cells synthesizing SgmX-mVenus was followed manually.
For the analysis of single cell cdGreen2 fluorescence, cells were segmented using Omnipose 70 and the segmentation manually curated using Oufti 71 .For normalization, the average cellular fluorescence of each cell in the green channel (cdGreen2) was divided by the red channel (mScarlet-I).
C-di-GMP quantification.C-di-GMP levels were determined as described 74 .Briefly, 4 ml of exponentially growing cells were harvested by centrifugation (20 min, 2,500 g, 4°C).Cells were mixed with 300 µl ice-cold extraction buffer (high-pressure liquid chromatography [HPLC]-grade acetonitrile-methanol-water [2:2:1, v:v:v]), and incubated 15 min at 4°C to quench metabolism.Extraction was performed at 95°C for 10 min, samples were centrifuged (10 min, 21,130 g, 4°C) and the supernatant containing extracted metabolites transferred to a new Eppendorf tube.The pellet was washed with 200 µl extraction buffer and centrifuged (10 min, 21,130 g, 4°C).This step was repeated.The residual pellet containing proteins was kept, and the three supernatants from the extraction and the two washing steps containing cdi-GMP were pooled and evaporated to dryness in a vacuum centrifuge.Subsequently, the samples with c-di-GMP were dissolved in HPLC-grade water for analysis by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS).In parallel, to determine the protein concentration for each sample, the residual pellets were resuspended in 800 µl 0.1 M NaOH, and heated for 15 min at 95°C until dissolution.Protein levels were determined using a 660 nm Protein Assay (Pierce) following the manufacturer's instructions.
DGC activity assay.DGC activity assays were performed, using the EnzCheck®Pyrophosphate Assay Kit (Thermo) as described 76 .Briefly, the release of inorganic pyrophosphate during c-di-GMP synthesis was followed by measuring the absorbance change at 360 nm in a Tecan M200 pro, in 30 s intervals for 1 h.Reactions In vitro nucleotide binding assay.C-di-GMP binding was determined by Bio-Layer Interferometry using the BLItz system (ForteBio) 77 and a Streptavidin SA biosensor (ForteBio).Briefly, 500 nM biotinylated c-di-GMP (Biolog) in SEC-buffer supplemented with 0.1% (v/v) Tween-20 was immobilized onto the biosensors for 120 s, and unbound molecules washed off for 30 s. Association and dissociation of a protein were carried out for 120 and 120 s respectively.Binding was fitted to the "One site -Total" binding model in GraphPad Prism.
The beads were washed three times with 1 ml RIPA buffer, twice with 1 ml 1 M KCl, and three times with 1 ml 50 mM Tris-HCl pH 7.6.Finally, proteins were eluted using on-bead digest as described 69 .Briefly, 100 µl elution buffer 1 (100 mM ammonium bicarbonate, 1 µg trypsin (Promega)) was added to each sample.After 30 min incubation at 30°C, the supernatant containing the digested proteins was collected.Beads were washed twice with elution buffer 2 (10 mM ammonium bicarbonate, 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)) and added to the first elution fraction.Digestion continued overnight at 30°C.Next, the peptides were incubated with 10 mM iodoacetamide for 30 min at 25°C in the dark.Prior to LC-MS analysis, peptide samples were desalted using C18 solid phase extraction spin columns (Macherey-Nagel).Peptide mixtures were then analysed using LC-MS on an Exploris 480 instrument connected to an Ultimate 3000 RSLCnano and a nanospray flex ion source (all Thermo Scientific).A detailed description of the LC-MS parameters are described in 78 .The following separating gradient was used: 98% solvent A (0.15% formic acid) and 2% solvent B (99.85% acetonitrile, 0.15% formic acid) to 30% solvent B over 40 min at a flow rate of 300 nl/min.Peptides were ionized at a spray voltage of 2.3 kV, and ion transfer tube temperature set at 275°C, 445.12003 m/z was used as internal calibrant.The data acquisition mode was set to obtain one high-resolution MS scan at a resolution of 60,000 full width at half maximum (at m/z 200) followed by MS/MS scans of the most intense ions within 1 s (cycle 1s).The ion accumulation time was set to 50 ms (MS) and 50 ms at 17,500 resolution (MS/MS).The automatic gain control (AGC) was set to 3×10 6 for MS survey scan and 2×10 5 for MS/MS scans.MS raw data was then analysed with MaxQuant 79 , and an M. xanthus UniProt database 80 .MaxQuant was executed in standard settings without "match between runs" option.The search criteria were set as follows: full tryptic specificity was required (cleavage after lysine or arginine residues); two missed cleavages were allowed; carbamidomethylation (C) was set as fixed modification; oxidation (M) and deamidation (N,Q) as variable modifications.The MaxQuant proteinGroups.txtfile was further processed by the SafeQuant R package for statistical analysis 81 .
Total proteome analysis.The total proteome of M. xanthus cells grown in suspension culture was determined following a slightly modified protocol of 69 .Briefly, 2 ml of exponentially growing suspension cultures were harvested (8,000 g, 3 min, RT).Cells were resuspended in 1 ml PBS and harvested again.Subsequently, the supernatant was discarded and the pellet snap-frozen in liquid nitrogen.The pellet was suspended in 150 µl 2% Sodium Lauryl Sulfate (SLS) and proteins precipitated using acetone.For digestion, samples were resuspended in 0.5% SLS with 1 μg trypsin (Promega) and incubated for 30 min at 30˚C, subsequently 5 mM TCEP were added to the suspension and further incubated overnight.
Following, acetylation using 10 mM iodoacetamide for 30 min at 25˚C in the dark, the peptides were desalted using C18 solid phase extraction.For label-free protein quantification, peptide mixtures were analysed using LC-MS.The data was acquired in dataindependent acquisition mode and the MS raw data analysed by DIA-NN as described 82,83 .
Data were further analysed and plotted using Python (3.7).The mass spectrometry proteomics data of whole cell proteomics and proximity labelling experiments have been deposited to the ProteomeXchange Consortium 84 via the PRIDE 85 partner repository with the dataset identifier PXD049046 (username: reviewer_pxd049046@ebi.ac.uk; password: WxYzAbMr).
Bioinformatics.The KEGG database 86 was used to assign functions to proteins, identify orthologs of M. xanthus proteins using a reciprocal best BlastP hit method and collect the 16s ribosomal RNA sequence of fully sequenced myxobacteria (Table S6).Protein domains were identified using InterPro 87 , SMART 88 , and the predicted AlphaFold structures.The DmxA protein sequence without the N-terminal transmembrane helices (amino acid 1-209) was used for AlphaFold-Multimer modelling via ColabFold (1.5.0) [89][90][91]  The phylogenetic tree was prepared using the 16s ribosomal RNA sequence of fully sequenced myxobacteria in MEGA7 92 using the Neighbor-Joining method 93 .Bootstrap values (500 replicates) are shown next to the branches 94 .RNA-seq.data were plotted using the BioMap function in Matlab.The base-by-base alignment coverage of RNA-seq and Cappable-seq reads of 95 were plotted for each position.
, Metamorph® v 7.5 (Molecular Devices), Matlab and ImageJ 67 .DmxA-mVenus clusters and constrictions were identified manually.To identify and analyse polar clusters in snapshots, we used a custom-made Matlab script 26 .Briefly, cells were segmented, and polar clusters were identified as having an average fluorescence signal of 1.5 SD (MglA) or 2 SD (all other proteins), above the mean cytoplasmic fluorescence and a size of three or more pixels.For each cell with polar clusters, an asymmetry index (ω) was calculated as: ω total fluorescence at pole 1 total fluorescence at pole 2 total fluorescence at pole 1 total fluorescence at pole 2 Pole 1 was assigned to the pole with the highest fluorescence.The localization patterns

contained 1
µM protein, and 50 µM GTP.Inhibition reactions were fit in GraphPad Prism to the equation V[cdG]=V0/(1/1+([cdG]/Ki) h ), where V0 represents the reaction velocity in the absence of c-di-GMP, [cdG] the concentration of c-di-GMP in the reaction, Ki the inhibitory constant and h the Hill coefficient.
Statistics.Colony expansion and c-di-GMP measurements were analysed using Student's ttest in GraphPad Prism.Single cell speed and cell length distributions were analysed using the Mann-Whitney test in GraphPad Prism.Single cell reversal assays were analysed using the One-Way ANOVA function with Fishers LSD post-hoc test in GraphPad Prism.Asymmetry indexes (ω) were analysed using the rank-sum function (Mann-Whitney test) in Matlab.MAD was used as a measure of data variability and calculated based on the formula MAD=median(|xi -x͂ |).

Figure 1 .
Figure 1.DmxA has DGC activity and a low-affinity I-site.a, Domain architecture of DmxA.b, SEC of MalE-DmxA variants.Domain architecture of DmxA variants are shown above chromatograms.Grey arrowheads indicate void volume, and black arrowheads elution volume with the corresponding calculated molecular weight.c, AlphaFold-Multimer structural model of dimeric DmxA.The transmembrane helices were removed before generating the model, residue numbers are indicated.d, In vitro DGC assay of the indicated MalE-DmxA variants.The relative amount of c-di-GMP synthesized after 1 h was determined by measuring released inorganic pyrophosphate.Measurements from three technical replicates are shown relative to the mean (black lines) of MalE-DmxA WT .e, Bio-Layer Interferometric analysis of c-di-GMP binding by MalE-DmxA variants.Streptavidincoated sensors were loaded with 500 nM biotinylated c-di-GMP and probed with 10 µM of the indicated proteins.The interaction kinetics were followed by monitoring the wavelength shift during the association and dissociation of the analyte.f, Determination of KD of MalE-DmxA for c-di-GMP.Plot shows the equilibrium levels measured at the indicated MalE-DmxA WT concentrations (see also Fig S1d).The data were fitted to a non-cooperative onesite specific-binding model.g, Determination of Ki of MalE-DmxA for c-di-GMP.Inhibition of the specific activity of DmxA WT and DmxA R615A DGC activity over time was measured as in d in the presence of different c-di-GMP concentrations.Points and error bars represent the mean ± standard deviation (SD) calculated from three biological replicates.The data were fitted to an inhibition model with variable slope.

Figure 2 .
Figure 2. The ΔdmxA mutant has an aberrant motility behaviour with aberrant reversals and a cell polarity defect a, Population based motility assays.T4P-dependent motility and gliding were analysed on 0.5% and 1.5% agar, respectively.Numbers indicate the colony expansion in 24 h as mean ± SD (n=3 biological replicates).In the complementation strain, dmxA was expressed from its native promoter from a plasmid integrated in a single copy at the Mx8 attB site.Black, white and red asterisks indicate p<0.05,Student's t-test against WT (black, white) or the ΔpilA or ΔgltB controls (red).Scale bars, 1 mm (left), 1 mm (middle), 50 μm (right).b-c, Single cell-based motility assays.T4P-dependent motility was measured for cells on a polystyrene surface covered with 1% methylcellulose (b) and gliding on 1.5% agar supplemented with 0.5% CTT (c).Cells were imaged for 15 min with 30 s intervals.Speed (n=20 in each of three biological replicates indicated in different colours) and number of reversals (n=30 in each of three biological replicates).Only cells moving during the entire recording interval were included.For speed, error bars represent mean ± SD calculated from all data points.Reversals are represented as histograms based on all three replicates, error

Figure 3 .
Figure 3. DmxA is recruited to the division site during cytokinesis by the divisome and its function depends on DGC activity a, Localization of DmxA-mVenus in the presence and absence of cephalexin.The percentage of cells with a DmxA-mVenus cluster (white) or a constriction (pink) is indicated (n=200 from one biological replicate).Scale bars, 5µm.b, DmxA-mVenus localization during the cell cycle.Left panels, epifluorescence and phase-contrast images from time-lapse microscopy of cells expressing DmxA-mVenus.Images were recorded every 5 min; black arrowhead indicates completion of cytokinesis (defined as the first frame at which daughters were clearly separated); right panels, analysis of the appearance relative to completion of cytokinesis and lifetime of DmxA-mVenus clusters.The first time point after completion of cytokinesis is defined as t=0 and indicated by the grey vertical bar.Box plots show the

Figure 4 .
Figure 4. DmxA DGC activity is switched on upon recruitment to the division site a, Analysis of cdGreen2 and mScarlet-I fluorescence in the indicated strains.Upper panels, epifluorescence snapshot images of cells expressing cdGreen2 and mScarlet-I.White arrowheads indicate cells with high cdGreen2 fluorescence.The cdGreen2-mScarlet-I operon was expressed from the constitutively active pilA promoter.Scale bar, 5 μm.Lower panels, scatter plots of the cdGreen2/mScarlet-I fluorescence ratio of each cell relative to its cell length.Colours indicate the density of points according to the scale on the right.Total number of cells from one biological replicate indicated.b-c, cdGreen2 fluorescence in Δ10 (b) and ΔdmxA (c) cells during the cell cycle.Left panels, epifluorescence and phasecontrast images from time-lapse microscopy of cells expressing cdGreen2.Images were recorded every 10 min; arrowheads indicate completion of cytokinesis.In b, right panels, analysis of the appearance relative to completion of cytokinesis and lifetime of the high cdGreen2 fluorescence.The first time point after completion of cytokinesis is defined as t=0 and indicated by the grey vertical bar.Box plots as in Fig. 3b based on three biological replicates with the number of cells per replicate and the corresponding data points in different colours.d, FRAP experiment on predivisional Δ10 cells expressing cdGreen2 and mScarlet-I.The mScarlet-I signal of one half of a cell was bleached.Post-bleached images

Figure 5 .
Figure 5. DmxA is essential for the symmetric incorporation and allocation of the core T4PM proteins at the nascent and new cell poles a, PilQ-sfGFP localization during the cell cycle.Epifluorescence and phase-contrast images from time-lapse microscopy of WT (left panels) and ΔdmxA cells (right panels).Images were recorded every 30 min; arrowheads indicate completion of cytokinesis.Schematics show dominant localization patterns of PilQ-sfGFP in WT and ΔdmxA cells during the cell cycle.b, Localization of PilQ-sfGFP, mCherry-PilM, PilB-mCherry and mCherry-PilT in WT and ΔdmxA cells.Left panels, representative snapshot images.Scale bar, 5 μm.Middle panels, histograms of the distribution of the fraction of the total cellular fluorescence in polar clusters

Figure 6 .
Figure 6.DmxA is essential for the symmetric allocation of polarity proteins to the daughters during cytokinesis a, RomR-mCherry localization during the cell cycle.Epifluorescence and phase-contrast images from time-lapse microscopy of WT and ΔdmxA cells.Images were recorded every 10 min; black arrowheads indicate completion of cytokinesis.Schematics show localization patterns of RomR-mCherry in WT and ΔdmxA cells during the cell cycle.b, Localization of RomR-mCherry, MglB-mVenus, MglA-mVenus in WT and ΔdmxA cells.Left panels, representative snapshot images.Analysis of snapshot images of WT (grey) and ΔdmxA (blue) were done as in Fig. 5b.c, SgmX-mVenus localization in moving WT and ΔdmxA cells.Images were recorded every 30 s. White arrows indicate reversals.

Figure 7 .
Figure 7. Deployment of DGC and/or PDEs to distinct subcellular locations establishes deterministic genetic programs hardwired into the cell cycle to generate or minimize phenotypic heterogeneity In M. xanthus, DmxA (blue) localizes to and is switched on at the division site creating a c-di-GMP burst that ensures similar daughter cells.In C. crescentus, the flagellated, swarmer cell has low c-di-GMP due to the activity of the PDE PdeA at the flagellated pole (green).Upon differentiation to the surface-associated stalked cell, the c-di-GMP level increases due to the activity of the DGC PleD at the stalked pole (blue).In predivisional cells, PdeA and PleD localize to opposite poles, giving rise to a swarmer cell with low c-di-GMP and a stalked cell with high c-di-GMP upon division.In P. aeruginosa/S.putrefaciens, the flagellated, swimming cell has low c-di-GMP due to the activity of the PDE Pch/PdeB at the flagellated pole (green).Upon surface contact, c-di-GMP increases but the involved DGC(s) remain to be identified.High c-di-GMP stimulates T4P formation and surface adhesion.During division, the flagellated pole inherits the PDE, creating a flagellated, swimming daughter with low c-di-GMP and a surface-adhered, piliated daughter with high c-di-GMP.
89The predicted Local Distance Difference Test (pLDDT) and predicted Alignment Error (pAE) graphs of the five models generated were made using a custom Matlab script.Models were ranked based on combined pLDDT and pAE values, with the best-ranked models used for further analysis and presentation.Per residue model accuracy was estimated based on pLDDT values (>90, high accuracy; 70-90, generally good accuracy; 50-70, low accuracy; <50, should not be interpreted)89.Relative domain positions were validated by pAE.The pAE graphs indicate the expected position error at residue X if the predicted and true structures were aligned on residue Y; the lower the pAE value, the higher the accuracy of the relative position of residue pairs and, consequently, the relative position of domains/subunits/proteins89.PyMOL version 2.4.1 (http://www.pymol.org/pymol)was used to analyse and visualize the models.