Summary
Myxococcus xanthus, a soil bacterium, predates collectively using motility to invade prey colonies. Prey lysis is mostly thought to rely on secreted factors, cocktails of antibiotics and enzymes, and direct contac with Myxococcus cells. In this study, we show that on surfaces the coupling of A-motility and contact-dependent killing is the central predatory mechanism driving effective prey colony invasion and consumption. At the molecular level, contact-dependent killing involves a newly discovered type IV filament-like machinery (Kil) that both promotes motility arrest and prey cell plasmolysis. In this process, Kil proteins assemble at the predator-prey contact site, suggesting that they allow tight contact with prey cells for their intoxication. Kil-like systems form a new class of Tad-like machineries in predatory bacteria, suggesting a conserved function in predator-prey interactions. This study further reveals a novel cell-cell interaction function for bacterial pili-like assemblages.
Introduction
Bacterial predators have evolved strategies to consume other microbes as a nutrient source. Despite the suspected importance of predation on microbial ecology1, a limited number of bacterial species are currently reported as predatory. Amongst them, obligate intracellular predators collectively known as BALOs (eg Bdellovibrio bacteriovorus)1, penetrate their bacterial prey cell wall and multiply in the periplasm, escaping and killing the host bacteria2. Quite differently, facultative predators (meaning that they can be cultured in absence of prey if nutrient media are provided, ie Myxococcus, Lysobacter and Herpetosiphon1) attack their preys extracellularly, presumably by secreting antimicrobial substances and digesting the resulting products. Among these organisms and studied here, Myxococcus xanthus, a delta-proteobacterium, is of particular interest because it uses large-scale collective movements to attack prey bacteria in a so-called “wolf-pack” mechanism3.
A tremendous body of work describes how Myxococcus cells move and respond to signals in pure culture4. In contrast, mechanistic studies of the predatory cycle have been limited. Currently, it is considered that coordinated group movements allow Myxococcus cells to invade prey colonies and consume them via the secretion of a number of diffusible factors, extracellular enzymes, antibiotics and outer membrane vesicles3,5,6. While each of these processes could contribute to predation, evidence for their requirement is still missing3. In addition, Myxococcus cells have also been observed to induce prey cell plasmolysis upon contact7. While a number of contact-dependent mechanisms could be involved, including Type VI secretion8 and Outer Membrane Exchange (OME9, see below), none have yet been implicated in predation. In this study, we analyzed the importance of motility and contact-dependent killing in the Myxococcus predation cycle.
To explore these central questions, we first developed a sufficiently resolved imaging assay where the Myxococcus predation cycle can be imaged stably at the single cell level over periods of time encompassing several hours with a temporal resolution of seconds. The exact methodology underlying this technique is described in a dedicated manuscript10; briefly, the system relates predatory patterns observed at the mesoscale with single cell resolution, obtained by zooming in and out on the same microscopy specimen (Figure 1). Here, we employed it to study how Myxococcus cells invade and grow over Escherichia coli prey cells during the initial invasion stage (Figure 1, Movie S1).
A-motility is required for prey colony invasion
Although the function of motility in prey invasion is generally accepted, Myxococcus xanthus possesses two independent motility systems and the relative contribution of each system to the invasion process is unknown. Social (S)-motility is a form of bacterial “twitching” motility that uses so-called Type IV pili (TFP) acting at the bacterial pole11. In this process, polymerized TFPs act like “grappling hooks” that retract and pull the cell forward. S-motility promotes the coordinated movements of Myxococcus cells within large cell groups due to interaction with a self-secreted extracellular matrix formed of Exo-Polysaccharide (EPS)12–14. A(Adventurous)-motility promotes the movement of Myxococcus single cells at the colony edges. A-motility is driven by a mobile cell-envelope motor complex (named Agl-Glt) that traffics in helical trajectories along the cell axis, driving rotational propulsion of the cell when it becomes tethered to the underlying surface at so-called bacterial Focal Adhesions (bFAs)15. We tested the relative contribution of each motility system to prey invasion by comparing the relative predatory performances of WT, A+S-(ΔpilA16) and A-(ΔaglQ16) S+ (Figure 1). Interestingly, although A+S- cells were defective in the late developmental steps (fruiting body formation), they were still proficient at prey invasion (Figure 1b). On the contrary, the A-S+ strain was very defective at prey colony invasion (Figure 1c). Zooming at the prey colony border, it was apparent that the A-S+ cells were able to expand and contact the prey colony, but they were unable to penetrate it efficiently, suggesting that Type IV pili on their own are not sufficient for invasion (Figure 1c, Movie S2). Conversely, A-motile cells were observed to penetrate the tightly-knitted E. coli colony with single Myxococcus cells moving into the prey colony, followed by larger cell groups (Figure 1a). Similar motility requirements were also observed in a predatory assay where predatory and prey cells are pre-mixed before they are spotted on an agar surface (Figure S1a). Thus, A-motility is the main driver of prey invasion on surfaces.
Invading Myxococcus cells kill prey cells upon contact
To further determine how A-motile cells invade the prey colony, we shot single cell time-lapse movies of the invasion process. First, we localized a bFA marker, the AglZ protein17 fused to Neon-Green (AglZ-NG) in Myxococcus cells as they penetrate the prey colony. AglZ-NG binds to the cytoplasmic face of the Agl-Glt complex and has long been used as a bFA localization marker; it generally forms fixed fluorescent clusters on the ventral side of the cell that retain fixed positions in gliding cells17. As Myxococcus cells invaded prey colonies, they often formed “arrow-shaped” cell groups, in which the cells within the arrow assembled focal adhesions (Figure 2a, Movie S3). E. coli cells lysed in the vicinity of the Myxococcus cells, suggesting that a contact-dependent killing mechanism as reported by Zhang et al.7 occurs during prey colony invasion (Figure 2a). To observe this activity directly, we set up a Myxococcus - E. coli interaction microscopy assay where predator - prey interactions can be easily studied, isolated from a larger multicellular context (see Methods). In this system, A-motile Myxococcus cells were observed to mark a pause and disassemble bFAs when contacting E. coli cells (Figure 2b, Movie S4, further quantified below); this pause was invariably followed by the rapid death of E. coli, as detected by the instantaneous dispersal of a cytosolic fluorescent protein (mCherry or GFP, Figure 2b-2c, observed in n=20 cells). This observation suggest that the killing occurs by plasmolysis, a process which is likely to be the same as that described by Zhang et al.7. To demonstrate this, we mixed Myxococcus cells with E. coli cells in which peptidoglycan (PG) had been labeled by fluorescent D-amino Acids (TADA15). TADA is covalently incorporated into the PG pentapeptide backbone and it does not diffuse laterally18. We first observed contraction of the E. coli cytosolic dense region at the pole by phase contrast (Figure 2d), which was followed by the appearance of a dark area in the PG TADA staining exactly at the predator-prey contact site (Figure 2d). It is unlikely that this dark area forms due to the new incorporation of unlabeled prey PG, because it was detected immediately upon prey cell death and propagated bi-directionally afterwards (Figure 2d-e). Thus, these observations suggest that, upon contact, Myxococcus induces degradation of the E. coli PG, which provokes cell lysis due to loss of turgor pressure and hyper osmotic shock7. The bi-directional propagation of PG hydrolysis (as detected by loss of TADA signal) suggests that PG hydrolysis could be driven by the activity of PG hydrolase(s) disseminating from the predator-prey contact site.
A predicted Tad-pilus is required for contact-dependent killing
We next aimed to identify the molecular system that underlies contact-dependent killing. Although motility appears to be essential during the predation process (Figure S1a), at the microscopic level, direct transplantation of A-S- (aglQ pilA) in E. coli prey colonies still exhibit contact-dependent killing (Figure S1a-b), demonstrating that the killing activity is not carried by the motility complexes themselves. Myxococcus xanthus also expresses a functional Type VI secretion system (T6SS), which appears to act as a factor modulating population homeostasis and mediating Kin discrimination between M. xanthus strains8,19. A T6SS deletion strain (Δt6ss) had no observable defect in contact-dependent killing of prey cells (Figure S1a and S1c). In addition, the Myxococcus T6SS assembled in a prey-independent manner as observed using a functional VipA-GFP strain that marks the T6SS contractile sheath20 (Figure S1d-f), confirming that T6SS is not involved in predatory killing on surfaces.
To identify the genes (directly or indirectly) involved in the contact-dependent killing mechanism, we designed an assay where contact-dependent killing can be directly monitored in liquid cultures and observed via a simple colorimetric assay. In this system, the lysis of E. coli cells can be directly monitored when intracellular β-galactosidase is released in buffer containing ChloroPhenol Red-β-D-Galactopyranoside (CPRG), which acts as a substrate for the enzyme and generates a dark red hydrolysis reaction product21. Indeed, while Myxococcus or E. coli cells incubated alone did not produce color during a 120-hour incubation, their mixing produced red color indicative of E. coli lysis after 24 h (Figure S2a). In this assay, a t6SS mutant was still able to lyse E. coli cells, demonstrating that it does not report on T6SS-dependent killing (Figure S2a-b). CPRG hydrolysis was not detected when Myxococcus and E. coli were separated by a semi-permeable membrane that allows diffusion of soluble molecules, showing that the assay reports contact-dependent killing (Figure S2a). In this liquid assay, the Myxococcus - E. coli contacts are very distinct from contacts on solid surfaces and thus, the genetic requirements are likely quite distinct. Indeed, in the liquid CPRG assay, we observed that TFPs are essential for killing while the Agl/Glt system is dispensable (Figure S2b). In this condition, TFPs promote a prey-induced aggregation of cells (Figure S2c) and thus probably mediate the necessary tight contacts between Myxococcus and E. coli cells. As shown below, the killing process itself is the same in liquid as the one observed on surfaces and it is not directly mediated by the TFPs.
Given the probable indirect effect of TFPs, we next searched additional systems involved in CPRG contact-dependent killing, using a targeted approach and testing the effect of mutations in genome annotated cell-envelope complexes, on contact-dependent killing in liquid cultures. Doing so, we identified two critical genetic regions, the MXAN_3102-3108 and the MXAN_4648-4661 (Figure 3). Functional annotations indicate that both genetic regions carry a complementary set of genes encoding proteins that assemble a so-called Tight adherence (Tad) pilus. Bacterial Tad pili are members of the type IV filament superfamily (also including Type IV pili, a and b types, and Type II secretion systems) and extrude polymeric pilin filaments assembled via inner membrane associated motors through an OM secretin22. Tad pili have been generally involved in bacterial adhesion and more recently, in contact-dependent regulation of adhesion23. Within the MXAN_3102-3108 cluster, genes with annotated functions encode a predicted pre-pilin peptidase (CpaA and renamed KilA) following the Caulobacter crescentus Tad pilus encoding cpa genes nomenclature), a secretin homolog (CpaC/KilC) and a cytoplasmic hexameric ATPase (CpaF/KilF) (Figure 3a, Figure S3a-b, Table S1). All the other genes encode proteins of unknown function, with two predicted OM lipoproteins and several proteins containing predicted ForkHead-Associated domains (FHA24, Table S1, see discussion). The second genetic region, MXAN_4648-4661, contains up to 14 predicted open-reading frames of which the only functionally annotated genes encode homologs of the Tad IM platform proteins (CpaG/KilG and CpaH/KilH), OM protein (CpaB/KilB), major pilin (Flp/KilK) and two pseudo-pilin subunits (KilL, M) (Figure 3a, Figure S3c-d, Table S1). However, the splitting of Tad homologs in distinct genetic clusters is a unique situation22 and asks whether these genes encode proteins involved in the same function.
Expression analysis suggests that the cluster 1 and cluster 2 genes are expressed together and induced in starvation conditions (Figure S4a). We systematically deleted all the predicted Tad components in cluster 1 and 2 alone or in combination and measured the ability of each mutant to lyse E. coli in the CPRG colorimetric assay (Figure 3b). All the predicted core genes, IM platform, OM secretin and associated CpaB homolog are essential for prey lysis, with the exception of the putative pre-pilin peptidase, KilA. Deletion of the genes encoding predicted pseudo-pilins KilL and M did not affect E. coli killing; in these conditions, pilin fibers are only partially required because deletion of KilK, the major pilin subunit, reduces the lytic activity significantly but not fully (Figure 3b). Given that the genes are organized into potential operon structures, we confirmed that the CPRG-killing phenotypes of predicted cluster 1 and cluster 2 core genes were not caused by potential polar effects (Figure S4b). In liquid, predicted core gene mutants had the same propensity as wild-type to form biofilms in presence of the prey suggesting that they act downstream in the interaction process (Figure S2c).
We next tested whether liquid killing and contact-dependent killing on surfaces reflected the same process. For this, we analyzed selected kil mutants, predicted secretin (KilC), IM platform (KilH and KilG), OM-CpaB homolog (KilB), pilin and pseudopilins (KilK, L, M) in contact-dependent killing at the single cell level. Prey recognition is first revealed by the induction of a motility pause upon prey cell contact (Figure 2). This recognition was severely impaired although not fully in secretin (kilC), IM platform protein (kilG) and triple pilin (ΔkilKMN) mutants (Figure 3c, ∼8% of the contacts led to motility pauses vs ∼30% for the WT). In contrast, recognition was not impaired to significant levels in IM platform protein (kilH), CpaB-homolog (kilB) and pilin (kilK) mutants (Figure 3c). The potential basis of this differential impact is further analyzed in the discussion. On the contrary, prey cell plasmolysis was dramatically impacted in all predicted core components (∼2% of the contacts led to prey lysis vs ∼26% for the WT), the only exception being the single pilin (kilK) mutant in which prey cell lysis was reduced but still present (∼13%, Figure 3d). Deletion of all three genes encoding pilin-like proteins nevertheless affected in prey cell killing to levels observed in core component mutants. This is not observed to such extent in the CPRG assay, which could be explained by different cell-cell interaction requirements, perhaps compensation by TFPs in liquid cultures. Given the prominent role of the pilins at the single cell level, the predicted pre-pilin peptidase KilA would have been expected to be essential. However, expression of the kilA gene is very low under all tested conditions (Figure S4a). Prepilin peptidases are known to be promiscuous25 and thus another peptidase (ie PilD, the Type IV pilus peptidase26) could also process the Kil-associated pilins. This hypothesis could however not be tested because PilD appears essential for reasons that remain to be determined26. Altogether, the data supports that the proteins from the two clusters function in starvation conditions and that they could make up a Tad-like core structure, for prey cell recognition, regulating motility in contact with prey cells, and prey killing, allowing contact-dependent plasmolysis.
Kil proteins assemble at contact sites and mediate motility regulation and killing
We next determined if the Kil proteins indeed form a single Tad-like system in contact with prey cells. To do so, the predicted ATPAse (KilF) (Figure 3a) was N-terminally fused to the Neon Green (NG) and expressed from the native chromosomal locus. The corresponding fusion appeared fully functional (Figure S4c). In absence of prey cells, NG-KilF was diffuse in the cytoplasm. Remarkably, when Myxoccocus cells established contact with prey cells, NG-KilF rapidly formed a fluorescent-bright cluster at the prey contact site. Cluster formation was invariably followed by a motility pause and cell lysis. Observed clusters did not localize to any specific cellular site but they formed where Myxococcus cells touched prey cells. Cluster formation was correlated to motility arrest and their dispersal coincided with motility resumption (Figure 4a, Movie S5). To confirm that the KilF clusters reflect assembly of a full Tad-like apparatus, we next attempted to label a component of the IM platform KilG (Cluster 2), expressing a KilG-NG fusion this time ectopically from a pilA promoter (PpilA) in a kilG mutant background. The fusion was partially functional (Figure S4d), but nevertheless KilG-NG clusters could also be observed forming at the prey contact site immediately after lysis (Figure 4b, Movie S6). These results strongly suggest that a Tad-apparatus assembled from the products of the cluster 1 and cluster 2 genes.
Additional non-core proteins are also recruited at the contact sites: downstream from kilF and likely co-transcribed, the MXAN_3108 gene (kilD, Figures 3a and S4a) encodes a predicted cytoplasmic multidomain protein also required for killing and thus functionally associated with the Kil apparatus (Figure 3b). An NG-KilD fusion was fully functional, also forming a fluorescent-bright cluster at a prey contact site, followed by motility arrest and prey cell lysis (Figure 4c and S4c, Movie S7). Given that this protein is the most downstream component of the cluster 1 region, which facilitates further genetic manipulations (see below), we next used it as a reporter for Kil system-associated functions for further characterization and in-depth quantifications. First, to confirm that prey intoxication occurs at sites where the Kil proteins are recruited, we imaged NG-KilD in the presence of E. coli cells labeled with TADA. As expected, PG degradation was detection at the points where the clusters are formed, showing that cluster formation correlates with contact dependent killing (Figure 4d). Using cluster assembly as a proxy for activation of the Kil system, we measured that killing is observed within ∼2 min after assembly, a rapid effect which suggests that Kil system assembly is tightly connected to a prey cell lytic activity (Figure S4e).
We next used NG-KilD as a proxy to monitor the function of the Kil Tad apparatus in prey recognition and killing. For this, NG-KilD was stably expressed from the native chromosomal locus in different genetic backgrounds (Figure S4f). In WT cells, NG-KilD clusters only formed in the presence of prey cells and ∼30% contacts were productive for cluster formation (Figure 4e). In kil mutants, NG-KilD clusters still formed upon prey cell contact with a minor reduction (up to ∼2 fold in the KilC and KilK), suggesting theTad-like apparatus is not directly responsible for initial prey cell sensing (Figure 4e, Movie S8). Nevertheless, cluster assembly was highly correlated to motility pauses (Figure 4f); which was impaired (up to 60%) in the kil mutants (except in the pilin, kilK mutant) and most strongly in the kilC (secretin), kilG (IM platform) and triple pilin (kilKLM) mutants (Figure 4f). Strikingly and contrarily to WT cells, cluster formation was not followed by cell lysis in all kil mutants, except in the major pilin kilK mutant (or very rarely, ∼4% of the time versus more than 80% in WT, Figure 4g). Altogether, these results indicate the Tad-like Kil system is dispensable for immediate prey recognition, but functions downstream to induce a motility pause and critically, provoke prey cell lysis.
The Kil apparatus is central for Myxococcus predation
We next tested the exact contribution of the kil genes to predation and prey consumption. This question is especially relevant because a number of mechanisms have been proposed to contribute to Myxococcus predation and all involve the extracellular secretion of toxic cargos9,11,12. In pure cultures, deletion of the kil genes is not linked to detectable motility and growth phenotypes, suggesting that the Tad-like Kil system mostly operates in predatory context (Figure S4g-h). Critically, core kil mutants where unable to predate colonies on plate, which could be fully complemented when corresponding kil genes were expressed ectopically (Figure 5a). When observed by time lapse, a kil mutant (here ΔkilACF) can invade a prey colony, but no prey killing is observed, showing that the prey killing phenotype is indeed due to the loss of contact-dependent killing (Figure 5b, Movie S9). To measure the impact of this defect quantitatively, we developed a FACS-based assay that directly measures the relative proportion of Myxococcus cells and E. coli cells in the prey colony across time (Figure 5c, see methods). In this assay, we observed that WT Myxococcus cells completely take over the E. coli population after 72h (Figure 5c). In contrast, the E. coli population remained fully viable when in contact with the kilACF triple mutant, even after 72h (Figure 5c). Predatory-null phenotypes were also observed in absence of selected Tad structural components, including the secretin (KilC), the ATPase (KilF) and the IM platform protein (KilH) (Figure 5d). A partial defect was observed in the pilin (KilK) but a triple pilin deletion mutant (kilKLM) was however completely deficient (Figure 5d).
To further test whether Kil-dependent prey killing provides the necessary nutrient source, we directly imaged Myxococcus growth in prey colonies, tracking single cells over the course of 6 hours (see methods). This analysis revealed that invading Myxococcus cell grow actively during prey invasion. The Myxococcus cell cycle could be imaged directly in single cells: cell size increased linearly up to a certain length, which was followed by a motility pause and cytokinesis (Figure 5e, Movie S10). The daughter cells immediately resumed growth at the same speed (Figure 5e). Cell size and cell age are therefore linearly correlated allowing estimation of a ∼5.5 hours generation time from a compilation of traces (Figure 5f, n=16). When the ΔkilACF mutant was similarly observed, cell size tended to decrease with time and cell division was not observed (Figures 5e-5f, n=20). Cell shortening could be a consequence of starvation, as observed for example in Bacillus subtilis27 (although this remains to be documented in Myxococcus). Taken together, these results demonstrate the central function of the Kil Tad apparatus in prey killing and consumption.
The Kil system promotes killing of phylogenetically diverse prey bacteria
Myxococcus is a versatile predator and can attack and digest a large number of preys28,29. We therefore tested if the Kil system also mediates predation by contact-dependent killing of other bacterial species. To this aim, we tested evolutionarily-distant preys, diderm bacteria, Caulobacter crescentus, Salmonella typhimurium and Pseudomonas aeruginosa, and monoderm, Bacillus subtilis. In plate assays, M. xanthus was able to lyse all tested preys, except P. aeruginosa (Figure 6a-f). When the Kil system was deleted, the predation ability of M. xanthus was severely diminished in all cases (Figure 6a-f). Consistently, Myxococcus assembled NG-KilD clusters in contact with Caulobacter, Salmonella and Bacillus cells, which in all cases led to cell plasmolysis (Figure 6g-i, Movie S11-13). Myxococcus cells were however unable to form lethal clusters when mixed with Pseudomonas cells (Movie S14), suggesting that, although the Kil system has a large spectrum of target species, it is not universally effective and resistance/evasion mechanisms must exist.
The kil genes evolved in predatory bacteria
We next explored bacterial genomes for the presence of kil-like genes. Phylogenetic analysis indicates that the ATPase (KilF), IM platform proteins (KilH and KilG) and CpaB protein (KilB) share similar evolutionary trajectories, allowing the construction of a well-supported phylogenetic tree based on a supermatrix (Figure 7, see methods). This analysis reveals that Kil-like systems are indeed related to Tad systems (ie Tad systems from alpha-proteobacteria, Figure 7) but they form specific clades in deltaproteobacteria, specifically in Myxococcales, in Bdellovibrionales and in the recently discovered Bradymonadales. In these bacteria, predicted Kil machineries are very similar to the Myxococcus Kil system, suggesting a similar function (Figure 7, Table S2). Remarkably, these bacteria are all predatory; the predatory cycle of Bradymonadales is yet poorly described but it is thought to be quite similar to the Myxococcus predatory cycle, involving surface motility and extracellular prey attack1. At first glance, Bdellovibrio species use a distinct predatory process, penetrating the prey cell to actively replicate in their periplasmic space2. However, this cycle involves a number of processes that are similar to Myxobacteria: Bdellovibrio cells also attack prey cells using gliding motility30 and attach to them using Type IV pili and a number of common regulatory proteins31. Prey cell penetration follows from the ability of the predatory cell to drill a hole into the prey PG at the attachment site32. While there is currently no direct evidence that the Bdellovibrio Kil-like system is involved in this process, multiple genetic evidence suggest that the Kil homolog are important for prey invasion and attachment33,34. It is therefore possible that acquisition of a Tad-like system in deltaproteobacteria was key to the emergence of predation, following its specialization in a possible ancestor of the Myxococcales, Bdellovibrionales and Bradymonadales.
Discussion
Prior to this work, Myxococcus predation was thought to be multifactorial and involve motility, secreted proteins, Outer Membrane Vesicles (OMVs) and antibiotics (ie Myxovirescin and Myxoprincomide) to kill and digest preys extracellularly3,5. While a contribution of these processes is not to be ruled out, most likely for prey cell digestion rather than killing (for example by degradative enzymes3), we show here that contact-dependent killing is the major prey killing mechanism. In Myxococcus, contact-dependent killing can be mediated by several processes, now including T6SS, OME and Kil. We exclude a function for the T6SS, for which a function in Myxococcus interspecies interactions has yet to be demonstrated. Rather, it appears that together with OME, Type VI secretion controls a phenomenon called social compatibility, in which the exchange of toxins between Myxococcus cells prevents immune cells from mixing with non-immune cells19. We have not tested a possible function of OME in prey killing because OME allows transfer of OM protein and lipids between Myxococcus cells when contact is established between identical outer membrane receptors, TraA9. OME is therefore highly Myxococcus species and even strain-specific and mediates social compatibility when SitA lipoprotein toxins are delivered to non-immune TraA-carrying Myxococcus target cells35.
The Kil system is both required for contact-dependent killing in liquid and on surfaces. Remarkably, proteins belonging to each motility systems show distinct requirement in liquid or on solid media. In liquid, Type-IV pili mediate prey-induced biofilm formation, which likely brings Myxococcus in close contact with the prey cells. This intriguing process likely requires EPS (since pilA mutants also lack EPS36), which deserves further exploration. On surfaces, likely a more biologically relevant context, contact-dependent killing is coupled to A-motility to penetrate prey colonies and interact with individual prey cells. The prey recognition mechanism is especially intriguing because dynamic assembly of a Tad-like system at the prey contact site is a novel observation; in general, these machineries and other Type IV filamentous systems22, such as TFPs tend to assemble at fixed cellular sites, often a cell pole11,23,37. KilD clusters do not require a functional Tad-like system to form in contact with prey cells, suggesting prey contact induces Tad assembly via an upstream signaling cascade. Such sensory system could be encoded within the clusters 1 and 2, which contain a large number of conserved genes with unknown predicted functions (up to 11 proteins of unknown functions just considering cluster 1 and 2, Figure 3a, Table S1). In particular, the large number of predicted proteins with FHA24 type domains (Table S1) suggests a function in a potential signaling cascade. In Pseudomonas aeruginosa, FHA domain-proteins act downstream from a phosphorylation cascade triggered by contact, allowing Pseudomonas to fire its T6SS upon contact38. This mechanism is triggered by general perturbation of the Pseudomonas membrane39, which could also be the case for the Kil system. Kil assembly is provoked both by monoderm and diderm bacteria, which suggests that prey-specific determinants are unlikely. Recognition is nevertheless non-universal and does not occur in contact with Pseudomonas or Myxococcus itself. Therefore, evasion mechanisms must exist, perhaps in the form of genetic determinants that shield cells from recognition.
The Kil Tad-like system itself is required to pause A-motility and prey cell killing. Motility regulation could be indirect because differential effects are observed depending on kil gene deletions (Figures 3 and 4), suggesting that assembly of a functional Tad apparatus is not strictly required for regulation. In contrast, prey killing requires a functional Tad apparatus. In particular, the pilin proteins are required during prey invasion but they are dispensable (partially) in liquid cultures showing that they do not promote toxicity. In liquid, direct contacts may be enforced by TFPs in the biofilm, perhaps rendering the Tad pilins partially redundant. Tad Pilin function would however become essential to onto prey cells on surfaces where the pili are dispensable. How the pilins organize to form polymers and whether they do, remains to be determined; the lack of the major pilin (KilK) is compensated by the remaining pseudo-pilins KilL and M, which is somewhat surprising given that pseudopilins are generally considered to prime assembly of major pilin polymers22. It is currently unclear if the Kil system is also a toxin-secretion device; for example, if it also functioned as a Type II secretion system. Alternatively, the Kil complex might recruit a toxin delivery system at the prey contact site. This latter hypothesis is in fact suggested by the remaining low (but still detectable) contact-dependent toxicity of kil mutants (Figures 3 and 4). Given that Myxococcus induces prey PG degradation locally, we hypothesize that a secreted cell wall hydrolase becomes active at the prey contact site. This is not unprecedented: Bdellovibrio cells secrete a sophisticated set of PG modifying enzymes, D,D-endopeptidases40, L,D transpeptidases32 and Lysozyme-like enzymes41 to penetrate prey cells, carve them into bdelloplasts and escape. In Myxococcus, deleting potential D,D-endopeptidases42 (ΔdacB) did not affect predation (Figure S5) which might not be surprising given that Myxococcus simply lyses its preys while Bdellovibrio needs to penetrate them while avoiding their lysis to support its intracellular cycle. The Myxococcus toxin remains to be discovered, bearing in mind, that similar to synergistic toxic T6SS effectors43, several toxic effectors could be injected, perhaps explaining how Myxococcus is able to kill both monoderm and diderm preys.
The Myxobacteria are potential keystone taxa in the soil microbial food web44, meaning that Kil-dependent mechanisms could have a major impact in shaping soil eco-systems. While the Kil proteins are most similar to proteins from Tad systems, there are a number of key differences that suggest profound diversification: (i), the Kil system involves a single ATPase and other Tad proteins such as assembly proteins TadG, RcpB and pilotin TadD are missing22; (ii), several Kil proteins have unique signatures, the large number of associated genes of unknown function; in particular, the over-representation of associated FHA domain proteins, including the central hexameric ATPase KilF itself fused to an N-terminal FHA domain. The KilC secretin is also uniquely short and lacks the N0 domain, canonically found in secretin proteins45, which could be linked to increased propensity for dynamic recruitment at prey contact sites. Future studies of the Kil machinery could therefore reveal how the contact-dependent properties of Tad pili were adapted to prey cell interaction and intoxication, likely a key evolutionary process in predatory bacteria.
Methods
Bacterial strains, growth conditions, motility plates, western blotting and genetic constructs
See Tables S3-S5 for strains, plasmids, and primers. E. coli cells were grown under standard laboratory conditions in Luria-Bertani (LB) broth supplemented with antibiotics, if necessary. M. xanthus strains were grown at 32°C in CYE (Casitone Yeast Extract) rich media as previously described42. S. enterica Typhimurium, B. subtilis and P. aeruginosa were grown overnight at 37°C in LB. C. crescentus strain NA1000 was grown overnight at 30°C in liquid PYE (Peptone Yeast Extract). Motility plate assays were conducted as previously described on soft (0.3%) or hard (1.5%) agar CYE plates46.
The deletion strains and the strains expressing the different Neon Green fusions were obtained using a double-recombination strategy as previously described46,47. Briefly, the kil deletion alleles (carrying ∼700-nucleotide long 5’ and 3’ flanking sequences of M. xanthus locus tags) were Gibson assembled into the suicide plasmid pBJ114 (galK, KanR) and used for allelic exchange. Plasmids were introduced in M. xanthus by electroporation. After selection, clones containing the deletion alleles were identified by PCR. Using the same strategy, “Neon Green fusion” alleles were introduced at kilD and kilF loci. The corresponding strains expressed, under the control of their native promoters, C-terminal Neon Green fusions of KilD and KilF.
For complementation of ΔkilC, ΔkilF, ΔkilG and ΔkilH strains, we used the pSWU19 plasmid (KanR) allowing ectopic expression of the corresponding genes from the pilA promoter at Mx8-att site. The same strains transformed with the empty vector were used as controls.
To express KilG C-terminally fused to Neon Green, a pSWU19-PpilA-kilG-NG was created and transformed in the ΔkilG strain.
Western blotting was performed as previously described46 using a commercial polyclonal anti Neon-Green antibody (Chromotek).
Growth in liquid cultures
To compare growth rates of M. xanthus WT and ΔkilACF strains, overnight CYE cultures were used to inoculate 25 ml of CYE at OD600= 0.05. Cultures were then incubated at 32°C with a shaking speed of 160 rpm. To avoid measuring cell densities at night, a second set of cultures were inoculated 12 hours later at OD600= 0.05. Every 4 hours, 1 ml sample of each culture was used to measure optical densities at 600 nm with a spectrophotometer. The different measurements were then combined into a single growth curve. This experiment was performed with three independent cultures per strain.
Predation assay on agar plates
Prey colony invasion on CF agar plates
M. xanthus and the different prey cells (except C. crescentus) were respectively grown overnight in 20 ml of CYE at 32°C and in 20 ml of LB at 37°C. C. crescentus was grown in 20 ml of PYE at 30°C. The next day, cells were pelleted and resuspended in CF medium (MOPS 10 mM pH 7.6; KH2PO4 1 mM; MgSO4 8 mM; (NH4)2SO4 0.02%; Na citrate 0.2%; Bacto Casitone 0.015%) to a final OD600 of 5. 10 µl of M. xanthus and prey cell suspensions were then spotted next to each other (leaving less than 1-mm gap between each spot) on CF 1.5% agar plates with or without 0.07% glucose (to allow minimal growth of the prey cells) and incubated at 32°C. After 48-hours incubation, pictures of the plates were taken using a Nikon Olympus SZ61 binocular loupe (10x magnification) equipped with a camera and an oblique filter. ImageJ software was used to measure the surface of the prey spot lysed by M. xanthus.
Spotting predator-prey mixes on CF agar plates
to force the contact between M. xanthus and a prey, mixes of predator/prey were made and spotted of CF agar plates. 200 µl of a prey cell suspension (in CF, OD600 = 5) were mixed with 25 µl of a M. xanthus cell suspension (in CF, OD600 = 5) and 10 µl of this mix were spotted on CF agar plates supplemented with 0.07% glucose. As described above, pictures of the plates were taken after 24-hour incubation.
Microscope invasion predation assay and contact-dependent killing
Prey colony invasion on CF agar pads
prey invasion was imaged by microscopy using the Bacto-Hubble system (the specific details of the Method are described elsewhere10). Briefly, cell suspensions concentrated to OD600=5 were spotted at 1 mm distance onto CF 1.5% agar pads and a Gene Frame (Thermo Fisher Scientific) was used to sandwich the pad between the slide and the coverslip and limit evaporation of the sample. Slides were incubated at 32°C for 6 hours before imaging, allowing Myxococcus and E. coli to form microcolonies. Time-lapse of the predation process was taken at 40x or 100x magnification. Movies were taken at the invasion front where Myxococcus cells enter the E. coli colony. To facilitate tracking, M. xanthus cells were labeled with fluorescence48. Fluorescence images were acquired every 30 seconds for up to 10 hours, at room temperature.
Spotting predator-prey mixes on CF agar pads
to image contact-dependent killing between M. xanthus and prey cells (E. coli, C. crescentus, B. subtilis, S. typhimurium and P. aeruginosa), cells were grown as described above, pelleted and resuspended in CF medium to a final OD600 of 1. Equal volumes of M. xanthus and prey cell suspensions were then mixed together and 1 µl of the mix was spotted on a freshly made CF 1.5% agar pad on a microscope slide. After the spot has dried, the agar pad was covered with a glass coverslip, and incubated in the dark at room temperature for 20-30 minutes before imaging.
Time-lapses experiments were performed using two automated and inverted epifluorescence microscope: a TE2000-E-PFS (Nikon), with a ×100/1.4 DLL objective and an ORCA Flash 4.0LT camera (Hamamatsu) or a Ti Nikon microscope equipped with an ORCA Flash 4.0LT camera (Hamamatsu). Theses microscopes are equipped with the “Perfect Focus System” (PFS) that automatically maintains focus so that the point of interest within a specimen is always kept in sharp focus at all times, despite any mechanical or thermal perturbations. Images were recorded with NIS software from Nikon. All fluorescence images were acquired with appropriate filters with a minimal exposure time to minimize photo-bleaching and phototoxicity effects: 30-minute long time-lapses (one image acquired every 30 seconds) of the predation process were taken at 100x magnification. DIA images were acquired using a 5 ms light exposure and GFP fluorescent images were acquired using a 100 ms fluorescence exposure with power intensity set to 50% (excitation wavelength 470 nm) to avoid phototoxicity.
Labelling E. coli cells with the fluorescent D-Amino Acid TADA
Lyophilized TADA (MW = 381.2g/mol, laboratory stock15) was re-suspended in DMSO at 150 mM and conserved at −20°C. The labeling was performed, for 2 h in the dark at room temperature, using 2 μl of the TADA solution for 1ml of cells culture (OD600 = 2). Cells were then washed four times with 1ml of CF and directly used for predation assays on agar pad.
Image Analysis
Image analysis was performed under FIJI49 and MicrobeJ50 an ImageJ plug-in for the analysis of bacterial cells.
Semantic segmentation of Myxococcus cells
was obtained using the newly developed MiSiC system, a deep learning based bacterial cell segmentation tool10. The system was used in semantic segmentation mode and annotated manually to reveal E. coli lysing cells.
Kymograph construction
Kymographs were obtained after manual measurements of fluorescence intensities along FIJI hand-drawn segments and the FIJI-Plot profile tool. The measurements were then exported into the Prism software (Graphpad, Prism 8) to construct kymographs.
Cell tracking
Cell tracking and associated morphometrics were obtained using MicrobeJ. Image stacks were first processed stabilized and filtered with a moderate Gaussian blur and cells were detected by thresholding and fitted with the Plug-in “medial axis” model. Trajectories were systematically verified and corrected by hand when necessary.
Tracking Myxococcus pauses in contact with a prey, NG-KilD foci formation and prey cell lysis
in 30-minute time-lapses, contacts between prey cells and Myxococcus cells were scored. Pauses were counted when the predatory cell stopped all movement upon contact with the prey. We also counted if these contacts lead to the formation of NG-KilD foci and to cell lysis. Thus, for a determined E. coli cell, we scored the number of contacts with Myxococcus, the number of pauses these contacts induces in M. xanthus motility, the number of NG-KilD foci formed upon contacts and, ultimately, the lysis of the cell. Five independent movies were analyzed for each strain and the percentage of contacts leading to a pause in motility, NG-KilD foci formation and cell lysis was calculated. We also estimated the percentage of NG-KilD clusters leading to cell lysis
Tracking cluster time to lysis
Time to lysis measures the elapsed time between cluster appearance to prey cell death. Data were obtained from two biological replicates.
CPRG assay for contact-dependent killing in liquid
CPRG assay in 24-well plates
M. xanthus and E. coli cultures were grown overnight, pelleted and resuspended in CF at OD600 ∼5. 100 µl of M. xanthus cell suspension (WT and mutants) were mixed with 100 µl of E. coli cell suspension in a 24-well plate containing, in each well, 2 ml of CF medium supplemented with CPRG (Sigma Aldrich, 20 µg/ml) and IPTG (Euromedex, 50 µM) to induce lacZ expression. The plates were then incubated at 32°C with shaking and pictures were taken after 24 and 48 hours of incubation. To test the contact-dependance, a two-chamber assay was carried out in a Corning 24 well-plates containing a 0.4-µm pore polycarbonate membrane insert (Corning Transwell 3413). This membrane is permeable to small metabolites and proteins and impermeable to cells. E. coli cells were inoculated into the top chamber and M. xanthus cells into the bottom chamber.
CPRG assay in 96-well plates
To evaluate the predation efficiency of the different kil mutants, the CPRG assay was adapted as follow: wild-type M. xanthus and the kil mutant strains were grown overnight in 15 ml of CYE. E. coli was grown overnight in 15 ml of LB. The next morning, M. xanthus and E. coli cells were pelleted and resuspended in CF at OD600 = 0.5 and 10, respectively. To induce expression of the β-galactosidase, IPTG (100 µM final) was added to the E. coli cell suspension.
In a 96-well plate, 100 µl of M. xanthus cell suspension were mixed with 100 µl of E. coli cell suspension. Wells containing only M. xanthus, E. coli or CF were used as controls. The lid of the 96-well plate was then sealed with a breathable tape (Greiner bio-one) and the plate was incubated for 24 hours at 32°C while shaking at 160 rpm. In this setup, we observed that M. xanthus and E.coli cells aggregate at the bottom of the well and therefore come in direct contact, favoring predation in liquid.
The next day, the plate was centrifuged 10 minutes at 4800 rpm and 25 µl of the supernatant were transferred in a new 96-well plate containing 125 µl of Z-buffer (Na2HPO4 60 mM, NaH2PO4 40 mM, KCl 10 mM pH7) supplemented with 20 µg/ml of CPRG. After 15-30 minutes of incubation at 37°C, the enzymatic reaction was stopped with 65 µl of Na2CO3 (1 M) and the absorbance at 576 nm was measured using a TECAN Spark plate reader.
This experiment was performed independently four times. For Miller unit calculation, after absorbance of the blank (with CF) reaction was subtracted, the absorbances measured at 576 nm were divided by the incubation time and the volume of cell lysate used for reaction. The resulting number was then multiplied by 1000.
Crystal violet biofilm staining
In a 96-well plate, 100 µl of M. xanthus cell suspension (in CF, OD600 = 0.5) were mixed with 100 µl of E. coli cell suspension (in CF, OD600 = 10) and incubated for 24 hours at 32°C while shaking at 160 rpm. The next day, the supernatant was carefully removed and the wells were washed with 200 µl of CF twice. Then, 100 µl of a 0.01% crystal violet solution were added to each well and incubated for 5 minutes. Wells were washed twice with 200 µl of water before imaging.
Prey CFU counting after predation
E. coli, S. typhi, P. aeruginosa and B. subtilis kanamycin resistant strains were grown at 37°C in liquid LB supplemented with kanamycin (50 or 10 µg/ml). C. crescentus kanamycin resistant strain was grown at 30°C in liquid PYE supplemented with kanamycin (25 µg/ml). Wild-type and ΔkilACF M. xanthus strains were grown at 32°C in liquid CYE. Cells were then centrifuged and pellets were resuspended in CF at an OD600 of 5. 25 µl of M. xanthus cell suspension and 200 µl of prey cell suspension were then mixed together and 10 µl were spotted on CF agar plates supplemented with 0.07% glucose. After drying, plates were incubated at 32°C. At 0, 8, 24, and 48-hour time points, spots were harvested with a loop and resuspended in 500 µl of CF. This solution was then used to make 10-fold serial dilutions in a 96-well plate containing CF. At the exception of C. crescentus, 5 µl of each dilution were spotted on LB agar plates supplemented with 10 µg/ml of kanamycin and incubated at 37°C for 24 hours. C. crescentus dilutions were spotted on PYE agar plates supplemented with 25 µg/ml of kanamycin and incubated at 30°C for 24 hours. The next day, colony-forming units were counted and the number of prey cells that survived in the predator/prey spot was calculated.
Fluorescence-Activated Cell Sorting (FACS) measurements of E. coli killing
M. xanthus strains (wild-type and kil mutants) constitutively expressing GFP were grown overnight in liquid CYE without antibiotics. E. coli mCherry (prey) was grown overnight in liquid LB supplemented with ampicillin (100 µg/ml). The next morning, optical densities of the cultures were adjusted in CF medium to OD600= 5. M. xanthus GFP and E. coli mCherry cell suspensions were then spotted onto fresh CF 1.5% agar plates as previously described46. Briefly, 10-µl drops of the prey and the predator cell suspensions were placed next to each other and let dry. Inoculated plates were then incubated at 32°C. Time 0 corresponds to the time at which the prey and the predator spots were set on the CF agar plate. At time 0, 24, 48 and 72 hours (post predation) and for each M. xanthus strain, two predator/prey spot couples were harvested with a loop and resuspended in 750 µl of TPM. To fix the samples, paraformaldehyde (32% in distilled water, Electron Microscopy Sciences) was then added to the samples to a final concentration of 4%. After 10-min incubation at room temperature, samples were centrifuged (8 min, 7500 rpm), cell pellets were then resuspended in fresh TPM and optical densities were adjusted to OD600 ∼0.1.
Samples were then analyzed by flow cytometry. Flow cytometry data were acquired on a Bio-Rad S3e Cell Sorter and analyzed using the ProSort software, version 1.6. For each sample, a total population of 500,000 events was used and events corresponding to the sum of M. xanthus-GFP and E. coli-mCherry. A blue laser (488 nm, 100mW) was used for detection of forward scatter (FSC) and side scatter (SSC) and for excitation of GFP. A yellow-green laser (561 nm, 100 mW) was used for excitation of mCherry. GFP and mCherry signals were collected using, respectively, the emission filters FL1 (525/30 nm) and FL3 (615/25 nm) and a compensation was applied on the mCherry signal. Samples were run using the low-pressure mode (10,000 particles/s). To calibrate the instrument and reduce background noise, suspensions of fluorescent and non-fluorescent M. xanthus and E. coli cells were used: a threshold was applied on the FSC signal, and voltages of the photomultipliers for FSC, SSC, FL1 and FL3 were also adjusted. The density plots obtained (small angle scattering FSC versus wide angle scattering SSC signal) were first gated on the overlapped population of M. xanthus and E. coli, filtered to remove the multiple events and finally gated for high FL1 signal (M. xanthus-GFP) and high FL3 signal (E. coli-mCherry).
Bioinformatic analyses
Homology search strategy
we used several search strategies to identify all potential homologous proteins of the Kil system: we first used BLAST51,52 to search for reciprocal best hits (RBH) between the M. xanthus and the B. bacteriovorus and B. Sediminis Kil systems, as well as the C. crescentus Tad system, identifying bona fide orthologs between the three species. We limited the search space to the respective proteomes of the three species. We then used HHPRED53 to search for remotely conserved homologs in B. bacteriovorus using the proteins from the two operons identified in M. xanthus. Finally, we performed domain comparisons between proteins from the B. bacteriovorus and B. sediminis Kil operons and C. crescentus Tad system to identify proteins with similar domain compositions in M. xanthus. Identified orthologs or homologs between the three species, the employed search strategy, as well as resulting e-values are shown in Table S2.
Structure predictions
tertiary structural models of secretin and cytoplasmic ATPase were done using Phyre254or SWISS-MODEL55, in both cases using default parameters. Quaternary models were generated using SWISS-MODEL. Structural models were displayed using Chimera56 and further processed in Illustrator ™.
Phylogenetic analyses
we used the four well-conserved Kil system components for phylogenetic analysis. To collect species with secretion systems similar to the Kil system, we first used MultiGeneBLAST57 with default parameters. Orthologs of the four proteins from B. bacteriovorus, B. Sediminis and C. crescentus from closely related species were added manually. We aligned each of the four proteins separately using MAFFT58 and created a supermatrix from the four individual alignments. Gblocks58 using relaxed parameters was used prior to tree reconstruction to remove badly aligned or extended gap regions. The resulting alignment is shown in Suppl. File 1. Alignments of individual trees were also trimmed using Gblocks. PhyML59 was used for tree reconstruction, using the JTT model and 100 bootstrap iterations. Trees were displayed with Dendroscope60 and further processed in Illustrator ™.
Author contributions
SS, JH and TM conceived the experiments and analyzed the data. SS, JH and DR performed most experiments. GB ran FACS experiments and analyzed data. PDB and BH performed bioinformatic analysis, homology searches, structure predictions and phylogenetic analysis. LM, EC, SS and TM conceived and analyzed T6SS experiments. RM provided data with the A-S- motility mutant. TM and JH wrote the paper.
Extended Figures and Legends
Legends to Supplemental Movies
Movie S1: Invasion of E. coli colonies by WT Myxococcus cells. This movie was taken at the interface between the two colonies during invasion. The movie is a 8x compression of an original movie that was shot for 10 hours with a frame taken every 30s at 40x magnification. To facilitate Myxococcus cells tracking, the wild-type strain was labeled with the mCherry fluorescent protein.
Movie S2: A-motility is required for prey invasion. This movie was taken at the interface between the two colonies during invasion. The movie is a compression of an original movie that was shot for 10 hours with a frame taken every 30s at 40x magnification. To facilitate Myxococcus cells tracking, the A-S+ (ΔaglQ) strain was labeled with the mCherry fluorescent protein.
Movie S3: Prey invasion by A-motile cells in “arrowhead” formations. Focal adhesions and thus active A-motility complexes were detected with an AglZ-Neon green fusion. The movie contains 51 frames taken every 30 seconds at 100x magnification. Shown side-by-side are fluorescence images, fluorescence overlaid with phase contrast and MiSiC segmentation (lysing E. coli cells are colored magenta and blue).
Movie S4: A Myxococcus cell kills an E. coli cell by contact. The Myxococcus cell expresses AglZ-nG and the E. coli cell expresses mCherry. Shown side-by-side are fluorescence images and MiSiC segmentation (Myxococcus: green, E. coli: magenta). The movie contains 20 frames taken every 30 seconds at 100x magnification.
Movie S5: NG-KilF cluster formation in contact with E. coli prey cells. Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with three E. coli cells. The movie was shot at 100x magnification objective for 30 minutes. Pictures were taken every 30 seconds.
Movie S6: KilG-NG cluster formation in contact with E. coli prey cells. Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with three E. coli cells. The movie was shot at 100x magnification objective for 9 minutes. Pictures were taken every 30 seconds.
Movie S7: NG-KilD cluster formation in contact with E. coli prey cells. Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with three E. coli cells. The movie was shot at 100x magnification objective for 15 minutes. Pictures were taken every 30 seconds.
Movie S8: NG-KilD clusters form in a kilC mutant but no motility pauses and prey cell lysis can be observed. Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with three E. coli cells. The movie was shot at 100x magnification objective for 8 minutes. Pictures were taken every 30 seconds.
Movie S9: a ΔkilACF still invades but does not kill E. coli prey cells. This movie was taken at the interface between the two colonies during invasion. The movie is a 4x compression of an original movie that was shot for 4.5 hours with a frame taken every 30s at 40x magnification. To facilitate Myxococcus ΔkilACF cells are labeled with the mCherry fluorescent protein.
Movie S10: Predatory cells division and tracking during invasion of prey colony. To follow cell growth and division at the single cell level during prey invasion, WT cells were mixed with a WT strain expressing the mCherry at a 50:1 ratio and imaged every 30 seconds at 40x magnification for up to 10 hours within non-labeled prey colonies. Cell growth was measured by fitting cell contours to medial axis model followed by tracking under Microbe-J. Real time of the track for the example cell: 95 min.
Movie S11: NG-KilD cluster formation in contact with Caulobacter crescentus prey cells. Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with a C. crescentus cell. The movie was shot at 100x magnification objective for 7 minutes. Pictures were taken every 30 seconds.
Movie S12: NG-KilD cluster formation in contact with Salmonella typhimurium prey cells. Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with an S. enterica Typhimurium cell. The movie was shot at 100x magnification objective for 20 minutes. Pictures were taken every 30 seconds.
Movie S13: NG-KilD cluster formation in contact with Bacillus subtilis prey cells. Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cell in predatory contact with a B. subtilis cell. The movie was shot at 100x magnification objective for 30 minutes. Pictures were taken every 30 seconds.
Movie S14: Pseudomonas aeruginosa is not lysed by Myxococcus and does not induce NG-KilD cluster formation. Shown is an overlay of the fluorescence and phase contrast images of a motile Myxococcus cells mixed with Pseudomonas cells. The movie was shot at 100x magnification objective for 30 minutes. Pictures were taken every 30 seconds.
Acknowledgements
We thank Lotte Søgaard-Andersen and Anke Treuner-Lange for the gift of the VipA plasmid. We thank Laurent Aussel for E. coli plasmids, Anne Galinier lab for the Bacillus subtilis strains, Emanuele Biondi lab for the Caulobacter crescentus strains and Sophie Bleves for the Pseudomonas aeruginosa strain. We thank Dorothée Murat, Romé Voulhoux, Marcelo Nöllmann, Vladimir Pelicic and Friedhelm Pfeiffer for discussions.
Research in TM lab was supported by a 2019 CNRS 80-Prime allowance on bacterial predation and pattern formation. SS and PDB are supported by an MENRT thesis grant from the ministry of research.