A bipartite, low-affinity roadblock domain-containing GAP complex regulates bacterial front-rear polarity.

The Ras-like GTPase MglA is a key regulator of front-rear polarity in the rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two machineries for type IV pili-dependent motility and gliding motility. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. Paradoxically, RomR/RomX and MglB localize similarly with low and high concentrations at the leading and lagging poles, respectively. Yet, GEF activity dominates at the leading and GAP activity at the lagging pole by unknown mechanisms. Here, we identify RomY and show that it stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting formation of the bipartite MglB/RomY GAP complex almost exclusively to the lagging pole with the high MglB concentration. Our data support a model wherein RomY, by forming a low-affinity complex with MglB, ensures that the high MglB/RomY GAP activity is confined to the lagging pole where it dominates and outcompetes the GEF activity of the RomR/RomX complex. Thereby, MglA-GTP localization is constrained to the leading pole establishing front-rear polarity.

The predatory soil bacterium Myxococcus xanthus combines a Tad- and an atypical type 3-like protein secretion system to kill bacterial cells.

Predatory Myxobacteria employ a multilayered predation strategy to kill and lyse soil microorganisms. Aiming to dissect the mechanism of contact-dependent killing of bacteria, we analyze four protein secretion systems in Myxococcus xanthus and investigate the predation of mutant strains on different timescales. We find that a Tad-like and a type 3-like secretion system (Tad and T3SS∗) fulfill distinct functions during contact-dependent prey killing: the Tad-like system is necessary to induce prey cell death, while the needle-less T3SS∗ initiates prey lysis. Fluorescence microscopy reveals that components of both systems interdependently localize to the predator-prey contact site prior to killing. Swarm expansion assays show that both Tad and T3SS∗ are required to handle live prey and that nutrient extraction from prey bacteria is sufficient to power M. xanthus motility. In conclusion, our observations indicate the functional interplay of two types of secretion systems for killing and lysis of bacterial cells.

The differential expression of PilY1 proteins by the HsfBA phosphorelay allows twitching motility in the absence of exopolysaccharides.

Type Four Pili (T4P) are extracellular appendages mediating several bacterial functions such as motility, biofilm formation and infection. The ability to adhere to substrates is essential for all these functions. In Myxococcus xanthus, during twitching motility, the binding of polar T4P to exopolysaccharides (EPS), induces pilus retraction and the forward cell movement. EPS are produced, secreted and weakly associated to the M. xanthus cell surface or deposited on the substrate. In this study, a genetic screen allowed us to identify two factors involved in EPS-independent T4P-dependent twitching motility: the PilY1.1 protein and the HsfBA phosphorelay. Transcriptomic analyses show that HsfBA differentially regulates the expression of PilY1 proteins and that the down-regulation of pilY1.1 together with the accumulation of its homologue pilY1.3, allows twitching motility in the absence of EPS. The genetic and bioinformatic dissection of the PilY1.1 domains shows that PilY1.1 might be a bi-functional protein with a role in priming T4P extension mediated by its conserved N-terminal domain and roles in EPS-dependent motility mediated by an N-terminal DUF4114 domain activated upon binding to Ca2+. We speculate that the differential transcriptional regulation of PilY1 homologs by HsfBA in response to unknown signals, might allow accessorizing T4P tips with different modules allowing twitching motility in the presence of alternative substrates and environmental conditions.

Cell density, alignment, and orientation correlate with C-signal-dependent gene expression during Myxococcus xanthus development.

Starving Myxococcus xanthus bacteria use short-range C-signaling to coordinate their movements and construct multicellular mounds, which mature into fruiting bodies as rods differentiate into spherical spores. Differentiation requires efficient C-signaling to drive the expression of developmental genes, but how the arrangement of cells within nascent fruiting bodies (NFBs) affects C-signaling is not fully understood. Here, we used confocal microscopy and cell segmentation to visualize and quantify the arrangement, morphology, and gene expression of cells near the bottom of NFBs at much higher resolution than previously achieved. We discovered that "transitioning cells" (TCs), intermediate in morphology between rods and spores, comprised 10 to 15% of the total population. Spores appeared midway between the center and the edge of NFBs early in their development and near the center as maturation progressed. The developmental pattern, as well as C-signal-dependent gene expression in TCs and spores, were correlated with cell density, the alignment of neighboring rods, and the tangential orientation of rods early in the development of NFBs. These dynamic radial patterns support a model in which the arrangement of cells within the NFBs affects C-signaling efficiency to regulate precisely the expression of developmental genes and cellular differentiation in space and time. Developmental patterns in other bacterial biofilms may likewise rely on short-range signaling to communicate multiple aspects of cellular arrangement, analogous to juxtacrine and paracrine signaling during animal development.

PomX, a ParA/MinD ATPase activating protein, is a triple regulator of cell division in Myxococcus xanthus.

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

Linking single-cell decisions to collective behaviours in social bacteria.

Social bacteria display complex behaviours whereby thousands of cells collectively and dramatically change their form and function in response to nutrient availability and changing environmental conditions. In this review, we focus on Myxococcus xanthus motility, which supports spectacular transitions based on prey availability across its life cycle. A large body of work suggests that these behaviours require sensory capacity implemented at the single-cell level. Focusing on recent genetic work on a core cellular pathway required for single-cell directional decisions, we argue that signal integration, multi-modal sensing and memory are at the root of decision making leading to multicellular behaviours. Hence, Myxococcus may be a powerful biological system to elucidate how cellular building blocks cooperate to form sensory multicellular assemblages, a possible origin of cognitive mechanisms in biological systems. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.

Four different mechanisms for switching cell polarity.

The mechanisms and design principles of regulatory systems establishing stable polarized protein patterns within cells are well studied. However, cells can also dynamically control their cell polarity. Here, we ask how an upstream signaling system can switch the orientation of a polarized pattern. We use a mathematical model of a core polarity system based on three proteins as the basis to study different mechanisms of signal-induced polarity switching. The analysis of this model reveals four general classes of switching mechanisms with qualitatively distinct behaviors: the transient oscillator switch, the reset switch, the prime-release switch, and the push switch. Each of these regulatory mechanisms effectively implements the function of a spatial toggle switch, however with different characteristics in their nonlinear and stochastic dynamics. We identify these characteristics and also discuss experimental signatures of each type of switching mechanism.

Global gene expression analysis of the Myxococcus xanthus developmental time course.

To accurately identify the genes and pathways involved in the initiation of the Myxococcus xanthus multicellular developmental program, we have previously reported a method of growing vegetative populations as biofilms within a controllable environment. Using a modified approach to remove up to ~90% rRNAs, we report a comprehensive transcriptional analysis of the M. xanthus developmental cycle while comparing it with the vegetative biofilms grown in rich and poor nutrients. This study identified 1522 differentially regulated genes distributed within eight clusters during development. It also provided a comprehensive overview of genes expressed during a nutrient-stress response, specific development time points, and during development initiation and regulation. We identified several differentially expressed genes involved in key central metabolic pathways suggesting their role in regulating myxobacterial development. Overall, this study will prove an important resource for myxobacterial researchers to delineate the regulatory and functional pathways responsible for development from those of the general nutrient stress response.

An ambruticin-sensing complex modulates Myxococcus xanthus development and mediates myxobacterial interspecies communication.

Starvation induces cell aggregation in the soil bacterium Myxococcus xanthus, followed by formation of fruiting bodies packed with myxospores. Sporulation in the absence of fruiting bodies can be artificially induced by high concentrations of glycerol through unclear mechanisms. Here, we show that a compound (ambruticin VS-3) produced by a different myxobacterium, Sorangium cellulosum, affects the development of M. xanthus in a similar manner. Both glycerol (at millimolar levels) and ambruticin VS-3 (at nanomolar concentrations) inhibit M. xanthus fruiting body formation under starvation, and induce sporulation in the presence of nutrients. The response is mediated in M. xanthus by three hybrid histidine kinases (AskA, AskB, AskC) that form complexes interacting with two major developmental regulators (MrpC, FruA). In addition, AskB binds directly to the mrpC promoter in vitro. Thus, our work indicates that the AskABC-dependent regulatory pathway mediates the responses to ambruticin VS-3 and glycerol. We hypothesize that production of ambruticin VS-3 may allow S. sorangium to outcompete M. xanthus under both starvation and growth conditions in soil.

The polar Ras-like GTPase MglA activates type IV pilus via SgmX to enable twitching motility in Myxococcus xanthus.

Type IV pili (Tfp) are highly conserved macromolecular structures that fulfill diverse cellular functions, such as adhesion to host cells, the import of extracellular DNA, kin recognition, and cell motility (twitching). Outstandingly, twitching motility enables a poorly understood process by which highly coordinated groups of hundreds of cells move in cooperative manner, providing a basis for multicellular behaviors, such as biofilm formation. In the social bacteria Myxococcus xanthus, we know that twitching motility is under the dependence of the small GTPase MglA, but the underlying molecular mechanisms remain elusive. Here we show that MglA complexed to GTP recruits a newly characterized Tfp regulator, termed SgmX, to activate Tfp machines at the bacterial cell pole. This mechanism also ensures spatial regulation of Tfp, explaining how MglA switching provokes directional reversals. This discovery paves the way to elucidate how polar Tfp machines are regulated to coordinate multicellular movements, a conserved feature in twitching bacteria.

PilY1 and minor pilins form a complex priming the type IVa pilus in Myxococcus xanthus.

Type IVa pili are ubiquitous and versatile bacterial cell surface filaments that undergo cycles of extension, adhesion and retraction powered by the cell-envelope spanning type IVa pilus machine (T4aPM). The overall architecture of the T4aPM and the location of 10 conserved core proteins within this architecture have been elucidated. Here, using genetics, cell biology, proteomics and cryo-electron tomography, we demonstrate that the PilY1 protein and four minor pilins, which are widely conserved in T4aP systems, are essential for pilus extension in Myxococcus xanthus and form a complex that is an integral part of the T4aPM. Moreover, these proteins are part of the extended pilus. Our data support a model whereby the PilY1/minor pilin complex functions as a priming complex in T4aPM for pilus extension, a tip complex in the extended pilus for adhesion, and a cork for terminating retraction to maintain a priming complex for the next round of extension.

Cooperation and Cheating among Germinating Spores.

Many microbes produce stress-resistant spores to survive unfavorable conditions [1-4] and enhance dispersal [1, 5]. Cooperative behavior is integral to the process of spore formation in some species [3, 6], but the degree to which germination of spore populations involves social interactions remains little explored. Myxococcus xanthus is a predatory soil bacterium that upon starvation forms spore-filled multicellular fruiting bodies that often harbor substantial diversity of endemic origin [7, 8]. Here we demonstrate that germination of M. xanthus spores formed during fruiting-body development is a social process involving at least two functionally distinct social molecules. Using pairs of natural isolates each derived from a single fruiting body that emerged on soil, we first show that spore germination exhibits positive density dependence due to a secreted "public-good" germination factor. Further, we find that a germination defect of one strain under saline stress in pure culture is complemented by addition of another strain that germinates well in saline environments and mediates cheating by the defective strain. Glycine betaine, an osmo-protectant utilized in all domains of life, is found to mediate saline-specific density dependence and cheating. Density dependence in non-saline conditions is mediated by a distinct factor, revealing socially complex spore germination involving multiple social molecules.

Protein-protein interaction network controlling establishment and maintenance of switchable cell polarity.

Cell polarity underlies key processes in all cells, including growth, differentiation and division. In the bacterium Myxococcus xanthus, front-rear polarity is crucial for motility. Notably, this polarity can be inverted, independent of the cell-cycle, by chemotactic signaling. However, a precise understanding of the protein network that establishes polarity and allows for its inversion has remained elusive. Here, we use a combination of quantitative experiments and data-driven theory to unravel the complex interplay between the three key components of the M. xanthus polarity module. By studying each of these components in isolation and their effects as we systematically reconstruct the system, we deduce the network of effective interactions between the polarity proteins. RomR lies at the root of this network, promoting polar localization of the other components, while polarity arises from interconnected negative and positive feedbacks mediated by the small GTPase MglA and its cognate GAP MglB, respectively. We rationalize this network topology as operating as a spatial toggle switch, providing stable polarity for persistent cell movement whilst remaining responsive to chemotactic signaling and thus capable of polarity inversions. Our results have implications not only for the understanding of polarity and motility in M. xanthus but also, more broadly, for dynamic cell polarity.

Conditional requirement of SglT for type IV pili function and S-motility in Myxococcus xanthus.

Myxobacteria exhibit complex social behaviors such as predation, outer membrane exchange and fruiting body formation. These behaviors depend on coordinated movements of cells on solid surfaces that involve social (S) motility. S-motility is powered by extension-retraction cycles of type 4 pili (Tfp) and exopolysaccharides (EPS) that provide a matrix for group cellular movement. Here, we characterized a new class of S-motility mutants in Myxococcus xanthus. These mutants have a distinctive phenotype: they lack S-motility even though they produce pili and EPS and the phenotype is temperature-sensitive. The point mutations were mapped to a single locus, MXAN_3284, named sglT. Similar to pilT mutants, sglT mutants are hyperpiliated and, strikingly, the temperature-sensitive phenotype is caused by null mutations. Our results indicate that SglT plays a critical role in Tfp function associated with pilus retraction and that the block in pili retraction is caused by a Tfp assembly defect in the absence of SglT at high-temperature growth.

Iron competition triggers antibiotic biosynthesis in Streptomyces coelicolor during coculture with Myxococcus xanthus.

Microbial coculture to mimic the ecological habitat has been suggested as an approach to elucidate the effect of microbial interaction on secondary metabolite biosynthesis of Streptomyces. However, because of chemical complexity during coculture, underlying mechanisms are largely unknown. Here, we found that iron competition triggered antibiotic biosynthesis in Streptomyces coelicolor during coculture with Myxococcus xanthus. During coculture, M. xanthus enhanced the production of a siderophore, myxochelin, leading M. xanthus to dominate iron scavenging and S. coelicolor to experience iron-restricted conditions. This chemical competition, but not physical contact, activated the actinorhodin biosynthetic gene cluster and the branched-chain amino acid degradation pathway which imply the potential to produce precursors, along with activation of a novel actinorhodin export system. Furthermore, we found that iron restriction increased the expression of 21 secondary metabolite biosynthetic gene clusters (smBGCs) in other Streptomyces species. These findings suggested that the availability for key ions stimulates specific smBGCs, which had the potential to enhance secondary metabolite biosynthesis in Streptomyces.

MYXO-CTERM sorting tag directs proteins to the cell surface via the type II secretion system.

Cells interact with their surrounding environment through surface proteins. However, knowledge gaps remain in understanding how these important types of proteins are transported and anchored on the cell surface. In the Gram-negative social bacterium, Myxococcus xanthus, a putative C-terminal sorting tag (MYXO-CTERM) is predicted to help direct 34 different proteins onto the cell surface. Here we investigate the sorting pathway for MYXO-CTERM proteins by using the TraA cell surface receptor as a paradigm. Deleting this motif from TraA abolishes the cell surface anchoring and results in extracellular secretion. Our findings indicate that conserved cysteines within the MYXO-CTERM are posttranslationally modified and are required for TraA cell surface localization and function. A region immediately upstream of these residues is predicted to be disordered and removing this motif caused a secretion defect and blocked cell surface anchoring. We further show that the type II secretion system is required for translocation across the outer membrane and that a cysteine-rich region directs TraA to the T2SS. Similar results were found with another MYXO-CTERM protein indicating our findings can be generalized. Further, we show the universal distribution of MXYO-CTERM motif across the Myxococcales order and provide a working model for sorting of these proteins.

Peripheral rods: a specialized developmental cell type in Myxococcus xanthus.

In response to nutrient deprivation, the ubiquitous Gram-negative soil bacterium Myxococcus xanthus undergoes a well-characterized developmental response, resulting in the formation of a multicellular fruiting body. The center of the fruiting body consists of myxospores; surrounding this structure are rod-shaped peripheral cells. Unlike spores, the peripheral rods are a metabolically active cell type that inhabits nutrient-deprived environments. The survival characteristics exhibited by peripheral rods, protection from oxidative stress and heat shock, are common survival characteristics exhibited by cells in stationary phase including modifications to morphology and metabolism. Vegetative M. xanthus cells undergo a number of physiological changes during the transition into stationary phase similar to other proteobacteria. In M. xanthus, stationary-phase cells are not considered a component of the developmental response and occur when cells are grown on nutrient-rich plates or in dispersed aqueous media. However, this cell type is not routinely studied and little of its physiology is known. Similarities between these two stress-induced cell types led to the question of whether peripheral rods are actually a distinct developmental cell type or simply cells in stationary phase. In this study, we examine the transcriptome of peripheral rods and its relationship to development. This work demonstrates that peripheral rods are in fact a distinct developmentally differentiated cell type. Although peripheral rods and stationary phase cells display similar characteristics, each transcriptomic pattern is unique and quite different from that of any other M. xanthus cell type.

Dynamics of Solitary Predation by Myxococcus xanthus on Escherichia coli Observed at the Single-Cell Level.

The predatory behavior of Myxococcus xanthus has attracted extensive attention due to its unique social traits and inherent biological activities. In addition to group hunting, individual M. xanthus cells are able to kill and lyse prey cells; however, there is little understanding of the dynamics of solitary predation. In this study, by employing a bacterial tracking technique, we investigated M. xanthus predatory dynamics on Escherichia coli at the single-cell level. The killing and lysis of E. coli by a single M. xanthus cell was monitored in real time by microscopic observation, and the plasmolysis of prey cells was identified at a relatively early stage of solitary predation. After quantitative characterization of their solitary predatory behavior, M. xanthus cells were found to respond more dramatically to direct contact with live E. coli cells than heat-killed or UV-killed cells, showing slower predator motion and faster lysing of prey. Among the three contact-dependent killing modes classified according to the major subareas of M. xanthus cells in contact with prey, leading pole contact was observed most. After killing the prey, approximately 72% of M. xanthus cells were found to leave without thorough degradation of the lysed prey, and this postresidence behavior is described as a lysis-leave pattern, indicating that solitary predation has low efficiency in terms of prey-cell consumption. Our results provide a detailed description of the single-cell level dynamics of M. xanthus solitary predation from both prey and predator perspectives.IMPORTANCE Bacterial predation plays multiple essential roles in bacterial selection and mortality within microbial ecosystems. In addition to its ecological and evolutionary importance, many potential applications of bacterial predation have been proposed. The myxobacterium Myxococcus xanthus is a well-known predatory member of the soil microbial community. Its predation is commonly considered a collective behavior comparable to a wolf pack attack; however, individual M. xanthus cells are also able to competently lead to the lysis of a prey cell. Using a bacterial tracking technique, we are able to observe and analyze solitary predation by M. xanthus on Escherichia coli at the single-cell level and reveal the dynamics of both predator and prey during the process. The present study will not only provide a comprehensive understanding of M. xanthus solitary predation but also help to explain why M. xanthus often displays multicellular characteristic predatory behaviors in nature, while a single cell is capable of predation.

A divergent CheW confers plasticity to nucleoid-associated chemosensory arrays.

Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein.