Recognizing self versus nonself is a crucial step in the development of multicellularity. The social bacterium Myxococcus xanthus is a tractable model organism for studying this transition from single-cell to multicellular life. The polymorphic cell-surface receptor TraA directs cooperative behaviors toward kin. TraA is a highly specific receptor, capable of recognizing other TraA proteins with identical or nearly identical sequences by homotypic binding, but the molecular basis of this specificity remains poorly understood. Here, we generated a targeted TraA mutant library comprising thousands of variants with substitutions at 10 predicted specificity-determining residues. Screening revealed variants with altered recognition profiles, often resulting in promiscuous and/or heterotypic TraA-TraA interactions. We further identified key residues that govern specificity, as substitutions at these positions rewired recognition outcomes. Finally, we propose an evolutionary model in which new TraA specificities arise through promiscuous intermediate states shaped by reward-punishment dynamics. Together, these findings demonstrate the malleability of TraA specificity and provide molecular and evolutionary insight into social recognition.

Disease outbreaks caused by numerous pathogens pose significant risks to the development of green agriculture. Combating pathogen invasion using predatory microbes offers an alternative to the common use of agrochemicals for agricultural disease management. However, a limited understanding of bacterial predator-prey interactions restricts their practical use. In this review, we summarize the expanding body of research of known predatory bacteria, with a focus on myxobacteria-prey interactions at both the cellular and population levels. We highlight the promising potential of bacterial predators to control plant disease by preying on pathogens, manipulating environmental microbiomes and suppressing pathogen populations. Finally, we discuss current challenges utilizing bacterial predators to control plant diseases, and address open research questions and possible future directions. We hope this review will provide new insights for understanding predator-prey interactions, and offers alternative schemes to reduce the dependency on agrochemicals.

Aggregative multicellularity is a cooperative strategy used by some microbes. Unlike plant and animal development, which proceeds through clonal expansion from a single progenitor cell, aggregation is susceptible to genetic conflict and cheating, threatening multicellular stability. Myxococcus xanthus and Dictyostelium spp. are soil-dwelling models that form spore-producing fruiting bodies by aggregation upon starvation. Studies of natural M. xanthus fruiting bodies show that this process is confined to clonemates or close relatives, whereas Dictyostelium spp. can form polyclonal fruiting bodies. Here, we examine kin recognition by co-culturing two distantly related M. xanthus strains under vegetative and starvation conditions. We show that strains segregate and antagonize via their type VI secretion systems (T6SS), yielding monoclonal fruiting bodies. In contrast, mixtures of T6SS mutant strains do not antagonize and form chimeric swarms and fruiting bodies with spores from both strains. Nevertheless, within these T6SS mutant swarms and fruiting bodies, strains segregate, thus revealing that nonlethal kin discrimination mechanisms also exist in this species. These findings further suggest that T6SS are a major mediator of lethal kin discrimination between distantly related strains. Thus, lethal kin discrimination ensures homogeneous populations and monoclonal fruiting body development, whereas nonlethal mechanisms play a more subtle and less discriminatory role similar to Dictyostelium spp.

Type IV pili (T4P) drive social (S) motility in Myxococcus xanthus through cycles of extension and retraction powered by the ATPases PilB and PilT. Although the canonical retraction ATPase PilT is essential for force generation, M. xanthus encodes four PilT-like paralogs whose contributions to motility remain unclear. Here, we identify MXAN_1995 as the long-sought PilU protein that serves as a second T4P retraction motor. A frameshift mutation or deletion of pilU abolishes S-motility while preserving pilus assembly and exopolysaccharide (EPS) production, phenocopying the pilT mutant. Single-cell analyses revealed that ΔpilU mutants exhibit rare, low-force movements, consistent with a role for PilU in force generation. Fluorescence microscopy showed that PilU localizes predominantly to cell poles, similar to PilT, and that PilU localization is independent of PilT but partly dependent on core T4P assembly proteins. Notably, calcium differentially modulates motility, enhancing movement in wild-type cells while suppressing motility in ΔpilU mutants, indicating a role for PilU under varying environmental conditions. Structural modeling, together with an intragenic suppressor, suggests a regulatory function for the intrinsically disordered C-terminal region of PilU. As found in other bacterial species, our findings establish PilU as a secondary retraction ATPase and uncover a dual-motor retraction system that is environmentally responsive and mechanically tunable in M. xanthus.IMPORTANCET4P are widespread motility and adhesion systems that enable bacteria to move, interact, and form multicellular communities. While the primary retraction ATPase PilT is well characterized, the function of additional PilT-like proteins remains unclear in many species. This work provides the first mechanistic characterization of PilU in Myxococcus xanthus, a model for multicellular behavior and T4P biology. We show that PilU is essential for productive T4P retraction, functioning as an accessory motor that enhances or stabilizes PilT-driven force generation. We further reveal that PilU activity is modulated by environmental calcium and depends on a flexible C-terminal region that influences motor dynamics. These findings uncover a dual-motor architecture that enables adaptive control of T4P retraction in response to environmental cues.

Myxobacteria are predatory soil bacteria with the largest known bacterial genomes, rich in biosynthetic gene clusters for specialized metabolites. Despite their ecological importance as potential keystone taxa in soil food webs, there is a disconnect between laboratory-isolated myxobacteria and abundant Myxococcota detected in environmental metagenomic studies. Here, we report the isolation and characterization of stable myxobacterial swarm consortia from rhizospheric soil, consisting of myxobacteria associated with novel Microvirga species. Using metagenomic sequencing, we assembled metagenome-assembled genomes for four consortia, revealing phylogenetically distinct yet stably associated bacterial partnerships. Comparative genomics identified evidence of horizontal gene transfer, including acyl-homoserine lactone synthases and ankyrin repeat (ANKYR) proteins shared between consortium members, and genome-scale metabolic modeling predicted complementary auxotrophies. Time-lapse microscopy revealed that Archangium exhibited reduced predation toward its Microvirga companion (0.7% predation rate) compared to nonsymbiotic Myxococcus xanthus (14.9% predation rate) but maintained robust predatory capacity against Escherichia coli prey. These findings indicate that predation avoidance and metabolic complementarity can drive stable interbacterial symbiosis in predatory myxobacterial communities, providing foundational insights into previously overlooked myxobacterial partnerships that may be prevalent in natural soil ecosystems.

Kin recognition, the ability to distinguish self from nonself at the cellular level is critical to multicellular life. Myxococcus xanthus is a multicellular bacterium that cooperates among genetically-related cells and reduces exploitation by nonkin through outer membrane exchange (OME) of common goods and toxins. The polymorphic cell surface receptor called TraA and its partner protein TraB mediate kin recognition by OME, but its molecular mechanism remains unknown. Here we used quantitative microscopy techniques to characterize the stoichiometry of the intracellular TraAB complexes and the intercellular TraA-TraA interactions. We visualized the OME of single protein particles between cells and revealed that OME depends on the free diffusion of outer membrane (OM) contents. Based on the predicted structures, we propose a model that TraAB overcomes the repulsion between OMs by stressing the membranes and reducing the contact area, analogous to the eukaryotic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which mediate plasma membrane fusion. Our working model provides a novel pathway that leads to an underlying conserved mechanism for membrane fusion that is a foundation process for multicellularity.

Cell surface proteins determine how cells interact with their biotic and abiotic environments. In social myxobacteria, a C-terminal protein sorting tag called MYXO-CTERM is universally found within the Myxococcota phylum, where their genomes typically contain dozens of proteins with this motif. MYXO-CTERM harbors a tripartite architecture: a short signature motif containing an invariant cysteine, followed by a transmembrane helix and a short arginine-rich C-terminal region localized in the cytoplasm. In Myxococcus xanthus, MYXO-CTERM is predicted to be posttranslationally lipidated and cleaved for subsequent cell surface localization by the type II secretion system. Here, following our bioinformatic discovery, we experimentally show that myxosortase (MrtX, MXAN_2755) is responsible for the C-terminal cleavage and cell surface anchoring of TraA, a prototypic cell surface receptor. The cleavage by MrtX depends on conserved cysteines within the MYXO-CTERM motif of TraA. M. xanthus mutants lacking myxosortase are defective in TraA-mediated outer membrane exchange and exhibit cell envelope defects. In a heterologous Escherichia coli expression system, the MYXO-CTERM motif is cleaved when MrtX is co-expressed. Therefore, MrtX represents a new family of sorting enzyme that enables cell surface localization of MYXO-CTERM proteins.IMPORTANCEThe CPBP (CaaX protease and bacteriocin processing) protease family is widespread across the three domains of life. Despite considerable research on eukaryotic homologs, prokaryotic CPBP family members remain largely unexplored. In this study, we experimentally reveal the function of a novel CPBP protease called myxosortase. Our findings show that myxosortase is responsible for the C-terminal cleavage and cell surface anchoring of substrate proteins containing MYXO-CTERM motifs in Myxococcus xanthus. MYXO-CTERM cleavage also occurred in a heterologous Escherichia coli host when myxosortase is co-expressed. This is the first report that a CPBP protease is involved in protein sorting in prokaryotes. This work provides important insights into the biogenesis and anchoring of cell surface proteins in gram-negative bacteria.

From the humblest of beginnings (i.e. a pile of dry cow dung) over 80 years ago, the Gram-negative bacterium Myxococcus xanthus has emerged as a premier model system for studying diverse fields of bacteriology, including multicellular development, sporulation, motility, cell-envelope biogenesis, spatiotemporal regulation, signaling, photoreception, kin recognition, social evolution, and predation. As the flagship representative of myxobacteria found in varied terrestrial and aquatic environments, M. xanthus research has evolved into a collaborative global effort, as reflected by the contributions to this article. In celebration of the upcoming 50th anniversary of the International Conference on the Biology of Myxobacteria, this review highlights the historical and ongoing contributions of M. xanthus as a multifaceted model bacterium.

Recognizing the difference between self and nonself is a crucial step in the development of multicellularity. Myxococcus xanthus is a model organism for studying these processes during the transition from single cell to multicellular life. The polymorphic cell surface receptor TraA helps to mediate these transitions by directing cooperative behavior toward kin or clonemates. TraA is a highly specific receptor, capable of recognizing other TraA proteins with identical or nearly identical sequences by homotypic binding, but the molecular basis of recognition is poorly understood. In this study, we designed a targeted TraA library, consisting of thousands of variants, which changed 10 predicted specificity residues. By screening this library, we identified TraA variants with different combinations of substitutions that resulted in altered recognition, often leading to promiscuous TraA-TraA binding. Additionally, we identified key residues that dictate specificity between distant TraA groups and showed that changing these residues altered the recognition specificity. Furthermore, we propose a model to explain how TraA recognition specificity evolved through the generation of intermediate promiscuous variants driven by reward/punishment interactions. Our results highlight the malleable nature of the TraA variable domain involved in specificity, shedding light on the molecular and evolutionary basis of social recognition in M. xanthus.

Microbial cell factories for the production of high-quality commercial-grade enzymes have accelerated the development of advanced bio-manufacturing approaches, which in turn are environmentally friendly and sustainable. Myxobacteria, a term commonly used to refer to a group within the Myxococcota phylum, are of great interest for their biotechnological applications due to their ability to synthesize a wide range of natural products and lytic enzymes. These traits are essential for the development of robust expression systems. However, myxobacteria have remained an underexploited resource with industrial relevance. Nevertheless, a growing number of food and industrial enzymes have been identified, highlighting myxobacteria as suitable platforms for exploring enzymes with commercial applications, including biomass conversion. Yet, the discovered lytic enzymes are just the tip of the iceberg given their large genomes and diversity across myxobacteria taxa. Despite holding much promise, challenges in genetic engineering, slow growth, and limitations in metabolic remodeling and expression strategies have limited the construction of myxobacterial cell factories. In this review, we highlight recent advances in the discovery of new myxobacterial enzymes and biomass conversion resources, focusing on their potential applications in agriculture and industry. We describe how myxobacteria and their enzymes can be identified through bioprospecting and computational approaches and summarize current biotechnological applications and synthetic biology strategies for bio-manufacturing. Finally, we discuss the promising potential of myxobacteria as industrial cell factories and address open research questions and future directions.

Introduction Outer membrane vesicles (OMVs) of Gram-negative bacteria mediate diverse functions in natural ecosystems. As a keystone taxon in soil, myxobacteria produce OMVs for cargo packing and microbial predation. However, the roles of OMVs in the interactions of myxobacteria with fungi remain poorly understood. Objectives This work aims to clarify the role of outer membrane vesicles in the interaction between myxobacteria and fungi and the regulatory mechanism during the interaction process. Methods We found that OMVs play a significant role in the interaction between Myxococcus sp. MP20 and Verticillium dahliae (Vd). We further identified the antifungal metabolites in OMVs and verified the regulatory mechanism of OMVs in the model strain Myxococcus xanthus DK 1622. Results We discover that Myxococcus sp. MP20 uses fungal networks and cotton root exudates for spatial dispersal. MP20 deploys the antifungal metabolites myxothiazole via OMVs to inhibit Vd growth by fusing the OMVs with fungal cells, thus restraining fungal invasion. Containment of myxothiazol within OMVs maintains an effective antifungal concentration on target cells. Furthermore, we demonstrate that the release of OMVs from MP20 was suppressed by iron via a newly discovered ABC transport system. In turn the lower number of OMVs reduced the antifungal behavior of MP20, suggesting that iron acquisition regulates OMV-mediated competition between myxobacteria and fungi. Our findings thus unravel a novel antifungal tactic employed by myxobacteria to suppress fungi prey and control Verticillium wilt, which also provides new insights for understanding predator–prey interactions.

Bacterial genomes contain a surprisingly large number of toxin systems that are neutralized by specific cognate antitoxins or immunity factors. Their high abundance is even apparent in common Escherichia coli K12 cloning strains, which contain at least 36 toxin-antitoxin systems, while other bacteria frequently contain more. These numbers raise two key questions: why are they so numerous, and to what extent do toxin systems interact or interfere with one another? Recently in mBio , Wang and co-workers addressed these questions in the social bacterium Myxococcus xanthus , where they investigated crosstalk between four homologous toxin-immunity loci involved in kin discrimination. Here, the type VI secretion system delivers toxins into neighboring myxobacterial cells (F. Wang, J. Luo, Z. Zhang, Y. Liu, et al., mBio e03902-24, 2025, https://doi.org/10.1128/mbio.03902-24 ). If the target cell is clonal and expresses a complete set of cognate immunity proteins—which are not themselves transferred—the cell is protected. However, if immunity is incomplete, the cell is poisoned.

Microbes face many physical, chemical, and biological insults from their environments. In response, cells adapt, but whether they do so cooperatively is poorly understood. Here, we use a model social bacterium, Myxococcus xanthus, to ask whether adapted traits are transferable to naïve kin. To do so we isolated cells adapted to detergent stresses and tested for trait transfer. In some cases, strain-mixing experiments increased sibling fitness by transferring adaptation traits. This cooperative behavior depended on a kin recognition system called outer membrane exchange (OME) because mutants defective in OME could not transfer adaptation traits. Strikingly, in mixed stressed populations, the transferred trait also benefited the adapted (actor) cells. This apparently occurred by alleviating a detergent-induced stress response in kin that otherwise killed actor cells. Additionally, this adaptation trait when transferred also conferred resistance against a lipoprotein toxin delivered to targeted kin. Based on these and other findings, we propose a model for stress adaptation and how OME in myxobacteria promotes cellular cooperation in response to environmental stresses.

Aggregative multicellularity is a cooperative strategy employed by some microorganisms. Unlike clonal expansion within protected environments during multicellular eukaryotic development, an aggregation strategy introduces the potential for genetic conflicts and exploitation by cheaters, threatening the stability of the social system. Myxococcus xanthus, a soil-dwelling bacterium, employs aggregative multicellularity to form multicellular fruiting bodies that produce spores in response to starvation. Studies of natural fruiting bodies show that this process is restricted to close kin or clonemates. Here, we investigate the mechanisms underlying kin recognition during development in M. xanthus. By co-culturing two distantly related M. xanthus strains under vegetative and starvation conditions, we observed that the strains segregate in both contexts. During vegetative growth, one strain antagonized the other using the type VI secretion system (T6SS). T6SS-mediated antagonism was also observed during development, resulting in monoclonal fruiting bodies when WT strains were mixed. In contrast, mixtures of T6SS knockout strains formed chimeric fruiting bodies, that produced viable spores from both strains. These findings suggest that T6SS is the primary mechanism of kin discrimination in distantly related M. xanthus strains, and its use ensures the development of monoclonal fruiting bodies and social integrity.

Social diversification in microbes is an evolutionary process where lineages bifurcate into distinct populations that cooperate with themselves but not with other groups. In bacteria, this is frequently driven by horizontal transfer of mobile genetic elements (MGEs). Here, the resulting acquisition of new genes changes the recipient's social traits and consequently how they interact with kin. These changes include discriminating behaviors mediated by newly acquired effectors. Since the producing cell is protected by cognate immunity factors, these selfish elements benefit from selective discrimination against recent ancestors, thus facilitating their proliferation and benefiting the host. Whether social diversification benefits the population at large is less obvious. The widespread use of next-generation sequencing has recently provided new insights into population dynamics in natural habitats and the roles MGEs play. MGEs belong to accessory genomes, which often constitute the majority of the pangenome of a taxon, and contain most of the kin-discriminating loci that fuel rapid social diversification. We further discuss mechanisms of diversification and its consequences to populations and conclude with a case study involving myxobacteria.

The synthesis of imidazole fused spirocyclic ketones as templates for acetyl-CoA carboxylase (ACC) inhibitors is reported. By completing the spirocyclic ring closure via divergent pathways, the synthesis of these regioisomers from common intermediates was developed. Through an aldehyde homologation/ transmetalation strategy, one isomer was formed selectively. The second desired isomer was obtained via an intramolecular aromatic homolytic substitution reaction. Preparation of these isomeric spiroketones provided templates which, upon elaboration, led to key structure-activity relationship (SAR) points for delivery of potent ACC inhibitors.

The melanocortin-4 receptor (MC4R) is a centrally expressed, class A GPCR that plays a key role in the regulation of appetite and food intake. Deficiencies in MC4R signaling result in hyperphagia and increased body mass in humans. Antagonism of MC4R signaling has the potential to mitigate decreased appetite and body weight loss in the setting of anorexia or cachexia due to underlying disease. Herein, we report on the identification of a series of orally bioavailable, small-molecule MC4R antagonists using a focused hit identification effort and the optimization of these antagonists to provide clinical candidate 23. Introduction of a spirocyclic conformational constraint allowed for simultaneous optimization of MC4R potency and ADME attributes while avoiding the production of hERG active metabolites observed in early series leads. Compound 23 is a potent and selective MC4R antagonist with robust efficacy in an aged rat model of cachexia and has progressed into clinical trials.

Myxobacteria are social microbial predators that use cell-cell contacts to identify bacterial or fungal prey and to differentiate kin relatives to initiate cellular responses. For prey killing, they assemble Tad-like and type III-like secretion systems at contact sites. For kin discrimination (KD), they assemble outer membrane exchange complexes composed of the TraA and TraB receptors at contacts sites. A type VI secretion system and Rhs proteins also mediate KD. Following cellular recognition, these systems deliver appropriate effectors into target cells. For prey, this leads to cell death and lysis for nutrient consumption by myxobacteria. In KD, a panel of effectors are delivered, and if adjacent cells are clonal cells, resistance ensues because they express a cognate panel of immunity factors; while nonkin lack complete immunity and are intoxicated. This review compares and contrasts recent findings from these systems in myxobacteria.

As social micropredators, myxobacteria are studied for their abilities to prey on bacteria and fungi. However, their predation of oomycetes has received little attention. Here, we show that Archangium sp. AC19 secretes a carbohydrate-active enzyme (CAZyme) cocktail during predation on oomycetes Phytophthora . These enzymes include three specialized β-1,3-glucanases (AcGlu13.1, –13.2 and –13.3) that act as a cooperative consortium to target β-1,3-glucans of Phytophthora . However, the CAZymes showed no hydrolytic effects on fungal cells, even though fungi contain β-1,3-glucans. Heterologous expression of AcGlu13.1, –13.2 or –13.3 enzymes in Myxococcus xanthus DK1622, a model myxobacterium that antagonizes but does not predate on P. sojae , conferred a cooperative and mycophagous ability that stably maintains myxobacteria populations as a mixture of engineered strains. Comparative genomic analyses suggest that these CAZymes arose from adaptive evolution among Cystobacteriaceae myxobacteria for a specific prey killing behavior, whereby the presence of Phytophthora promotes growth of myxobacterial taxa by nutrient release and consumption. Our findings demonstrate that this lethal combination of CAZymes transforms a non-predatory myxobacterium into a predator with the ability to feed on Phytophthora , and provides new insights for understanding predator-prey interactions. In summary, our work extends the repertoire of myxobacteria predatory strategies and their evolution, and suggests that these CAZymes can be engineered as a functional consortium into strains for biocontrol of Phytophothora diseases and hence crop protection.

Myxobacteria exhibit a variety of complex social behaviors that all depend on coordinated movement of cells on solid surfaces. The cooperative nature of cell movements is known as social (S)-motility. This system is powered by cycles of type IV pili (Tfp) extension and retraction. Exopolysaccharide (EPS) also serves as a matrix to hold cells together. Here, we characterized a new S-motility gene in Myxococcus xanthus. This mutant is temperature-sensitive (Ts–) for S-motility; however, Tfp and EPS are made. A 1 bp deletion was mapped to the MXAN_4099 locus and the gene was named sglS. Null mutations in sglS exhibit a synthetic enhanced phenotype with a null sglT mutation, a previously characterized S-motility gene that exhibits a similar Ts– phenotype. Our results suggest that SglS and SglT contribute toward Tfp function at high temperatures in redundant pathways. However, at low temperatures only one pathway is necessary for wild-type S-motility, while in the double mutant, motility is nearly abolished at low temperatures. Interestingly, the few cells that do move do so with a high reversal frequency. We suggest SglS and SglT play conditional roles facilitating Tfp retraction and hence motility in M. xanthus.