Myxococcus xanthus and Bacillus subtilis are common soil-dwelling bacteria that produce a wide range of secondary metabolites and sporulate under nutrient-limiting conditions. Both organisms affect the composition and dynamics of microbial communities in the soil. However, M. xanthus is known to be a predator, while B. subtilis is not. A screen of various prey led to the finding that M. xanthus is capable of consuming laboratory strains of B. subtilis, while the ancestral strain, NCIB3610, was resistant to predation. Based in part on recent characterization of several strains of B. subtilis, we were able to determine that the pks gene cluster, which is required for production of bacillaene, is the major factor allowing B. subtilis NCIB3610 cells to resist predation by M. xanthus. Furthermore, purified bacillaene was added exogenously to domesticated strains, resulting in resistance to predation. Lastly, we found that M. xanthus is incapable of consuming B. subtilis spores even from laboratory strains, indicating the evolutionary fitness of sporulation as a survival strategy. Together, the results suggest that bacillaene inhibits M. xanthus predation, allowing sufficient time for development of B. subtilis spores.

bBiofilm formation is a common mechanism for surviving environmental stress and can be triggered by both intraspecies and interspecies interactions. Prolonged predator-prey interactions between the soil bacterium Myxococcus xanthus and Bacillus subtilis were found to induce the formation of a new type of B. subtilis biofilm, termed megastructures. Megastructures are treelike brachiations that are as large as 500 m in diameter, are raised above the surface between 150 and 200 m, and are filled with viable endospores embedded within a dense matrix. Megastructure formation did not depend on TasA, EpsE, SinI, RemA, or surfactin production and thus is genetically distinguishable from colony biofilm formation on MSgg medium. As B. subtilis endospores are not susceptible to predation by M. xanthus, megastructures appear to provide an alternative mechanism for survival. In addition, M. xanthus fruiting bodies were found immediately adjacent to the megastructures in nearly all instances, suggesting that M. xanthus is unable to acquire sufficient nutrients from cells housed within the megastructures. Lastly, a B. subtilis mutant lacking the ability to defend itself via bacillaene production formed megastructures more rapidly than the parent. Together, the results indicate that production of the megastructure facilitates B. subtilis escape into dormancy via sporulation. Biofilm formation is part of the life cycle for many bacterial species, leading to the accumulation of cells or spores embedded within a matrix (1, 2). Biofilms can be multispecies in composition (3) or comprised of a single species, as observed for Bacillus subtilis and Myxococcus xanthus. For both B. subtilis and M. xanthus, biofilms arise in response to environmental challenges, such as interspecies interactions or competition for nutrients, and culminate in sporulation, an escape into dormancy as a long-term survival mechanism. Entry into the dormant state is an energyconsuming process, and thus, the decision whether to enter the dormant state is critical for cells and is controlled by complex regulatory networks (4). An advantage gained by B. subtilis is that spores are widely resistant to predation by the protozoan Tetrahymena thermophile, the bacterivorous nematode Caenorhabditis elegans (5, 6), as well as the predatory bacterium M. xanthus (7). An advantage gained by M. xanthus spore production within fruiting bodies is that subsequent germinating populations are at critical numbers for group behavior, including predation.For both B. subtilis and M. xanthus, the biofilms that house dormant spores display varied structural complexity. The various biofilms produced by myxobacteria range from tree-like structures in Stigmatella aurantiaca (8) to domes for M. xanthus (9). These structures are called fruiting bodies, as they contain quiescent spores capable of germinating after extended periods of dormancy. For B. subtilis, cells produce wrinkled biofilms containing spores on solid surfaces (colony biofilms or fruiting bodies) or at an air-liquid interfa...

Soil bacteria engage each other in competitive and cooperative ways to determine their microenvironments. In this study, we report the identification of a large number of genes required for Myxococcus xanthus to engage Bacillus subtilis in a predatorprey relationship. We generated and tested over 6,000 individual transposon insertion mutants of M. xanthus and found many new factors required to promote efficient predation, including the specialized metabolite myxoprincomide, an ATP-binding cassette (ABC) transporter permease, and a clustered regularly interspaced short palindromic repeat (CRISPR) locus encoding bacterial immunity. We also identified genes known to be involved in predation, including those required for the production of exopolysaccharides and type IV pilus (T4P)-dependent motility, as well as chemosensory and two-component systems. Furthermore, deletion of these genes confirmed their role during predation. Overall, M. xanthus predation appears to be a multifactorial process, with multiple determinants enhancing predation capacity. IMPORTANCESoil bacteria engage each other in complex environments and utilize multiple traits to ensure survival. Here, we report the identification of multiple traits that enable a common soil organism, Myxococcus xanthus, to prey upon and utilize nutrients from another common soil organism, Bacillus subtilis. We mutagenized the predator and carried out a screen to identify genes that were required to either enhance or diminish capacity to consume prey. We identified dozens of genes encoding factors that contribute to the overall repertoire for the predator to successfully engage its prey in the natural environment.