Bacillaene and sporulation protect Bacillus subtilis from predation by Myxococcus xanthus

Abstract

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.

FIG 1

M. xanthus predation of various prey strains. Shown are predation assays using different Gram-positive and Gram-negative strains as prey for M. xanthus predator cells. Efficient predation results in clearing of the prey spot, with M. xanthus fruiting body formation occurring at the edge of the original prey spot. The prey strains tested were E. coli DH5α and B2155, B. subtilis OI1085, 168, and NCIB3610, Rhodobacter capsulatus, Salmonella enterica, Pseudomonas aeruginosa, and Staphylococcus aureus. Strains resisting predation show only minimal lysis at the center of the prey spot. (A) Prey only; (B) prey with buffer spotted at center; (C) prey with heat-killed predator; (D) prey with predator. Pictures were taken at 48 h after spotting at a magnification of ×10 (A to D) or ×30 (E). Bars, 0.5 cm (A to D) and 0.1 cm (E).

FIG 2

Quantification of prey survival and predator growth. (A) Prey and predator cells were mixed in a ratio of 50:1, plated onto CFL agar plates, and incubated at 32°C for 24 h. Prey and predator alone were used as controls. CFU were determined, and percent prey survival and percent predator growth were calculated relative to the controls. The majority of E. coli β2155 and B. subtilis OI1085 and 168 were consumed, whereas about 68% of B. subtilis NCIB3610 survived. (B) M. xanthus was able to grow significantly on E. coli β2155 and B. subtilis OI1085 but not the ancestral B. subtilis NCIB3610 strain.

FIG 3

Bacillaene inhibits M. xanthus predation. (A) Predation assays using the domesticated, ancestral, and mutant strains of B. subtilis. Mutations in sfp and pksL reveal the requirement for bacillaene as the major factor inhibiting M. xanthus predation. Pictures were taken 24 h after spotting M. xanthus predator cells in the center of the prey source. Bar, 0.1 cm. (B) Prey survival was quantified as described for Fig. 2 and normalized to that for the NCIB3610 control spotted without the predator. (C) Growth of the M. xanthus predator was quantified after 24 h and normalized to that for M. xanthus cells spotted without prey.

FIG 4

Bacillaene protects sensitive prey. Predation assays were conducted using sensitive prey mixed with purified bacillaene (left), methanol (center), and MMC buffer (right) on CFL agar plates. Photographs were taken after 24 h. Sensitive prey were protected in the presence of bacillaene. Bar, 0.5 cm.

FIG 5

Bacillus subtilis spores resist predation. Spores were made and purified as described in Materials and Methods (36). Spores were spotted on MOPS agar plates lacking any nutrients to prevent spore germination. Predation assays were conducted as shown in Fig. 1. Shown are spores alone (A), spores with predator cells (B), and vegetative prey cells with the predator (C). Domesticated strains as well as the NCIB3610 pksL mutant cells are capable of producing predation-resistant spores. Bar, 0.5 cm.