Identification of Functions Affecting Predator-Prey Interactions between Myxococcus xanthus and Bacillus subtilis

Abstract

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 predator-prey 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.

Importance: Soil 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.

FIG 1

M. xanthus transposon screen outline. A library of 6,000 M. xanthus transposon mutants was generated using the EZ-Tn5 transposable element. (I) To select for single M. xanthus colonies, cells were plated into Top agar on solid CYE agar plates containing kanamycin for selection and incubated for 5 to 6 days. (II) Individual colonies were transferred onto CYE kanamycin agar plates. (III) B. subtilis NCIB3610 prey cells were spotted onto CFL agar plates (predation/starvation medium), and the M. xanthus transposon mutants were transferred into the middle, showing the inside-out assay. Screening for predation phenotypes occurred for up to 5 days. GOF and LOF strains were selected by comparing them to M. xanthus wild type (WT) and an LOF control strain (mglAB mutant), as seen in the lower part of the figure.

FIG 2

Phenotypic analysis of in-frame deletion mutants. M. xanthus transposon mutants were grown to mid-log phase, washed, and resuspended to a cell concentration of 250 KU. (A to C) Phenotypic analysis was performed to assess T4P-dependent motility on 0.5% CYE agar (A), gliding motility on 1.5% CYE agar plates (B), and fruiting body formation on CFL starvation/predation medium (C). (D and E) To investigate predatory ability, 2 μl of M. xanthus cells was spotted into the middle of B. subtilis NCIB3610 (E), or both predator and prey cells were mixed in a ratio of 1:50 and spread on plates to study megastructure formation on CFL agar plates (D). (F to H) Semiquantitative predation assays. Predator and prey were mixed in a ratio of 1:25 (F), 1:50 (G), or 1:100 (H). Pictures were taken after 24 h (B) and 48 h (A and C to H) at a magnification of ×10 (A, B, and E to H), ×30 (C and D), or ×100 (B). (D) Bar, 0.5 mm; arrows indicate individual megastructures.

FIG 3

Quantification of prey survival and predator growth. Predator M. xanthus wild-type or myxoprincomide mutant [Δ(MXAN_3778-MXAN_3779)] cells were mixed with B. subtilis NCIB3610 prey cells in a ratio of 1:50, and the mixture was spread plated onto CFL agar. After incubation for 24 h at 32°C, cells were harvested and serial dilutions were plated onto selective medium. Predator and prey alone were used as controls. CFU were used to calculate the percentage of prey survival and predator growth relative to the controls. (A) The myxoprincomide mutant shows a reduced ability to consume B. subtilis NCIB3610. (B) The M. xanthus wild type shows no significant growth, whereas the loss of the specialized metabolite myxoprincomide results in reduced CFU. The data represent the average of the results from 3 individual eating experiments, and the error bars show the standard deviation.

FIG 4

Factors and pathways involved M. xanthus predation. Our transposon mutagenesis screen identified 2 two-component systems, the Dif chemosensory system (left) and the Hsf system (right), that are involved in regulating predation. Each system utilizes a histidine kinase (blue) that becomes autophosphorylated upon activation to regulate outputs via response regulators. Upon activation, phosphoryl groups are transferred to response regulators (orange) to generate the physiological responses, such as EPS production, motility, and specialized metabolite production. Additionally, we found an ABC transporter/permease and the specialized metabolite myxoprincomide to be involved in the predation of B. subtilis NCIB3610. HTH, helix-turn-helix.