Thiocillin contributes to the ecological fitness of Bacillus cereus ATCC 14579 during interspecies interactions with Myxococcus xanthus

Abstract

The soil-dwelling delta-proteobacterium Myxococcus xanthus is a model organism to study predation and competition. M. xanthus preys on a broad range of bacteria mediated by lytic enzymes, exopolysaccharides, Type-IV pilus-based motility, and specialized metabolites. Competition between M. xanthus and prey bacterial strains with various specialized metabolite profiles indicates a range of fitness, suggesting that specialized metabolites contribute to prey survival. To expand our understanding of how specialized metabolites affect predator-prey dynamics, we assessed interspecies interactions between M. xanthus and two strains of Bacillus cereus. While strain ATCC 14579 resisted predation, strain T was found to be highly sensitive to M. xanthus predation. The interaction between B. cereus ATCC 14579 and M. xanthus appears to be competitive, resulting in population loss for both predator and prey. Genome analysis revealed that ATCC 14579 belongs to a clade that possesses the biosynthetic gene cluster for production of thiocillins, whereas B. cereus strain T lacks those genes. Further, purified thiocillin protects B. cereus strains unable to produce this specialized metabolite, strengthening the finding that thiocillin protects against predation and contributes to the ecological fitness of B. cereus ATCC 14579. Lastly, strains that produce thiocillin appear to confer some level of protection to their own antibiotic by encoding an additional copy of the L11 ribosomal protein, a known target for thiopeptides. This work highlights the importance of specialized metabolites affecting predator-prey dynamics in soil microenvironments.

Keywords: B. cereus; M. xanthus; competition; predator–prey interactions; specialized metabolites.

Figure 1

Thiocillin protects B. cereus ATCC 14579 from predation by M. xanthus. B. cereus strains (prey) were grown to a certain cell density (see Materials and Methods) and nutrients were removed by washing the cells multiple times. Prey cells were mixed with the predator M. xanthus in a ratio of 50:1, spotted on CFL Agar plates and incubated at 32°C. Pictures were taken after different times of incubation at 15x magnification. Predation is visible by cell lysis (*) whereas competition/predation resistance is indicated by minimal loss of cells (O). Fruiting body formation can be seen after 48 h as an indicator that predator M. xanthus sensed a drop down in nutrients and is starving (black square). Only B. cereus ATCC 14579 resists predation and mutations inhibiting thiocillin production or resulting in modifications of the thiocillin molecule render cells to be predation sensitive. B. cereus ATCC 14579 ∆tclE-H does not contain the thiocillin prepeptide encoding genes tclE-H and does therefore not make thiocillin. B. cereus ATCC 14579 ∆tclE-H::tclE (T4V) makes a variant of the thiocillin molecule that has lost the antimicrobial function of the molecule but is still able to induce matrix formation in B. subtilis. Strain ∆tclM produces a thiocillin-like molecule that has no closed ring structure.

Figure 2

Purified Thiocillin rescues sensitive strains from predation by M. xanthus. The predation sensitive strains B. cereus strain T (A), B. cereus ATCC 14579 ∆tclE-H (B) and E.coli DH5α (C) were tested in predation assays with the predator M. xanthus with and without purified thiocillin (dissolved in DMSO). B. cereus ATCC 14579 ∆tclE-H is not able to produce thiocillin anymore and B. cereus strain T does not contain the thiocillin BSG. All strains are sensitive to predation (comparing column III to I and II). The addition of purified thiocillin protected all sensitive strains from predation (column IV). Increasing concentrations of thiocillin enhanced the predation protective effect. Pictures were taken after 24 h.

Figure 3

Quantification of predator and prey survival reveals strong competition during interspecies interactions between M. xanthus and B. cereus ATCC 14579. Quantification of prey survival and predator survival/growth. (A) Prey B. cereus ATCC 14579 and mutant strains B. cereus ATCC 14579 ∆tclE-H or ∆tclE-H::tclE (T4V) were mix in a 50:1 ratio with M. xanthus and spotted onto CFL Agar plates. As controls the strains were incubated individually. After incubation at 32°C. for 24 h, cells were removed from the plates and CFU were determined to calculate prey survival calculated relative to controls. The majority of B. cereus ATCC 14579 ∆tclE-H and ∆tclE-H::tclE (T4V) were consumed by the predator M. xanthus, whereas about 23.5% of the wild type strain B. cereus ATCC 14579 survived. (B) M. xanthus was able to grow in the presence of B. cereus ATCC 14579 ∆tclE-H and ∆tclE-H::tclE (T4V) but only around 12% of M. xanthus could be recovered when mixed with B. cereus ATCC 14579 clearly showing this interspecies interaction is of competitive nature effecting the ecological fitness of both strains. (C) Purified thiocillin affects predator viability. Percent M. xanthus survival after 24 h. M. xanthus cells where washed and resuspended to 250 KU. Cells where mixed with either buffer, DMSO or thiocillin (thiocillin 300 ng) and spotted on CFL Agar. Cells were harvested after 24 h and serial dilutions were plates in CYE agar to calculated CFU’s.

Figure 4

Pangenomics of B. cereus strain T and ATCC 14579 reveals differences in specialized metabolite gene clusters. (A) Venn diagram of the shared genes and unique genes between B. cereus strain T and ATCC 14579. (B) Anvi’o plot of B. cereus strain T and ATCC 14579 genomes. Open reading frames (ORFs) for the two strains were compared using the anvio workflow for pangenomics. ORFs were called using prodigal and annotated using InterproScan for PFAM annotations. ORFs were aligned and compared using blastp and weak matches refined using minbit = 0.5 and MCL inflation parameter of 10. AntiSMASHv5.0 was used to annotate the complete specialized metabolite clusters, including thiocillin. Individual ORFs for all specialized metabolites are shown in green lines (Specialized Metabolite ORFs ring), many of which are specific to ATCC (green) and strain T (gold) as visible in the thiocillin ORFs ring. ORFs comprising the thiocillin operon are marked with red arrows. (C) Phylogenetic tree (Maximum Likelihood, midpoint root) based on a multiple sequence alignment of SCGs (single copy genes) using 109 B. cereus strains. Five thiocillin producing strains were identified that cluster together (green) indicating that they are closely related. Due to the close phylogenetic relationship of the thiocillin BGC containing strains it is most likely that a single common ancestor did gain the BGC by horizontal gene transfer.

Figure 5

Comparison of the thiocillin synthesis gene clusters of B. cereus ATCC 14579 and B. cereus T. The thiocillin synthesis gene cluster is composed of the precursor peptide encoding genes tclE-H (black) as well as biosynthesis genes (blue and orange). Genes encoding proteins functioning in posttranslational modification (green) and transport (yellow) are also part of the thiocillin synthesis gene cluster. Comparison of both B. cereus strains revealed that B. cereus T does not contain the central components of the thiocillin synthesis gene cluster tclA-tclU including the precursor peptide encoding genes tclE-H.

Figure 6

Model for Inhibition by and Protection from Thiocillin During Predation. Thiocillin is known to inhibit the L11 interaction with 23S rRNA within the 50S subunit of the ribosome to affect translation. B. cereus ATCC 14579, a thiocillin producer, encodes two L11 paralogs, TclQ and TclT, whereas M. xanthus and other B. cereus strains do not. TclQ and TclT most likely provide a mechanism for immunity from thiocillin within thiocillin-producing B. cereus strains. Thiocillin affects the fitness of M. xanthus and therefore provides an advantage against predation. “Created with BioRender.com.”