Differential response to prey quorum signals indicates predatory range of myxobacteria

A potential keystone taxa, myxobacteria contribute to the microbial food web as generalist predators. However, the extent of myxobacterial impact on microbial community structure remains unknown. The chemical ecology of these predator-prey interactions provides insight into myxobacterial production of biologically active specialized metabolites used to benefit consumption of prey as well as the perception of quorum signals secreted by prey. Using comparative transcriptomics and metabolomics, we compared how the predatory myxobacteria Myxococcus xanthus and Cystobacter ferrugineus respond to structurally distinct exogenous quorum signaling molecules. Investigating acylhomoserine lactone (AHL) and quinolone type quorum signals used by the clinical pathogen Pseudomonas aeruginosa, we identified a general response to AHL signals from both myxobacteria as well as a unique response from C. ferrugineus when exposed to the quinolone signal 4-hydroxy-2-heptylquinolone (HHQ). Oxidative detoxification of HHQ in C. ferrugineus was not observed from M. xanthus. Subsequent predation assays indicated P. aeruginosa to be more susceptible to C. ferrugineus predation. These data indicate that as generalist predators myxobacteria demonstrate a common response to the ubiquitous AHL quorum signal class, and we suggest this response likely involves recognition of the homoserine lactone moiety of AHLs. We also suggest that oxidation of HHQ and superior predation of P. aeruginosa observed from C. ferrugineus provides an example of how prey signaling molecules impact predatory specialization of myxobacteria by influencing prey range. Summary Multiomic analysis of transcriptional and metabolic responses from the predatory myxobacteria Myxococcus xanthus and Cystobacter ferrugineus exposed to prey signaling molecules of the acylhomoserine lactone and quinolone quorum signaling classes provided insight into myxobacterial specialization associated with predatory eavesdropping. We suggest that the general response observed from both myxobacteria exposed to acylhomoserine lactone quorum signals is likely due to the generalist predator lifestyles of myxobacteria and ubiquity of acylhomoserine lactone signals. We also provide data that indicates the core homoserine lactone moiety included in all acylhomoserine lactone scaffolds to be sufficient to induce this general response. Comparing both myxobacteria, unique transcriptional and metabolic responses were observed from Cystobacter ferrugineus exposed to the quinolone signal 4-hydroxy-2-heptylquinoline (HHQ) natively produced by Pseudomonas aeruginosa. We suggest that this unique response and ability to metabolize quinolone signals contribute to the superior predation of P. aeruginosa observed from C. ferrugineus. These results further demonstrate myxobacterial eavesdropping on prey signaling molecules and provide insight into how responses to exogenous signals might correlate with prey range of myxobacteria. Originality-Significance Statement This manuscript provides the first multiomic analysis of how predatory myxobacteria respond to exogenous prey signaling molecules and details the differences observed by comparing responses from two myxobacteria.

lifestyles of myxobacteria and ubiquity of acylhomoserine lactone signals. We also 24 provide data that indicates the core homoserine lactone moiety included in all 25 acylhomoserine lactone scaffolds to be sufficient to induce this general response. 26 Comparing both myxobacteria, unique transcriptional and metabolic responses were 27 observed from Cystobacter ferrugineus exposed to the quinolone signal 4-hydroxy-2-28 heptylquinoline (HHQ) natively produced by Pseudomonas aeruginosa. We suggest that 29 this unique response and ability to metabolize quinolone signals contribute to the superior 30 predation of P. aeruginosa observed from C. ferrugineus. These results further 31 demonstrate myxobacterial eavesdropping on prey signaling molecules and provide 32 insight into how responses to exogenous signals might correlate with prey range of 33 myxobacteria.

Introduction 56
The uniquely multicellular lifestyles of myxobacteria have motivated continued efforts to 57 explore the myxobacterium Myxococcus xanthus as a model organism for cooperative from C. ferrugineus. Only one gene was observed to be upregulated by M. xanthus when 158 exposed to C6-AHL, and 25 total upregulated genes were observed from C. ferrugineus 159 during C6-AHL exposure. 160 increased transcription of lytic enzymes and mobile genetic elements observed from C. 180 ferrugineus exposed to C6-AHL suggest a predatory response; however, these features 181 could also be associated with a defense response akin to phage defense. Transcription whereas HHQ elicits a distinct response from C. ferrugineus dissimilar from the more 226 general response observed from both myxobacteria when exposed to C6-AHL. with a conserved response to both C6-AHL and 3-oxo-C6-AHL. These data also revealed 255 a unique metabolic response from C. ferrugineus when exposed to HHQ similar to our 256 previous transcriptomic observation.

C. ferrugineus response to HHQ correlates with superior predation of P. aeruginosa 294
Predation assays using the lawn culture method were conducted in triplicate on lawns of 295 P. aeruginosa with both M. xanthus and C. ferrugineus (Morgan et al., 2010). These 296 assays confirmed that P. aeruginosa was comparatively more susceptible to predation by 297 C. ferrugineus (Figure 9). These results suggest the unique response to exogenous HHQ 298 observed from C. ferrugineus to be an evolved trait associated with exposure to quinolone 299 signals that correlates with a prey range which includes quinolone signal-producing 300 pseudomonads. 301

Discussion 302
Although the predatory lifestyles of myxobacteria have long been associated with their 303 capacity as a resource for natural products discovery, the chemical ecology of predator-304

Subsequent comparative metabolomics experiments indicated that C6-AHL and 3-oxo-331
C6-AHL elicit overlapping responses from both myxobacteria and that the core AHL 332 moiety L-HSL also elicits a similar response from C. ferrugineus. We conclude that the 333 overlap between C6-AHL, 3-oxo-C6-AHL, and L-HSL indicates an evolved recognition of 334 the homoserine lactone unit present in all AHL quorum signals (Papenfort and Bassler, 335 2016; Mukherjee and Bassler, 2019). As generalist predators, a more general process for 336 AHL perception that responds to a core moiety in the scaffold of AHLs might be preferred 337 to a specialized process associated with the variable N-acylamides of AHLs. We also 338 suspect this centralized response to L-HSL may relate to the absence of a LuxR-type 339 translation and turnover, cell wall biogenesis and maintenance, and specialized 348 metabolism when exposed to HHQ. Interestingly, an annotated FAD-dependent 349 oxidoreductase homologous to the monooxygenase PqsH from P. aeruginosa, which 350 hydroxylates HHQ to yield PQS was upregulated 31-fold in C. ferrugineus exposed to Braunschweig, and Myxococcus xanthus strain GJV1 were employed in this study. 375 Cystobacter ferrugineus was grown on VY/2 agar (5 g/L baker's yeast, 1.36 g/L CaCl2,   C. ferrugineus exposed to HHQ when compared to signal unexposed C. ferrugineus