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Protozoan predation drives rapid adaptive divergence by selecting for tuned cellulose production and amyloid fibrils in Pseudomonas fluorescens SBW25

ABSTRACT: Purpose: Protozoan predators affect the structure of bacterial communities, but investigations into how predation influences bacterial evolution and antagonistic behaviours are scarce. We performed a 20-day predator-prey evolution experiment on solid media to investigate the adaptive traits that arise in bacterial prey under continuous protozoan predation. Methods: Pseudomonas fluorescens SBW25 and a wild Acanthamoeba sp. isolate as a predator prey pair co-evolved for 20 days yielded both previously described (Wrinkly Spreader; WS) and novel colony morphotype (Wrinkly Fried Egg; WFE) isolates with conferred grazing resistance. These isolates were subjected to RNAseq profiling with and without predation to determine transcriptional changes contributing to grazing resistance. Results: For differential gene expression the WT SBW25 without predation was used as a baseline. For the WS condition, a total of 881 differentially expressed genes (DEGs) were identified, of which 424 were upregulated and 457 were downregulated. In the WFE condition, a total of 908 DEGs were identified, of which 475 were upregulated and 434 were downregulated. Among all DEGs, 335 upregulated and 313 downregulated genes were shared between the WS1 and WFE conditions Conclusions: Our findings suggest that protozoan predation can profoundly influence the course of genetic and phenotypic evolution in a short period of time. Together, the differential expression results suggest expression of features that would be expected to increase biofilm formation in WFE according to previous studies. However, increased expression of these traits may not lead to a stronger biofilm, but may still provide predation resistance. For example, fibrils may increase the effective profile size of a bacterial cell. Increased Fap-mediated biofilm formation also induces increased alginate synthesis in P. aeruginosa PA01, an exopolysaccharide that protects mucoid P. aeruginosa against macrophage killing. Interestingly, we found increased expression of alginate biosynthesis genes in both WFE and WS1 (algA, algF), suggesting alternate mechanisms leading to increased alginate production in these two strains.

ORGANISM(S): Acanthamoeba sp. Pseudomonas fluorescens

PROVIDER: GSE200723 | GEO | 2022/04/20

REPOSITORIES: GEO

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Project description:Purpose: Generalist protozoan predators change the pace of evolution in microbial communities, but investigations into the molecular consequences of predation for the genomic evolution of defence or coadaptation are scarce. We have previously performed a 90-day co-evolution experiment to address these effects using the bacterium Pseudomonas fluorescens SBW25 on solid media in the presence and absence of a wild Acanthamoeba sp. predator. Coevolution led to genomic divergence, altered phenotypes and resistance to predation in coevolved bacterial lineages. Strong parallel phenotypic evolution was observed among the coevolved replicates leading to Wrinkly Spreader, Volcano and Mountain colony phenotypes. Here, we describe the genomic mutations underlying this parallelism with a focus on these morphotypes. Methods: Pseudomonas fluorescens SBW25 and a wild Acanthamoeba sp. isolate as a predator prey pair co-evolved for 90 days yielded novel colony morphotype (Mountain; MNT, and Volcano; VOL) isolates with conferred grazing resistance. These isolates were subjected to RNAseq profiling with and without predation to determine transcriptional changes contributing to grazing resistance. Results: We made a profile of differentially expressed genes (DEG) for the coevolved Vol-L1 (Volcano L1; ∆fadD2-fadD1 & luxR T2675G) while predated by ancestral Acanthamoebae. WT P. fluorescens SBW25 was used as the baseline and significant differential expression was considered for genes with log2 FC ≥ 2, P-value ≤ 0.05 between conditions. We identified a total of 953 DEGs, of which 293 were upregulated and 660 were downregulated. Differentially expressed gene (DEG) profiles were made for the coevolved strains Mnt-L2 (Mountain L2; ∆4974-4975 & PFLU_0924 C790T) while under predation by ancestral Acanthamoebae. In this case we also used WT P. fluorescens SBW25 without predation as a baseline as described previously. In this condition and compared to the WT SBW25, we identified a total of 209 DEGs, of which 118 were upregulated and 91 were downregulated. Conclusions: In this study, from the DEG profile of Mnt-L2 and Vol-L1 while predated by the ancestral Acanthamoebae we found 12 and 21 upregulated genes in O-antigen biosynthesis, respectively. The polysaccharide capsule (LPS) is considered as one of the main virulence factors produced by pathogenic bacteria to protect bacteria against phagocytosis by the host’s immune system as well. We found enrichments in lipopolysaccharide-A production and three viscosin genes in Vol-L1 (a finding unique to the Vol-L1 strain). Vol-L1 had significant downregulation of seven efflux genes, most significantly PFLU_3263 (an efflux transporter), as well as a fatty acid transporter (PFLU4903). These results, taken with the finding of altered survivability only when evolved strains are grown with Acanthamoebae and the abundance of mutations found in FadD genes, led to our hypothesis that the evolved bacteria might be altering transporters to counter increased exposure to LC fatty acids from the Acanthamoeba by increased oxidative stress from LC fatty acid degradation, masking, etc. Furthermore, we found upregulation of an efflux RND transporter downstream of the deletion (PFLU_4976) and a unique enrichment (5 genes) in the cyclophilin-like domain (immunophilin) in Mnt-L2. Among all DEGs, 72 upregulated and 66 downregulated genes were shared between the Vol-L1 and Mnt-L2 conditions.

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Protozoan predation drives rapid adaptive divergence by selecting for tuned cellulose production and amyloid fibrils in Pseudomonas fluorescens SBW25

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