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Coevolution between bacteria and phages in soil: ecological and genetic bases of its specificity

Final Report Summary - SOILCOEV (Coevolution between bacteria and phages in soil: ecological and genetic bases of its specificity)

Antagonistic coevolution between hosts and parasites has far-reaching consequences for evolutionary ecology, agriculture and medicine. Coevolution between bacteria and virulent phages is likely to be of particularly broad significance because of the ubiquity of phages, the key role played by bacteria in ecosystem functioning, and the therapeutic use of phages as ‘evolving’ antibiotics, in both clinical and agricultural contexts. Despite the far-reaching consequences of bacteria-phage coevolution in laboratory populations, we know virtually nothing about its operation and significance in natural ecosystems. The aim of this project was to increase our understanding of evolutionary maintenance of host and parasites in a natural environment, and their coevolutionary dynamics within communities in natural environment.

I have performed two complimentary approaches in order to study how bacteria and phages coevolve in a natural environment. First, sterilized soil microcosms were inoculated with the well studied coevolutionary system: Pseudomonas fluorescens SBW25 and their virulent associated phage SBW25φ2, in both the presence and absence of the natural microbial community. Hereby, I carried out a more detailed phenotypic and genetic analyses of the impact of coevolution on the evolution of within-population bacteria and phage diversity, and community diversity (Objective 1). Results show that the SNPs average on the whole bacteria genome for bacterial genotypes coevolving with phages was slightly higher than rest of treatments. However, bacterial genotypes from different treatments are not fixing specific SNPs in soil. Specifically, bacterial genotypes have 3.21 SNPs on average, including mutations in 51 genes across 21 functional categories. The most commonly mutated categories are signalling/biological regulation and catalytic activity (mutated in eight and 10 genotypes). Furthermore, all bacterial genotypes were competed with wild type in the absence and presence of phages and microbial community in order to assess the relative fitness for each genotype under all combined selection pressures, leading to correlate phenotypic with genotypic data. These results show that genetic variation was not constrained by a lack of evolution, as soil environment and presence of phages and microbial community act as strength of selection, suggesting a pleiotropic effect of mutations.

Thereafter, I determined the simultaneous roles of a tightly coevolving phage and interactions with the rest of the natural microbial and viral community in driving the evolution of mutation rates of P. fluorescens SBW25 in soil (Objective 2). After 48 days evolution in soil, I examined the mutation rates of P. fluorescens, and results show that direct evolutionary interactions with viruses and the other members of soil microbial communities, appear to have a relatively minor roles in driving the evolution of bacterial mutators in soil. Moreover, competition experiments between wild type and mutator bacteria in soil revealed that other features of the soil environment could appear to select for relatively low mutation rates. These results may therefore help to explain why bacteria with high mutation rates are found at relatively low frequencies (<2%) in soil, but can be found at much higher frequencies in the lab and in clinical infections.
Then, I determined the extent to which natural selection acts on bacterial cooperation in soil (Objective 3). I examined the occurrence of the cooperative production of siderophore in the soil bacterium P. fluorescens in both acid and basic soils. Unlike in vitro, results suggest that siderophore non-producers (cheaters) were not able to out-compete producers (cooperators) as result of the soil spatial structure. This constraint on the cheater bacteria is likely to be common in natural communities, and to play a key role in structuring natural microbial communities, and therefore, in ecosystem functioning.

Finally, I assessed the impact of some ecologically relevant manipulations, such as soil productivity and mixing on the rate of coevolution (Objective 4). First, the population and evolutionary dynamics of P. fluorescens were followed in the absence and the presence of phage SBW25φ2 under different soil mix regimes. Results showed that in soil phage infectivity appears to be promoted by mixing populations and an unstructured soil resulted in cycles of bacterial resistance and phage infectivity similar to arms race dynamic, rather than fluctuating selection dynamic. The absence of fluctuating selection in a non-spatial structure may have resulted from an increased encounter rate between bacteria and phage populations, which likely reduced the costs of generalism in the short term. Our findings demonstrate that mixing bacteria and phage populations in a structured environment affect their selection dynamics, leading to a switch from fluctuating to directional selection. It is therefore that elimination of the spatial structure in places where hosts and parasites are coevolving could have potential implications shaping natural communities, and highlights the consequences of ecological manipulations on host-parasite antagonistic coevolution. Second, this objective was overlapped with the second approach (Objective 5) in order to investigate the effect of resource availability on bacteriaphage coevolution in soil. Thus, I measured the resistance and infectivity of natural communities of soil bacteria and phage in the presence and absence of nutrient providing plants. And subsequently, I followed real-time coevolution between defined bacteria and phage populations with resource availability manipulated by the addition or not of an artificial plant root exudate. Increased resource availability resulted in increases in bacterial resistance to phages, but without a concomitant increase in phage infectivity or a change in the qualitative coevolutionary dynamics. These results suggest that phages may have a reduced impact on the control of bacterial densities and community composition in stable, high resource environments.
(Please, see attached file)