Andrew J. Spiers

Antagonistic coevolution accelerates molecular evolution

The Red Queen hypothesis proposes that coevolution of interacting species (such as hosts and parasites) should drive molecular evolution through continual natural selection for adaptation and counter-adaptation. Although the divergence observed at some host-resistance and parasite-infectivity genes is consistent with this, the long time periods typically required to study coevolution have so far prevented any direct empirical test. Here we show, using experimental populations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage Φ2 (refs 10, 11), that the rate of molecular evolution in the phage was far higher when both bacterium and phage coevolved with each other than when phage evolved against a constant host genotype. Coevolution also resulted in far greater genetic divergence between replicate populations, which was correlated with the range of hosts that coevolved phage were able to infect. Consistent with this, the most rapidly evolving phage genes under coevolution were those involved in host infection. These results demonstrate, at both the genomic and phenotypic level, that antagonistic coevolution is a cause of rapid and divergent evolution, and is likely to be a major driver of evolutionary change within species.

Biofilm formation at the air–liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose

The wrinkly spreader (WS) genotype of Pseudomonas fluorescens SBW25 colonizes the air–liquid interface of spatially structured microcosms resulting in formation of a thick biofilm. Its ability to colonize this niche is largely due to overproduction of a cellulosic polymer, the product of the wss operon. Chemical analysis of the biofilm matrix shows that the cellulosic polymer is partially acetylated cellulose, which is consistent with predictions of gene function based on in silico analysis of wss. Both polar and non‐polar mutations in the sixth gene of the wss operon (wssF ) or adjacent downstream genes (wssGHIJ ) generated mutants that overproduce non‐acetylated cellulose, thus implicating WssFGHIJ in acetylation of cellulose. WssGHI are homologues of AlgFIJ from P. aeruginosa, which together are necessary and sufficient to acetylate alginate polymer. WssF belongs to a newly established Pfam family and is predicted to provide acyl groups to WssGHI. The role of WssJ is unclear, but its similarity to MinD‐like proteins suggests a role in polar localization of the acetylation complex. Fluorescent microscopy of Calcofluor‐stained biofilms revealed a matrix structure composed of networks of cellulose fibres, sheets and clumped material. Quantitative analyses of biofilm structure showed that acetylation of cellulose is important for effective colonization of the air–liquid interface: mutants identical to WS, but defective in enzymes required for acetylation produced biofilms with altered physical properties. In addition, mutants producing non‐acetylated cellulose were unable to spread rapidly across solid surfaces. Inclusion in these assays of a WS mutant with a defect in the GGDEF regulator (WspR) confirmed the requirement for this protein in expression of both acetylated cellulose polymer and bacterial attachment. These results suggest a model in which WspR regulation of cellulose expression and attachment plays a role in the co‐ordination of surface colonization.

The causes of Pseudomonas diversity

The genus Pseudomonas encompasses arguably the most diverse and ecologically significant group of bacteria on the planet. Members of the genus are found in large numbers in all of the major natural environments (terrestrial, freshwater and marine) and also form intimate associations with plants and animals. This universal distribution suggests a remarkable degree of physiological and genetic adaptability.

Adaptive divergence in experimental populations of Pseudomonas fluorescens. III. Mutational origins of wrinkly spreader diversity

Understanding the connections among genotype, phenotype, and fitness through evolutionary time is a central goal of evolutionary genetics. Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations and show substantial morphological and fitness differences. Previous work identified genes contributing to the evolutionary success of WS, in particular the di-guanylate cyclase response regulator, WspR. Here we scrutinize the Wsp signal transduction pathway of which WspR is the primary output component. The pathway has the hallmarks of a chemosensory pathway and genetic analyses show that regulation and function of Wsp is analogous to the Che chemotaxis pathway from Escherichia coli. Of significance is the methyltransferase (WspC) and methylesterase (WspF) whose opposing activities form an integral feedback loop that controls the activity of the kinase (WspE). Deductions based on the regulatory model suggested that mutations within wspF were a likely cause of WS. Analyses of independent WS genotypes revealed numerous simple mutations in this single open reading frame. Remarkably, different mutations have different phenotypic and fitness effects. We suggest that the negative feedback loop inherent in Wsp regulation allows the pathway to be tuned by mutation in a rheostat-like manner.

Genes encoding a cellulosic polymer contribute toward the ecological success of Pseudomonas fluorescens SBW25 on plant surfaces

Pseudomonas fluorescens SBW25 is a Gram‐negative bacterium that grows in close association with plants. In common with a broad range of functionally similar bacteria it plays an important role in the turnover of organic matter and certain isolates can promote plant growth. Despite its environmental significance, the causes of its ecological success are poorly understood. Here we describe the development and application of a simple promoter trapping strategy (IVET) to identify P. fluorescens SBW25 genes showing elevated levels of expression in the sugar beet rhizosphere. A total of 25 rhizosphere‐induced (rhi) fusions are reported with predicted roles in nutrient acquisition, stress responses, biosynthesis of phytohormones and antibiotics. One rhi fusion is to wss, an operon encoding an acetylated cellulose polymer. A mutant carrying a defective wss locus was competitively compromised (relative to the wild type) in the rhizosphere and in the phyllosphere, but not in bulk soil. The rhizosphere‐induced wss locus therefore contributes to the ecological performance of SBW25 in the plant environment and supports our conjecture that genes inactive in the laboratory environment, but active in the wild, are likely to be determinants of fitness in natural environments.

Microbial Distribution in Soils: Physics and Scaling

In a handful of fertile soil there are billions of microorganisms and yet, even with a conservative estimate, the surface area covered by these organisms is considerably less than 1%. What does this tell us about the function of the physical structure in which soil organisms reside and function, collecting, and separating micropopulations from each other and from resources? It would seem that most of the soil is akin to desert regions with little life been supported on its terrains, yet with vast communities of individuals, from an amazing array of species, supported in small-scale habitats, connected or disconnected by saturated or unsaturated pore space over relatively short time-scales. The biodiversity of these communities remains impressive yet overall functionally illusive, bar some considerations of inbuilt redundancy. What is far more impressive is the range of habitats on offer to populations with short-term evolutionary time frames. The availability of spatially and temporally diverse habitats probably gives rise to the biodiversity that we seeAbstract In a handful of fertile soil there are billions of microorganisms and yet, even with a conservative estimate, the surface area covered by these organisms is considerably less than 1%. What does this tell us about the function of the physical structure in which soil organisms reside and function, collecting, and separating micropopulations from each other and from resources? It would seem that most of the soil is akin to desert regions with little life been supported on its terrains, yet with vast communities of individuals, from an amazing array of species, supported in small-scale habitats, connected or disconnected by saturated or unsaturated pore space over relatively short time-scales. The biodiversity of these communities remains impressive yet overall functionally illusive, bar some considerations of inbuilt redundancy. What is far more impressive is the range of habitats on offer to populations with short-term evolutionary time frames. The availability of spatially and temporally diverse habitats probably gives rise to the biodiversity that we see in soil. It is not too far fetched to state that the majority of habitats on Earth (and indeed extraterrestrial) are revealed in that handful of soil. The key question is what is the functional consequence of such habitat heterogeneity? To answer this it is clear that we need to bring together a new discipline that combines the biology and physics of the soil ecosystem. This biophysical approach, combined, where required, with important mineral-microbe knowledge is needed to help us understand the mechanisms by which soils remain productive, and to identify the tipping-points at which there may be no return to sustainability. This review aims to highlight the importance of addressing the soil ecosystem as a dynamic heterogeneous system focusing on microbiota–habitat interactions.

Chapter 4 Microbial Distribution in Soils: Physics and Scaling

In a handful of fertile soil there are billions of microorganisms and yet, even with a conservative estimate, the surface area covered by these organisms is considerably less than 1%. What does this tell us about the function of the physical structure in which soil organisms reside and function, collecting, and separating micropopulations from each other and from resources? It would seem that most of the soil is akin to desert regions with little life been supported on its terrains, yet with vast communities of individuals, from an amazing array of species, supported in small-scale habitats, connected or disconnected by saturated or unsaturated pore space over relatively short time-scales. The biodiversity of these communities remains impressive yet overall functionally illusive, bar some considerations of inbuilt redundancy. What is far more impressive is the range of habitats on offer to populations with short-term evolutionary time frames. The availability of spatially and temporally diverse habitats probably gives rise to the biodiversity that we seeAbstract In a handful of fertile soil there are billions of microorganisms and yet, even with a conservative estimate, the surface area covered by these organisms is considerably less than 1%. What does this tell us about the function of the physical structure in which soil organisms reside and function, collecting, and separating micropopulations from each other and from resources? It would seem that most of the soil is akin to desert regions with little life been supported on its terrains, yet with vast communities of individuals, from an amazing array of species, supported in small-scale habitats, connected or disconnected by saturated or unsaturated pore space over relatively short time-scales. The biodiversity of these communities remains impressive yet overall functionally illusive, bar some considerations of inbuilt redundancy. What is far more impressive is the range of habitats on offer to populations with short-term evolutionary time frames. The availability of spatially and temporally diverse habitats probably gives rise to the biodiversity that we see in soil. It is not too far fetched to state that the majority of habitats on Earth (and indeed extraterrestrial) are revealed in that handful of soil. The key question is what is the functional consequence of such habitat heterogeneity? To answer this it is clear that we need to bring together a new discipline that combines the biology and physics of the soil ecosystem. This biophysical approach, combined, where required, with important mineral-microbe knowledge is needed to help us understand the mechanisms by which soils remain productive, and to identify the tipping-points at which there may be no return to sustainability. This review aims to highlight the importance of addressing the soil ecosystem as a dynamic heterogeneous system focusing on microbiota–habitat interactions.

Transparent Soil for Imaging the Rhizosphere

Understanding of soil processes is essential for addressing the global issues of food security, disease transmission and climate change. However, techniques for observing soil biology are lacking. We present a heterogeneous, porous, transparent substrate for in situ 3D imaging of living plants and root-associated microorganisms using particles of the transparent polymer, Nafion, and a solution with matching optical properties. Minerals and fluorescent dyes were adsorbed onto the Nafion particles for nutrient supply and imaging of pore size and geometry. Plant growth in transparent soil was similar to that in soil. We imaged colonization of lettuce roots by the human bacterial pathogen Escherichia coli O157:H7 showing micro-colony development. Micro-colonies may contribute to bacterial survival in soil. Transparent soil has applications in root biology, crop genetics and soil microbiology.

Adaptive Divergence in Experimental Populations of Pseudomonas fluorescens. II. Role of the GGDEF Regulator WspR in Evolution and Development of the Wrinkly Spreader Phenotype

Wrinkly spreader (WS) genotypes evolve repeatedly in model Pseudomonas populations undergoing adaptive radiation. Previous work identified genes contributing to the evolutionary success of WS. Here we scrutinize the GGDEF response regulator protein WspR and show that it is both necessary and sufficient for WS. Activation of WspR occurs by phosphorylation and different levels of activation generate phenotypic differences among WS genotypes. Five alleles of wspR, each encoding a protein with a single amino acid substitution, were generated by mutagenesis. Two alleles are constitutively active and cause the ancestral genotype to develop a WS phenotype; the phenotypic effects are allele specific and independent of phosphorylation. Three alleles contain changes in the GGDEF domain and when overexpressed in WS cause reversion to the ancestral phenotype. Ability to mimic this effect by overexpression of a liberated N-terminal domain shows that in WS, regulatory components upstream of WspR are overactive. To connect changes at the nucleotide level with fitness, the effects of variant alleles were examined in both structured and unstructured environments: alleles had adaptive and deleterious effects with trade-offs evident across environments. Despite the proclivity of mutations within wspR to generate WS, sequence analysis of wspR from 53 independently obtained WS showed no evidence of sequence change in this gene.

Characterization of a novel air-liquid interface biofilm of Pseudomonas fluorescens SBW25

Pseudomonads are able to form a variety of biofilms that colonize the air-liquid (A-L) interface of static liquid microcosms, and differ in matrix composition, strength, resilience and degrees of attachment to the microcosm walls. From Pseudomonas fluorescens SBW25, mutants have evolved during prolonged adaptation-evolution experiments which produce robust biofilms of the physically cohesive class at the A-L interface, and which have been well characterized. In this study we describe a novel A-L interface biofilm produced by SBW25 that is categorized as a viscous mass (VM)-class biofilm. Several metals were found to induce this biofilm in static Kings B microcosms, including copper, iron, lead and manganese, and we have used iron to allow further examination of this structure. Iron was demonstrated to induce SBW25 to express cellulose, which provided the matrix of the biofilm, a weak structure that was readily destroyed by physical disturbance. This was confirmed in situ by a low (0.023-0.047 g) maximum deformation mass and relatively poor attachment as measured by crystal violet staining. Biofilm strength increased with increasing iron concentration, in contrast to attachment levels, which decreased with increasing iron. Furthermore, iron added to mature biofilms significantly increased strength, suggesting that iron also promotes interactions between cellulose fibres that increase matrix interconnectivity. Whilst weak attachment is important in maintaining the biofilm at the A-L interface, surface-interaction effects involving cellulose, which reduced surface tension by approximately 3.8 mN m(-1), may also contribute towards this localization. The fragility and viscoelastic nature of the biofilm were confirmed by controlled-stress amplitude sweep tests to characterize critical rheological parameters, which included a shear modulus of 0.75 Pa, a zero shear viscosity of 0.24 Pa s(-1) and a flow point of 0.028 Pa. Growth and morphological data thus far support a non-specific metal-associated physiological, rather than mutational, origin for production of the SBW25 VM biofilm, which is an example of the versatility of bacteria to inhabit optimal niches within their environment.