Anti-CRISPR Phages Cooperate to Overcome CRISPR-Cas Immunity.

Some phages encode anti-CRISPR (acr) genes, which antagonize bacterial CRISPR-Cas immune systems by binding components of its machinery, but it is less clear how deployment of these acr genes impacts phage replication and epidemiology. Here, we demonstrate that bacteria with CRISPR-Cas resistance are still partially immune to Acr-encoding phage. As a consequence, Acr-phages often need to cooperate in order to overcome CRISPR resistance, with a first phage blocking the host CRISPR-Cas immune system to allow a second Acr-phage to successfully replicate. This cooperation leads to epidemiological tipping points in which the initial density of Acr-phage tips the balance from phage extinction to a phage epidemic. Furthermore, both higher levels of CRISPR-Cas immunity and weaker Acr activities shift the tipping points toward higher initial phage densities. Collectively, these data help elucidate how interactions between phage-encoded immune suppressors and the CRISPR systems they target shape bacteria-phage population dynamics.

The host phylogeny determines viral infectivity and replication across Staphylococcus host species.

Virus host shifts, where a virus transmits to and infects a novel host species, are a major source of emerging infectious disease. Genetic similarity between eukaryotic host species has been shown to be an important determinant of the outcome of virus host shifts, but it is unclear if this is the case for prokaryotes where anti-virus defences can be transmitted by horizontal gene transfer and evolve rapidly. Here, we measure the susceptibility of 64 strains of Staphylococcaceae bacteria (48 strains of Staphylococcus aureus and 16 non-S. aureus species spanning 2 genera) to the bacteriophage ISP, which is currently under investigation for use in phage therapy. Using three methods-plaque assays, optical density (OD) assays, and quantitative (q)PCR-we find that the host phylogeny explains a large proportion of the variation in susceptibility to ISP across the host panel. These patterns were consistent in models of only S. aureus strains and models with a single representative from each Staphylococcaceae species, suggesting that these phylogenetic effects are conserved both within and among host species. We find positive correlations between susceptibility assessed using OD and qPCR and variable correlations between plaque assays and either OD or qPCR, suggesting that plaque assays alone may be inadequate to assess host range. Furthermore, we demonstrate that the phylogenetic relationships between bacterial hosts can generally be used to predict the susceptibility of bacterial strains to phage infection when the susceptibility of closely related hosts is known, although this approach produced large prediction errors in multiple strains where phylogeny was uninformative. Together, our results demonstrate the ability of bacterial host evolutionary relatedness to explain differences in susceptibility to phage infection, with implications for the development of ISP both as a phage therapy treatment and as an experimental system for the study of virus host shifts.

Fitness effects of plasmids shape the structure of bacteria-plasmid interaction networks.

Antimicrobial resistance (AMR) genes are often carried on broad host range plasmids, and the spread of AMR within microbial communities will therefore depend on the structure of bacteria­plasmid networks. Empirical and theoretical studies of ecological interaction networks suggest that network structure differs between communities that are predominantly mutualistic versus antagonistic, with the former showing more generalized interactions (i.e., species interact with many others to a similar extent). This suggests that mutualistic bacteria­plasmid networks­where antibiotics are present and plasmids carry AMR genes­will be more generalized than antagonistic interactions, where plasmids do not confer benefits to their hosts. We first develop a simple theory to explain this link: fitness benefits of harboring a mutualistic symbiont promote the spread of the symbiont to other species. We find support for this theory using an experimental bacteria­symbiont (plasmid) community, where the same plasmid can be mutualistic or antagonistic depending on the presence of antibiotics. This short-term and parsimonious mechanism complements a longer-term mechanism (coevolution and stability) explaining the link between mutualistic and antagonistic interactions and network structure.

Increased copy number couples the evolution of plasmid horizontal transmission and plasmid-encoded antibiotic resistance.

Conjugative plasmids are mobile elements that spread horizontally between bacterial hosts and often confer adaptive phenotypes, including antimicrobial resistance (AMR). Theory suggests that opportunities for horizontal transmission favor plasmids with higher transfer rates, whereas selection for plasmid carriage favors less-mobile plasmids. However, little is known about the mechanisms leading to variation in transmission rates in natural plasmids or the resultant effects on their bacterial host. We investigated the evolution of AMR plasmids confronted with different immigration rates of susceptible hosts. Plasmid RP4 did not evolve in response to the manipulations, but plasmid R1 rapidly evolved up to 1,000-fold increased transfer rates in the presence of susceptible hosts. Most evolved plasmids also conferred on their hosts the ability to grow at high concentrations of antibiotics. This was because plasmids evolved greater copy numbers as a function of mutations in the copA gene controlling plasmid replication, causing both higher transfer rates and AMR. Reciprocally, plasmids with increased conjugation rates also evolved when selecting for high levels of AMR, despite the absence of susceptible hosts. Such correlated selection between plasmid transfer and AMR could increase the spread of AMR within populations and communities.

Diversity loss from multiple interacting disturbances is regime-dependent.

Anthropogenic activities expose many ecosystems to multiple novel disturbances simultaneously. Despite this, how biodiversity responds to simultaneous disturbances remains unclear, with conflicting empirical results on their interactive effects. Here, we experimentally test how one disturbance (an invasive species) affects the diversity of a community over multiple levels of another disturbance regime (pulse mortality). Specifically, we invade stably coexisting bacterial communities under four different pulse frequencies, and compare their final resident diversity to uninvaded communities under the same pulse mortality regimes. Our experiment shows that the disturbances synergistically interact, such that the invader significantly reduces resident diversity at high pulse frequency, but not at low. This work therefore highlights the need to study simultaneous disturbance effects over multiple disturbance regimes as well as to carefully document unmanipulated disturbances, and may help explain the conflicting results seen in previous multiple-disturbance work.

Testing for the fitness benefits of natural transformation during community-embedded evolution.

Natural transformation is a process where bacteria actively take up DNA from the environment and recombine it into their genome or reconvert it into extra-chromosomal genetic elements. The evolutionary benefits of transformation are still under debate. One main explanation is that foreign allele and gene uptake facilitates natural selection by increasing genetic variation, analogous to meiotic sex. However, previous experimental evolution studies comparing fitness gains of evolved transforming- and isogenic non-transforming strains have yielded mixed support for the 'sex hypothesis.' Previous studies testing the sex hypothesis for natural transformation have largely ignored species interactions, which theory predicts provide conditions favourable to sex. To test for the adaptive benefits of bacterial transformation, the naturally transformable wild-type Acinetobacter baylyi and a transformation-deficient ∆comA mutant were evolved for 5 weeks. To provide strong and potentially fluctuating selection, A. baylyi was embedded in a community of five other bacterial species. DNA from a pool of different Acinetobacter strains was provided as a substrate for transformation. No effect of transformation ability on the fitness of evolved populations was found, with fitness increasing non-significantly in most treatments. Populations showed fitness improvement in their respective environments, with no apparent costs of adaptation to competing species. Despite the absence of fitness effects of transformation, wild-type populations evolved variable transformation frequencies that were slightly greater than their ancestor which potentially could be caused by genetic drift.

Comparative experimental evolution reveals species-specific idiosyncrasies in marine phytoplankton adaptation to warming.

A number of experimental studies have demonstrated that phytoplankton can display rapid thermal adaptation in response to warmed environments. While these studies provide insight into the evolutionary responses of single species, they tend to employ different experimental techniques. Consequently, our ability to compare the potential for thermal adaptation across different, ecologically relevant, species remains limited. Here, we address this limitation by conducting simultaneous long-term warming experiments with the same experimental design on clonal isolates of three phylogenetically diverse species of marine phytoplankton; the cyanobacterium Synechococcus sp., the prasinophyte Ostreococcus tauri and the diatom Phaeodoactylum tricornutum. Over the same experimental time period, we observed differing levels of thermal adaptation in response to stressful supra-optimal temperatures. Synechococcus sp. displayed the greatest improvement in fitness (i.e., growth rate) and thermal tolerance (i.e., temperature limits of growth). Ostreococcus tauri was able to improve fitness and thermal tolerance, but to a lesser extent. Finally, Phaeodoactylum tricornutum showed no signs of adaptation. These findings could help us understand how the structure of phytoplankton communities may change in response to warming, and possible biogeochemical implications, as some species show relatively more rapid adaptive shifts in their thermal tolerance.

Resident microbial communities inhibit growth and antibiotic-resistance evolution of Escherichia coli in human gut microbiome samples.

Countering the rise of antibiotic-resistant pathogens requires improved understanding of how resistance emerges and spreads in individual species, which are often embedded in complex microbial communities such as the human gut microbiome. Interactions with other microorganisms in such communities might suppress growth and resistance evolution of individual species (e.g., via resource competition) but could also potentially accelerate resistance evolution via horizontal transfer of resistance genes. It remains unclear how these different effects balance out, partly because it is difficult to observe them directly. Here, we used a gut microcosm approach to quantify the effect of three human gut microbiome communities on growth and resistance evolution of a focal strain of Escherichia coli. We found the resident microbial communities not only suppressed growth and colonisation by focal E. coli but also prevented it from evolving antibiotic resistance upon exposure to a beta-lactam antibiotic. With samples from all three human donors, our focal E. coli strain only evolved antibiotic resistance in the absence of the resident microbial community, even though we found resistance genes, including a highly effective resistance plasmid, in resident microbial communities. We identified physical constraints on plasmid transfer that can explain why our focal strain failed to acquire some of these beneficial resistance genes, and we found some chromosomal resistance mutations were only beneficial in the absence of the resident microbiota. This suggests, depending on in situ gene transfer dynamics, interactions with resident microbiota can inhibit antibiotic-resistance evolution of individual species.

Whole community invasions and the integration of novel ecosystems.

The impact of invasion by a single non-native species on the function and structure of ecological communities can be significant, and the effects can become more drastic-and harder to predict-when multiple species invade as a group. Here we modify a dynamic Boolean model of plant-pollinator community assembly to consider the invasion of native communities by multiple invasive species that are selected either randomly or such that the invaders constitute a stable community. We show that, compared to random invasion, whole community invasion leads to final stable communities (where the initial process of species turnover has given way to a static or near-static set of species in the community) including both native and non-native species that are larger, more likely to retain native species, and which experience smaller changes to the topological measures of nestedness and connectance. We consider the relationship between the prevalence of mutualistic interactions among native and invasive species in the final stable communities and demonstrate that mutualistic interactions may act as a buffer against significant disruptions to the native community.

A social life for discerning microbes.

Microbes are not only extremely social but also extremely discerning about whom they socialize with. Recent research has uncovered some of the evolutionary explanations behind these feats of social sophistication in bacteria (Ackermann et al., 2008; Diggle et al., 2007) and, most recently, has provided insights into the molecular mechanisms of discrimination in yeast (Smukalla et al., 2008).

Pulse and Press Disturbances Have Different Effects on Transient Community Dynamics.

AbstractDisturbances are important determinants of diversity, and the combination of their aspects (e.g., disturbance intensity, frequency) can result in complex diversity patterns. Here, we leverage an important approach to classifying disturbances in terms of temporal span to understand the implications for species coexistence: pulse disturbances are acute and discrete events, while press disturbances occur continuously through time. We incorporate the resultant mortality rates into a common framework involving disturbance frequency and intensity. Press disturbances can be encoded into models in two distinct ways, and we show that the appropriateness of each depends on the type of data available. Using this framework, we compare the effects of pulse versus press disturbance on both asymptotic and transient dynamics of a two-species Lotka-Volterra competition model to understand how they engage with equalizing mechanisms of coexistence. We show that press and pulse disturbances differ in transient behavior, though their asymptotic diversity patterns are similar. Our work shows that these differences depend on how the underlying disturbance aspects interact and that the two ways of characterizing press disturbances can lead to contrasting interpretations of disturbance-diversity relationships. Our work demonstrates how theoretical modeling can strategically guide and help the interpretation of empirical work.

Copper selects for siderophore-mediated virulence in Pseudomonas aeruginosa.

BACKGROUND: Iron is essential for almost all bacterial pathogens and consequently it is actively withheld by their hosts. However, the production of extracellular siderophores enables iron sequestration by pathogens, increasing their virulence. Another function of siderophores is extracellular detoxification of non-ferrous metals. Here, we experimentally link the detoxification and virulence roles of siderophores by testing whether the opportunistic pathogen Pseudomonas aeruginosa displays greater virulence after exposure to copper. To do this, we incubated P. aeruginosa under different environmentally relevant copper regimes for either two or twelve days. Subsequent growth in a copper-free environment removed phenotypic effects, before we quantified pyoverdine production (the primary siderophore produced by P. aeruginosa), and virulence using the Galleria mellonella infection model. RESULTS: Copper selected for increased pyoverdine production, which was positively correlated with virulence. This effect increased with time, such that populations incubated with high copper for twelve days were the most virulent. Replication of the experiment with a non-pyoverdine producing strain of P. aeruginosa demonstrated that pyoverdine production was largely responsible for the change in virulence. CONCLUSIONS: We here show a direct link between metal stress and bacterial virulence, highlighting another dimension of the detrimental effects of metal pollution on human health.

Rapid decline of adaptation of Pseudomonas fluorescens to soil biotic environment.

Interactions between microbes can both constrain and enhance their adaptation to the environment. However, most studies to date have employed simplified microbial communities and environmental conditions. We determined how the presence of a commercial potting compost microbial community affected adaptation of the soil bacterium Pseudomonas fluorescens SBW25 in potting compost. Pseudomonas fluorescens clones isolated from populations evolved in both the presence and absence of the community showed similar fitness increases when measured in the absence of the community. This suggests the presence of the community did not constrain adaptation. By contrast, fitness measured in the presence of the community increased for community-evolved populations, but decreased below the ancestral state for populations evolved in the absence of the community. This suggests some, but not all, mutations that were beneficial with respect to the abiotic environment were costly in the presence of the community, with the former selected against in the presence of the community. Whole-genome sequencing supports this interpretation: most mutations underpinning fitness changes were clone-specific, suggesting multiple genetic pathways to adaptation. Such extreme mutational effects have not been observed in comparable in vitro studies, suggesting that caution is needed when extrapolating results from simplified in vitro systems to natural contexts.

The diversity-generating benefits of a prokaryotic adaptive immune system.

Prokaryotic CRISPR-Cas adaptive immune systems insert spacers derived from viruses and other parasitic DNA elements into CRISPR loci to provide sequence-specific immunity. This frequently results in high within-population spacer diversity, but it is unclear if and why this is important. Here we show that, as a result of this spacer diversity, viruses can no longer evolve to overcome CRISPR-Cas by point mutation, which results in rapid virus extinction. This effect arises from synergy between spacer diversity and the high specificity of infection, which greatly increases overall population resistance. We propose that the resulting short-lived nature of CRISPR-dependent bacteria-virus coevolution has provided strong selection for the evolution of sophisticated virus-encoded anti-CRISPR mechanisms.

How disturbance history alters invasion success: biotic legacies and regime change.

Disturbance is a key factor shaping ecological communities, but little is understood about how the effects of disturbance processes accumulate over time. When disturbance regimes change, historical processes may influence future community structure, for example, by altering invasibility compared to communities with stable regimes. Here, we use an annual plant model to investigate how the history of disturbance alters invasion success. In particular, we show how two communities can have different outcomes from species introduction, solely due to past differences in disturbance regimes that generated different biotic legacies. We demonstrate that historical differences can enhance or suppress the persistence of introduced species, and that biotic legacies generated by stable disturbance history decay over time, though legacies can persist for unexpectedly long durations. This establishes a formal theoretical foundation for disturbance legacies having profound effects on communities, and highlights the value of further research on the biotic legacies of disturbance.

Stress causes interspecific facilitation within a compost community.

Ecological theory predicts interactions between species to become more positive under abiotic stress, while competition should prevail in more benign environments. However, experimental tests of this stress gradient hypothesis in natural microbial communities are lacking. We test this hypothesis by measuring interactions between 10 different members of a bacterial community inhabiting potting compost in the presence or absence of toxic copper stress. We found that copper stress caused significant net changes in species interaction signs, shifting the net balance towards more positive interactions. This pattern was at least in part driven by copper-sensitive isolates - that produced relatively small amounts of metal-detoxifying siderophores - benefitting from the presence of other species that produce extracellular detoxifying agents. As well as providing support for the stress gradient hypothesis, our results highlight the importance of community-wide public goods in shaping microbial community composition.

Compost spatial heterogeneity promotes evolutionary diversification of a bacterium.

Spatial resource heterogeneity is expected to be a key driver for the evolution of diversity. However, direct empirical support for this prediction is limited to studies carried out in simplified laboratory environments. Here, we investigate how altering spatial heterogeneity of potting compost-by the addition of water and mixing-affects the evolutionary diversification of a bacterial species, Pseudomonas fluorescens, that is naturally found in the environment. There was a greater propensity of resource specialists to evolve in the unmanipulated compost, while more generalist phenotypes dominated the compost-water mix. Genomic data were consistent with these phenotypic findings. Competition experiments strongly suggest these results are due to diversifying selection as a result of resource heterogeneity, as opposed to other covariables. Overall, our findings corroborate theoretical and in vitro findings, but in semi-natural, more realistic conditions.

Experimental (co)evolution in a multi-species microbial community results in local maladaptation.

Interspecific coevolutionary interactions can result in rapid biotic adaptation, but most studies have focused only on species pairs. Here, we (co)evolved five microbial species in replicate polycultures and monocultures and quantified local adaptation. Specifically, growth rate assays were used to determine adaptations of each species' populations to (1) the presence of the other four species in general and (2) sympatric vs. allopatric communities. We found that species did not show an increase in net biotic adaptation:ancestral, polyculture- and monoculture-evolved populations did not have significantly different growth rates within communities. However, 4/5 species' growth rates were significantly lower within the community they evolved in relative to an allopatric community. 'Local maladaptation' suggests that species evolved increased competitive interactions to sympatric species' populations. This increased competition did not affect community stability or productivity. Our results suggest that (co)evolution within communities can increase competitive interactions that are specific to (co)evolved community members.

Evolutionary temperature compensation of carbon fixation in marine phytoplankton.

The efficiency of carbon sequestration by the biological pump could decline in the coming decades because respiration tends to increase more with temperature than photosynthesis. Despite these differences in the short-term temperature sensitivities of photosynthesis and respiration, it remains unknown whether the long-term impacts of global warming on metabolic rates of phytoplankton can be modulated by evolutionary adaptation. We found that respiration was consistently more temperature dependent than photosynthesis across 18 diverse marine phytoplankton, resulting in universal declines in the rate of carbon fixation with short-term increases in temperature. Long-term experimental evolution under high temperature reversed the short-term stimulation of metabolic rates, resulting in increased rates of carbon fixation. Our findings suggest that thermal adaptation may therefore have an ameliorating impact on the efficiency of phytoplankton as primary mediators of the biological carbon pump.

Determining the prevalence, identity and possible origin of bacterial pathogens in soil.

Soil biomes are vast, exceptionally diverse and crucial to the health of ecosystems and societies. Soils also contain an appreciable, but understudied, diversity of opportunistic human pathogens. With climate change and other forms of environmental degradation potentially increasing exposure risks to soilborne pathogens, it is necessary to gain a better understanding of their ecological drivers. Here we use the Galleria mellonella insect virulence model to selectively isolate pathogenic bacteria from soils in Cornwall (UK). We find a high prevalence of pathogenic soil bacteria with two genera, Providencia and Serratia, being especially common. Providencia alcalifaciens, P. rustigianii, Serratia liquefaciens and S. plymuthica strains were studied in more detail using phenotypic virulence and antibiotic resistance assays and whole-genome sequencing. Both genera displayed low levels of antibiotic resistance and antibiotic resistance gene carriage. However, Serratia isolates were found to carry the recently characterized metallo-ß-lactamase blaSPR-1 that, although not conferring high levels of resistance in these strains, poses a potential risk of horizontal transfer to other pathogens where it could be fully functional. The Galleria assay can be a useful approach to uncover the distribution and identity of pathogenic bacteria in the environment, as well as uncover resistance genes with an environmental origin.