Evolution in host-parasite interaction between novel cyanophage and filamentous, nitrogen-fixing cyanobacterium

The thesis begins with an introduction to the characteristics of experimental organisms, cyanobacteria and cyanophages, and their role in the marine biogeochemical cycles and food-webs. Subsequently, the methodology of experimental evolution and models of host-parasite dynamics are presented. The aim of the experimental part is to test predictions concerning the effects of host-parasite interactions on the marine nitrogen cycle, food-webs, and host properties. Methods include batch culture growth experiments, liquid chromatography–mass spectrometry, an optical density based phage resistance assay, plaque assay, and microscopy. To the author’s knowledge, this is the first controlled study that demonstrates that viral lysis of a diazotrophic cyanobacterium results in the release of cellular nitrogen to the environment in a form that fuels phytoplankton growth. However, evolution with the phage alters the effect. These observations highlight the importance of host-parasite interactions in biogeochemical cycles and food-webs. Further, a novel phage resistant host genotype with short filaments compared to other sensitive and resistant genotypes was detected, with increased growth ability but decreased buoyancy. Reduced buoyancy is proposed as a novel fitness cost of resistance. Phage-mediated evolution resulted in increased diversity in host filament length, growth ability, and buoyancy, supporting the hypothesis that parasites act as drivers of host diversity

Low antibiotic concentrations and resistance in microbial communities

Antibiotics often occur in different environments at concentrations insufficient to inhibit the growth of susceptible bacteria. Subinhibitory concentrations can, nevertheless, select for chromosomal antibiotic resistance mutations and mobile genetic elements carrying antibiotic resistance genes, such as conjugative plasmids. However, it has been unclear whether this occurs in typical natural habitats where bacteria live in multispecies communities and interact with viral and protozoan consumers. In such settings, similar selection can also occur on other bacterial traits, potentially interfering with antibiotic resistance evolution. In this thesis, the combined effect of low antibiotic concentrations and ecological interactions on antibiotic resistance evolution was investigated. The thesis consists of four studies. In the first and second study, antibiotic and bacteriophage resistance evolution was examined at the phenotypic or whole-genome level when a bacterial population was exposed to one versus both factors. In the third study, the effect of antibiotics and protozoan predation on the spread and maintenance of a conjugative antibiotic resistance plasmid in a bacterial population was determined. The fourth study examined the effect of a low antibiotic concentration, protozoan predation, and spatial layout of habitat on community composition and horizontal transfer of a conjugative plasmid in a multispecies bacterial community. Antibiotic resistance evolution was shown to be promoted by low antibiotic concentrations independent of the presence of ecological interactions. The presence of protozoan predation also promoted the spread and maintenance of a resistance plasmid even in the absence of antibiotics. This demonstrates that ecological factors other than antibiotics can also play a role in the horizontal transfer of antibiotic resistance genes. Moreover, a low antibiotic concentration altered bacterial community composition, diversity, and the strains that received a resistance plasmid, although predation and spatial layout of habitat were equally strong or stronger drivers. Since the community effects of low antibiotic concentrations can be weaker or similar in strength to the effects of other ecological drivers, knowledge of antibiotic alone may be insufficient for predicting changes in the structure or diversity of a community.Antibiootteja esiintyy eri ympäristöissä usein niin alhaisina pitoisuuksina, etteivät ne estä edes niille herkkien bakteerien kasvua. Tällaisetkin pitoisuudet voivat kuitenkin tuottaa valintapainetta, jolloin antibiooteille vastustuskykyä aiheuttavat kromosomaaliset mutaatiot tai liikkuvat geneettiset elementit, kuten konjugatiiviset plasmidit, lisääntyvät bakteeripopulaatiossa. Vielä ei tiedetä, päteekö tämä bakteerien tyypillisissä elinympäristöissä, joissa bakteerit ovat osa monilajisia yhteisöjä ja vuorovaikutuksessa virusten sekä alkueläinsaalistajien kanssa. Tällaisissa olosuhteissa valintapaine voi kohdistua myös muihin bakteerin ominaisuuksiin, mikä voi häiritä antibioottiresistenssievoluutiota. Tässä työssä tutkittiin alhaisten antibioottipitoisuuksien ja ekologisten vuorovaikutusten yhteisvaikutusta antibioottiresistenssievoluutioon. Työ koostuu neljästä osatutkimuksesta. Ensimmäisessä ja toisessa osatutkimuksessa tarkasteltiin antibiootti- ja virusresistenssin evoluutiota bakteerin ilmiasussa tai perimässä altistamalla bakteeri joko antibiootille tai virukselle tai molemmille. Kolmannessa osatutkimuksessa tarkasteltiin antibiootin ja alkueläinsaalistuksen vaikutusta konjugatiivisen antibioottiresistenssiplasmidin leviämiseen ja säilymiseen bakteeripopulaatiossa. Neljännessä osatutkimuksessa tarkasteltiin alhaisen antibioottipitoisuuden, alkueläinsaalistuksen ja avaruudellisen rakenteen vaikutusta yhteisörakenteeseen sekä konjugatiivisen plasmidin leviämiseen monilajisessa bakteeriyhteisössä. Tutkimuksessa havaittiin, että alhaiset antibioottipitoisuudet lisäsivät antibioottiresistenssiä bakteeripopulaatiossa myös ekologisten vuorovaikutusten alaisuudessa. Lisäksi alkueläinsaalistus edisti resistenssiplasmidin leviämistä ja säilymistä bakteeripopulaatiossa antibiootista riippumatta. Tämä osoittaa, että myös muut ekologiset tekijät kuin antibiootit voivat olla merkityksellisiä antibioottiresistenssigeenien liikkuvuuden kannalta. Monilajisessa bakteeriyhteisössä matala antibioottipitoisuus muutti yhteisön rakennetta, monimuotoisuutta ja plasmidin vastaanottavia kantoja. Myös alkueläinsaalistus ja avaruudellinen rakenne muuttivat näitä tekijöitä vähintään yhtä voimakkaasti. Koska matalan antibioottipitoisuuden vaikutus yhteisöön voi olla samaa suuruusluokkaa tai pienempi kuin keskeisten ekologisten tekijöiden, tieto pelkästään antibiootista ei välttämättä riitä ennustamaan muutosta yhteisön koostumuksessa tai monimuotoisuudessa

Understanding cellular growth strategies via optimal control

Evolutionary prediction and control are increasingly interesting research topics that are expanding to new areas of application. Unravelling and anticipating successful adaptations to different selection pressures becomes crucial when steering rapidly evolving cancer or microbial populations towards a chosen target. Here we introduce and apply a rich theoretical framework of optimal control to understand adaptive use of traits, which in turn allows eco-evolutionarily informed population control. Using adaptive metabolism and microbial experimental evolution as a case study, we show how demographic stochasticity alone can lead to lag time evolution, which appears as an emergent property in our model. We further show that the cycle length used in serial transfer experiments has practical importance as it may cause unintentional selection for specific growth strategies and lag times. Finally, we show how frequency-dependent selection can be incorporated to the state-dependent optimal control framework allowing the modelling of complex eco-evolutionary dynamics. Our study demonstrates the utility of optimal control theory in elucidating organismal adaptations and the intrinsic decision making of cellular communities with high adaptive potential.Peer reviewe

Effect of mutation supply on population dynamics and trait evolution in an experimental microbial community

Mutation supply can influence evolutionary and thereby ecological dynamics in important ways which have received little attention. Mutation supply influences features of population genetics, such as the pool of adaptive mutations, evolutionary pathways and importance of processes, such as clonal interference. The resultant trait evolutionary dynamics, in turn, can alter population size and species interactions. However, controlled experiments testing for the importance of mutation supply on rapid adaptation and thereby population and community dynamics have primarily been restricted to the first of these aspects. To close this knowledge gap, we performed a serial passage experiment with wild-type Pseudomonas fluorescens and a mutant with reduced mutation rate. Bacteria were grown at two resource levels in combination with the presence of a ciliate predator. A higher mutation supply enabled faster adaptation to the low-resource environment and anti-predatory defence. This was associated with higher population size at the ecological level and better access to high-recurrence mutational targets at the genomic level with higher mutation supply. In contrast, mutation rate did not affect growth under high-resource level. Our results demonstrate that intrinsic mutation rate influences population dynamics and trait evolution particularly when population size is constrained by extrinsic conditions.Peer reviewe

Repeatable ecological dynamics govern the response of experimental communities to antibiotic pulse perturbation

By exposing an experimental 34-species bacterial community to different levels of pulse antibiotic disturbance with or without immigration, the authors identify a highly repeatable community response, the magnitude of which increases with increasing antibiotic levels. In an era of pervasive anthropogenic ecological disturbances, there is a pressing need to understand the factors that constitute community response and resilience. A detailed understanding of disturbance response needs to go beyond associations and incorporate features of disturbances, species traits, rapid evolution and dispersal. Multispecies microbial communities that experience antibiotic perturbation represent a key system with important medical dimensions. However, previous microbiome studies on this theme have relied on high-throughput sequencing data from uncultured species without the ability to explicitly account for the role of species traits and immigration. Here, we serially passage a 34-species defined bacterial community through different levels of pulse antibiotic disturbance, manipulating the presence or absence of species immigration. To understand the ecological community response measured using amplicon sequencing, we combine initial trait data measured for each species separately and metagenome sequencing data revealing adaptive mutations during the experiment. We found that the ecological community response was highly repeatable within the experimental treatments, which could be attributed in part to key species traits (antibiotic susceptibility and growth rate). Increasing antibiotic levels were also coupled with an increasing probability of species extinction, making species immigration critical for community resilience. Moreover, we detected signals of antibiotic-resistance evolution occurring within species at the same time scale, leaving evolutionary changes in communities despite recovery at the species compositional level. Together, these observations reveal a disturbance response that presents as classic species sorting, but is nevertheless accompanied by rapid within-species evolution.Peer reviewe

Strong selective environments determine evolutionary outcome in time-dependent fitness seascapes

The impact of fitness landscape features on evolutionary outcomes has attracted considerable interest in recent decades. However, evolution often occurs under time-dependent selection in so-called fitness seascapes where the landscape is under flux. Fitness seascapes are an inherent feature of natural environments, where the landscape changes owing both to the intrinsic fitness consequences of previous adaptations and extrinsic changes in selected traits caused by new environments. The complexity of such seascapes may curb the predictability of evolution. However, empirical efforts to test this question using a comprehensive set of regimes are lacking. Here, we employed an in vitro microbial model system to investigate differences in evolutionary outcomes between time-invariant and time-dependent environments, including all possible temporal permutations, with three subinhibitory antimicrobials and a viral parasite (phage) as selective agents. Expectedly, time-invariant environments caused stronger directional selection for resistances compared to time-dependent environments. Intriguingly, however, multidrug resistance outcomes in both cases were largely driven by two strong selective agents (rifampicin and phage) out of four agents in total. These agents either caused cross-resistance or obscured the phenotypic effect of other resistance mutations, modulating the evolutionary outcome overall in time-invariant environments and as a function of exposure epoch in time-dependent environments. This suggests that identifying strong selective agents and their pleiotropic effects is critical for predicting evolution in fitness seascapes, with ramifications for evolutionarily informed strategies to mitigate drug resistance evolution.Peer reviewe

Effects of phenotypic variation on consumer coexistence and prey community structure

A popular idea in ecology is that trait variation among individuals from the same species may promote the coexistence of competing species. However, theoretical and empirical tests of this idea have yielded inconsistent findings. We manipulated intraspecific trait diversity in a ciliate competing with a nematode for bacterial prey in experimental microcosms. We found that intraspecific trait variation inverted the original competitive hierarchy to favour the consumer with variable traits, ultimately resulting in competitive exclusion. This competitive outcome was driven by foraging traits (size, speed and directionality) that increased the ciliate's fitness ratio and niche overlap with the nematode. The interplay between consumer trait variation and competition resulted in non-additive cascading effects-mediated through prey defence traits-on prey community assembly. Our results suggest that predicting consumer competitive population dynamics and the assembly of prey communities will require understanding the complexities of trait variation within consumer species.Peer reviewe

Host range of antibiotic resistance genes in wastewater treatment plant influent and effluent

10.1093/femsec/fiy038Wastewater treatment plants (WWTPs) collect wastewater from various sources for a multi-step treatment process. By mixing a large variety of bacteria and promoting their proximity, WWTPs constitute potential hotspots for the emergence of antibiotic resistant bacteria. Concerns have been expressed regarding the potential of WWTPs to spread antibiotic resistance genes (ARGs) from environmental reservoirs to human pathogens. We utilized epicPCR (Emulsion, Paired Isolation and Concatenation PCR) to detect the bacterial hosts of ARGs in two WWTPs. We identified the host distribution of four resistance-associated genes (tetM, int1, qacE Delta 1 and bla(OXA-58)) in influent and effluent. The bacterial hosts of these resistance genes varied between the WWTP influent and effluent, with a generally decreasing host range in the effluent. Through 16S rRNA gene sequencing, it was determined that the resistance gene carrying bacteria include both abundant and rare taxa. Our results suggest that the studied WWTPs mostly succeed in decreasing the host range of the resistance genes during the treatment process. Still, there were instances where effluent contained resistance genes in bacterial groups not carrying these genes in the influent. By permitting exhaustive profiling of resistance-associated gene hosts in WWTP bacterial communities, the application of epicPCR provides a new level of precision to our resistance gene risk estimates.Peer reviewe

Ecology determines how low antibiotic concentration impacts community composition and horizontal transfer of resistance genes

Peer reviewe