Multispecies coinfections and presence of antibiotics shape resistance and fitness costs in a pathogenic bacterium.

Increasing antimicrobial resistance (AMR) poses a challenge for treatment of bacterial diseases. In real life, bacterial infections are typically embedded within complex multispecies communities and influenced by the environment, which can shape costs and benefits of AMR. However, knowledge of such interactions and their implications for AMR in vivo is limited. To address this knowledge gap, we investigated fitness-related traits of a pathogenic bacterium (Flavobacterium columnare) in its fish host, capturing the effects of bacterial antibiotic resistance, coinfections between bacterial strains and metazoan parasites (fluke Diplostomum pseudospathaceum) and antibiotic exposure. We quantified real-time replication and virulence of sensitive and resistant bacteria and demonstrate that both bacteria can benefit from coinfection in terms of persistence and replication, depending on the coinfecting partner and antibiotic presence. We also show that antibiotics can benefit resistant bacteria by increasing bacterial replication under coinfection with flukes. These results emphasize the importance of diverse, inter-kingdom coinfection interactions and antibiotic exposure in shaping costs and benefits of AMR, supporting their role as significant contributors to spread and long-term persistence of resistance.

The effect of a temperature-sensitive prophage on the evolution of virulence in an opportunistic bacterial pathogen.

Viruses are key actors of ecosystems and have major impacts on global biogeochemical cycles. Prophages deserve particular attention as they are ubiquitous in bacterial genomes and can enter a lytic cycle when triggered by environmental conditions. We explored how temperature affects the interactions between prophages and other biological levels using an opportunistic pathogen, the bacterium Serratia marcescens, which harbours several prophages and that had undergone an evolution experiment under several temperature regimes. We found that the release of one of the prophages was temperature-sensitive and malleable to evolutionary changes. We further discovered that the virulence of the bacterium in an insect model also evolved and was positively correlated with phage release rates. We determined through analysis of genetic and epigenetic data that changes in the bacterial outer cell wall structure possibly explain this phenomenon. We hypothezise that the temperature-dependent phage release rate acted as a selection pressure on S. marcescens and that it resulted in modified bacterial virulence in the insect host. Our study system illustrates how viruses can mediate the influence of abiotic environmental changes to other biological levels and thus be involved in ecosystem feedback loops.

Roles of adenine methylation and genetic mutations in adaptation to different temperatures in Serratia marcescens.

Epigenetic modifications can contribute to adaptation, but the relative contributions of genetic and epigenetic variation are unknown. Previous studies on the role of epigenetic changes in adaptation in eukaryotes have nearly exclusively focused on cytosine methylation (m5C), while prokaryotes exhibit a richer system of methyltransferases targetting adenines (m6A) or cytosines (m4C, m5C). DNA methylation in prokaryotes has many roles, but its potential role in adaptation still needs further investigation. We collected phenotypic, genetic, and epigenetic data using single molecule real-time sequencing of clones of the bacterium Serratia marcescens that had undergone experimental evolution in contrasting temperatures to investigate the relationship between environment and genetic, epigenetic, and phenotypic changes. The genomic distribution of GATC motifs, which were the main target for m6A methylation, and of variable m6A epiloci pointed to a potential link between m6A methylation and regulation of gene expression in S. marcescens. Evolved strains, while genetically homogeneous, exhibited many polymorphic m6A epiloci. There was no strong support for a genetic control of methylation changes in our experiment, and no clear evidence of parallel environmentally induced or environmentally selected methylation changes at specific epiloci was found. Both genetic and epigenetic variants were associated with some phenotypic traits. Overall, our results suggest that both genetic and adenine methylation changes have the potential to contribute to phenotypic adaptation in S. marcescens, but that any environmentally induced epigenetic change occurring in our experiment would probably have been quite labile.

A New Method to Reconstruct Quantitative Food Webs and Nutrient Flows from Isotope Tracer Addition Experiments.

Understanding how nutrients flow through food webs is central in ecosystem ecology. Tracer addition experiments are powerful tools to reconstruct nutrient flows by adding an isotopically enriched element into an ecosystem and tracking its fate through time. Historically, the design and analysis of tracer studies have varied widely, ranging from descriptive studies to modeling approaches of varying complexity. Increasingly, isotope tracer data are being used to compare ecosystems and analyze experimental manipulations. Currently, a formal statistical framework for analyzing such experiments is lacking, making it impossible to calculate the estimation errors associated with the model fit, the interdependence of compartments, and the uncertainty in the diet of consumers. In this article we develop a method based on Bayesian hidden Markov models and apply it to the analysis of N15-NH4+ tracer additions in two Trinidadian streams in which light was experimentally manipulated. Through this case study, we illustrate how to estimate N fluxes between ecosystem compartments, turnover rates of N within those compartments, and the associated uncertainty. We also show how the method can be used to compare alternative models of food web structure, calculate the error around derived parameters, and make statistical comparisons between sites or treatments.

Spatio-temporal dynamics of density-dependent dispersal during a population colonisation.

Predicting population colonisations requires understanding how spatio-temporal changes in density affect dispersal. Density can inform on fitness prospects, acting as a cue for either habitat quality, or competition over resources. However, when escaping competition, high local density should only increase emigration if lower-density patches are available elsewhere. Few empirical studies on dispersal have considered the effects of density at the local and landscape scale simultaneously. To explore this, we analyze 5 years of individual-based data from an experimental introduction of wild guppies Poecilia reticulata. Natal dispersal showed a decrease in local density dependence as density at the landscape level increased. Landscape density did not affect dispersal among adults, but local density-dependent dispersal switched from negative (conspecific attraction) to positive (conspecific avoidance), as the colonisation progressed. This study demonstrates that densities at various scales interact to determine dispersal, and suggests that dispersal trade-offs differ across life stages.

Broad thermal tolerance is negatively correlated with virulence in an opportunistic bacterial pathogen.

Predicting the effects of global increase in temperatures on disease virulence is challenging, especially for environmental opportunistic bacteria, because pathogen fitness may be differentially affected by temperature within and outside host environment. So far, there is very little empirical evidence on the connections between optimal temperature range and virulence in environmentally growing pathogens. Here, we explored whether the virulence of an environmentally growing opportunistic fish pathogen, Flavobacterium columnare, is malleable to evolutionary changes via correlated selection on thermal tolerance. To this end, we experimentally quantified the thermal performance curves (TPCs) for maximum biomass yield of 49 F. columnare isolates from eight different geographic locations in Finland over ten years (2003-2012). We also characterized virulence profiles of these strains in a zebra fish (Danio rerio) infection model. We show that virulence among the strains increased over the years, but thermal generalism, and in particular tolerance to higher temperatures, was negatively associated with virulence. Our data suggest that temperature has a strong effect on the pathogen genetic diversity and therefore presumably also on disease dynamics. However, the observed increase in frequency and severity of F. columnare epidemics over the last decade cannot be directly linked to bacterial evolution due to increased mean temperature, but is most likely associated with factors related to increased length of growing season, or other time-dependent change in environment. Our study demonstrates that complex interactions between the host, the pathogen and the environment influence disease virulence of an environmentally growing opportunistic pathogen.

Telomere length and antioxidant defense associate with parasite-induced retarded growth in wild brown trout.

Early growth conditions can have profound impacts on individuals' development, growth and physiology, with subsequent long-term consequences for individuals' fitness and life expectancy. Telomere length (TL) has been suggested to indicate both individual fitness and life expectancy in wide range of species, as the telomere attrition rate at early age can be accelerated due to exposure to various stressors, including parasites and inflammatory diseases, which increase production of reactive oxygen species (ROS) and influence antioxidant (AO) levels. We investigated impacts of Tetracapsuloides bryosalmonae infection, a causative agent of proliferative kidney disease (PKD), on AO status and TL in a natural population of juvenile brown trout (Salmo trutta). The fish with higher parasite load showed more severe kidney hyperplasia, anemia and smaller body size compared to less parasitized fish. Furthermore, fish with severe PKD symptoms had lower SOD-, CAT- and GST activity than fish with milder kidney hyperplasia. However, parasite load was not directly correlated either with AOs or with TL. Smaller fish showed shorter TLs, potentially reflecting lower individual quality. The fish, which were less sensitive to parasite-induced impaired growth, quantified as parasite load-adjusted fork length, showed also longer TLs, lower GR- and GST activity and less GSHtot compared to more sensitive fish. These results provide novel knowledge about the impacts of the PKD in brown trout at the molecular level and support the idea that TL may reflect individual quality and ability to cope with parasitic infections.

Prediction of harmful variants on mitochondrial genes: Test of habitat-dependent and demographic effects in a euryhaline fish.

Both effective population size and life history may influence the efficacy of purifying selection, but it remains unclear if the environment affects the accumulation of weakly deleterious nonsynonymous polymorphisms. We hypothesize that the reduced energetic cost of osmoregulation in brackish water habitat may cause relaxation of selective constraints at mitochondrial oxidative phosphorylation (OXPHOS) genes. To test this hypothesis, we analyzed 57 complete mitochondrial genomes of Pungitius pungitius collected from brackish and freshwater habitats. Based on inter- and intraspecific comparisons, we estimated that 84% and 68% of the nonsynonymous polymorphisms in the freshwater and brackish water populations, respectively, are weakly or moderately deleterious. Using in silico prediction tools (MutPred, SNAP2), we subsequently identified nonsynonymous polymorphisms with potentially harmful effect. Both prediction methods indicated that the functional effects of the fixed nonsynonymous substitutions between nine- and three-spined stickleback were weaker than for polymorphisms within species, indicating that harmful nonsynonymous polymorphisms within populations rarely become fixed between species. No significant differences in mean estimated functional effects were identified between freshwater and brackish water nine-spined stickleback to support the hypothesis that reduced osmoregulatory energy demand in the brackish water environment reduces the strength of purifying selection at OXPHOS genes. Instead, elevated frequency of nonsynonymous polymorphisms in the freshwater environment (Pn/Ps = 0.549 vs. 0.283; Fisher's exact test p = .032) suggested that purifying selection is less efficient in small freshwater populations. This study shows the utility of in silico functional prediction tools in population genetic and evolutionary research in a nonmammalian vertebrate and demonstrates that mitochondrial energy production genes represent a promising system to characterize the demographic, life history and potential habitat-dependent effects of segregating amino acid variants.

Application of high resolution melting assay (HRM) to study temperature-dependent intraspecific competition in a pathogenic bacterium.

Studies on species' responses to climate change have focused largely on the direct effect of abiotic factors and in particular temperature, neglecting the effects of biotic interactions in determining the outcome of climate change projections. Many microbes rely on strong interference competition; hence the fitness of many pathogenic bacteria could be a function of both their growth properties and intraspecific competition. However, due to technical challenges in distinguishing and tracking individual strains, experimental evidence on intraspecific competition has been limited so far. Here, we developed a robust application of the high-resolution melting (HRM) assay to study head-to-head competition between mixed genotype co-cultures of a waterborne bacterial pathogen of fish, Flavobacterium columnare, at two different temperatures. We found that competition outcome in liquid cultures seemed to be well predicted by growth yield of isolated strains, but was mostly inconsistent with interference competition results measured in inhibition tests on solid agar, especially as no growth inhibition between strain pairs was detected at the higher temperature. These results suggest that, for a given temperature, the factors driving competition outcome differ between liquid and solid environments.

Differences in the metabolic response to temperature acclimation in nine-spined stickleback (Pungitius pungitius) populations from contrasting thermal environments.

Metabolic responses to temperature changes are crucial for maintaining the energy balance of an individual under seasonal temperature fluctuations. To understand how such responses differ in recently isolated populations (<11,000 years), we studied four Baltic populations of the nine-spined stickleback (Pungitius pungitius L.) from coastal locations (seasonal temperature range, 0-29°C) and from colder, more thermally stable spring-fed ponds (1-19°C). Salinity and predation pressure also differed between these locations. We acclimatized wild-caught fish to 6, 11, and 19°C in common garden conditions for 4-6 months and determined their aerobic scope and hepatosomatic index (HSI). The freshwater fish from the colder (2-14°C), predator-free pond population exhibited complete temperature compensation for their aerobic scope, whereas the coastal populations underwent metabolic rate reduction during the cold treatment. Coastal populations had higher HSI than the colder pond population at all temperatures, with cold acclimation accentuating this effect. The metabolic rates and HSI for freshwater fish from the pond with higher predation pressure were more similar to those of the coastal ones. Our results suggest that ontogenic effects and/or genetic differentiation are responsible for differential energy storage and metabolic responses between these populations. This work demonstrates the metabolic versatility of the nine-spined stickleback and the pertinence of an energetic framework to better understand potential local adaptations. It also demonstrates that instead of using a single acclimation temperature thermal reaction norms should be compared when studying individuals originating from different thermal environments in a common garden setting.

Gene pleiotropy constrains gene expression changes in fish adapted to different thermal conditions.

Understanding the factors that shape the evolution of gene expression is a central goal in biology, but the molecular mechanisms behind this remain controversial. A related major goal is ascertaining how such factors may affect the adaptive potential of a species or population. Here we demonstrate that temperature-driven gene expression changes in fish adapted to differing thermal environments are constrained by the level of gene pleiotropy estimated by either the number of protein interactions or gene biological processes. Genes with low pleiotropy levels were the main drivers of both plastic and evolutionary global expression profile changes, while highly pleiotropic genes had limited expression response to temperature treatment. Our study provides critical insights into the molecular mechanisms by which natural populations can adapt to changing environments. In addition to having important implications for climate change adaptation, these results suggest that gene pleiotropy should be considered more carefully when interpreting expression profiling data.

Detection and characterisation of mutations responsible for allele-specific protein thermostabilities at the Mn-superoxide dismutase gene in the deep-sea hydrothermal vent polychaete Alvinella pompejana.

Alvinella pompejana (Polychaeta, Alvinellidae) is one of the most thermotolerant marine eukaryotes known to date. It inhabits chimney walls of deep-sea hydrothermal vents along the East Pacific Rise (EPR) and is exposed to various challenging conditions (e.g. high temperature, hypoxia and the presence of sulphides, heavy metals and radiations), which increase the production of dangerous reactive oxygen species (ROS). Two different allelic forms of a manganese-superoxide dismutase involved in ROS detoxification, ApMnSOD1 and ApMnSOD2, and differing only by two substitutions (M110L and A138G) were identified in an A. pompejana cDNA library. RFLP screening of 60 individuals from different localities along the EPR showed that ApMnSOD2 was rare (2 %) and only found in the heterozygous state. Dynamic light scattering measurements and residual enzymatic activity experiments showed that the most frequent form (ApMnSOD1) was the most resistant to temperature. Their half-lives were similarly long at 65 °C (>110 min) but exhibited a twofold difference at 80 °C (20.8 vs 9.8 min). Those properties are likely to be explained by the occurrence of an additional sulphur-containing hydrogen bond involving the M110 residue and the effect of the A138 residue on the backbone entropy. Our results confirm the thermophily of A. pompejana and suggest that this locus is a good model to study how the extreme thermal heterogeneity of the vent conditions may help to maintain old rare variants in those populations.

Molecular evolutionary and population genomic analysis of the nine-spined stickleback using a modified restriction-site-associated DNA tag approach.

In recent years, the explosion of affordable next generation sequencing technology has provided an unprecedented opportunity to conduct genome-wide studies of adaptive evolution in organisms previously lacking extensive genomic resources. Here, we characterize genome-wide patterns of variability and differentiation using pooled DNA from eight populations of the nine-spined stickleback (Pungitius pungitius L.) from marine, lake and pond environments. We developed a novel genome complexity reduction protocol, defined as paired-end double restriction-site-associated DNA (PE dRAD), to maximize read coverage at sequenced locations. This allowed us to identify over 114,000 short consensus sequences and 15,000 SNPs throughout the genome. A total of 6834 SNPs mapped to a single position on the related three-spined stickleback genome, allowing the detection of genomic regions affected by divergent and balancing selection, both between species and between freshwater and marine populations of the nine-spined stickleback. Gene ontology analysis revealed 15 genomic regions with elevated diversity, enriched for genes involved in functions including immunity, chemical stimulus response, lipid metabolism and signalling pathways. Comparisons of marine and freshwater populations identified nine regions with elevated differentiation related to kidney development, immunity and MAP kinase pathways. In addition, our analysis revealed that a large proportion of the identified SNPs mapping to LG XII is likely to represent alternative alleles from divergent X and Y chromosomes, rather than true autosomal markers following Mendelian segregation. Our work demonstrates how population-wide sequencing and combining inter- and intra-specific RAD analysis can uncover genome-wide patterns of differentiation and adaptations in a non-model species.

Biochemical and biophysical characterization of the Mg2+-induced 90-kDa heat shock protein oligomers.

The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg(2+)-induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.

Structural characterization of hemoglobins from Monilifera and Frenulata tubeworms (Siboglinids): first discovery of giant hexagonal-bilayer hemoglobin in the former "Pogonophora" group.

Siboglinids are symbiotic polychete annelids having hemoglobins as essential oxygen- and sulfide-carriers for their endosymbiotic bacteria. We analyzed the structure of the hemoglobins from two species of siboglinids: the monilifera Sclerolinum contortum and the frenulata Oligobrachia webbi (i.e. haakonmosbiensis) from Norwegian cold seeps. Measured by Multi-Angle Laser Light Scattering (MALLS), Sclerolinum shows a 3190+/-50 kDa hexagonal bilayer hemoglobin (HBL-Hb) and a 461+/-46 kDa ring-Hb, just as vestimentifera, whereas Oligobrachia has a 409+/-3.7 kDa ring-Hb only. Electrospray Ionization-Mass Spectrometry (ESI-MS) showed Sclerolinum HBL-Hb composed of seven monomeric globins (15-16 kDa), three disulfide-bonded globin heterodimers and three linkers. The heterodimers always contain globin-b (15814.4+/-1.5 Da). Sclerolinum ring-Hb is composed of globins and dimers with identical masses as its HBL-Hb, but lacks linkers. Oligobrachia ring-Hb has three globin monomers (14-15 kDa) only, with no disulfide-bonded dimers. Comparison of Sclerolinum hemoglobins between Storegga and Haakon Mosby Mud Volcano, using the normalized height of deconvoluted ESI-MS peaks, shows differences in globin monomers abundances that could reflect genetic differences or differential gene expression between distinct seep populations. The discovery of HBL-Hb in Sclerolinum is a new element supporting the hypothesis of monilifera being phylogenetically more closely related to vestimentifera, than to frenulata.

Structural study of Carcinus maenas hemocyanin by native ESI-MS: interaction with L-lactate and divalent cations.

The interaction of L-lactate and divalent cations with Carcinus maenas hemocyanin has been probed by electrospray ionization mass spectrometry under conditions preserving noncovalent interactions (native ESI-MS). C. maenas native hemocyanin in the hemolymph occurs mainly as dodecamers and to a lesser extent as hexamers. A progressive acidification with formic acid after alkaline dissociation resulted in the preferential recruitment of the two lightest subunits into light dodecamers, a molecular complex absent from native hemolymph, in addition to regular dodecamers and hexamers. Addition of L-lactic acid also induced the recruitment of these subunits, even at alkaline pH. A dodecamer-specific subunit is needed to enable aggregation over the hexameric state. Experiments with EDTA suggested the existence of different binding sites and association constants for divalent cations within hexameric structures and at the interface between two hexamers. L-lactic acid specific interaction with the lightest subunits was not inhibited by removal of the divalent cations.

The structural analysis of large noncovalent oxygen binding proteins by MALLS and ESI-MS: a review on annelid hexagonal bilayer hemoglobin and crustacean hemocyanin.

Understanding the function of macromolecular complexes is related to a precise knowledge of their structure. These large complexes are often fragile high molecular mass noncovalent multimeric proteins. Classical biochemical methods for determination of their native mass and subunit composition were used to resolve their quaternary structure, sometimes leading to different models. Recently, the development of mass spectrometry and multi-angle laser light scattering (MALLS) has enabled absolute determination of native masses and subunit masses. Electrospray ionization mass spectrometry (ESI-MS) was used in denaturing and native conditions to probe subunit composition and noncovalent assemblies masses up to 2.25 MDa. In a complementary way, MALLS provides mass and size estimation in various aqueous solvents. ESI-MS method can also give insights into post-translational modifications (glycosylation, disulfide bridges ). By combining native mass and subunit composition data, structural models can be proposed for large edifices such as annelid extracellular hexagonal bilayer hemoglobins (HBL Hb) and crustacean hemocyanins (Hc). Association/dissociation mechanisms, protein-protein interactions, structural diversity among species and environmental adaptations can also be addressed with these methods. With their absolute mass determination, the very high precision of spectrometry and the versatile nature of light scattering, ESI-MS and MALLS have provided a wealth of data helping to resolve parts of controversies for HBL-Hb models and opening access to new fields of investigation in structural diversity and molecular adaptation. In this review we will focus on annelid HBL-Hb and on crustacean Hc and on the original contributions of ESI-MS and MALLS in this field.