Strain Diversity and Evolution in Endosymbionts of Bathymodiolus Mussels
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Strain diversity and evolution in endosymbionts of Bathymodiolus mussels Dissertation Zur Erlangung des Grades eines Doktors der Naturwissenschaften - Dr. rer. nat. - dem Fachbereich Biologie/Chemie der Universität Bremen vorgelegt von Rebecca Ansorge Bremen Februar 2019 Die Untersuchungen zur vorliegenden Doktorarbeit wurden in der Abteilung Symbiose am Max-Planck-Institut für Marine Mikrobiologie unter der Leitung von Prof. Dr. Nicole Dubilier durchgeführt. Gutachter 1. Prof. Dr. Nicole Dubilier 2. Prof. Dr. Matthias Horn Prüfer 2. Dr. Bernhard Fuchs 3. Prof. Dr. Marko Rohlfs Tag des Promotionskolloquiums 26. März 2019 To Life. “It is not the mountain we conquer but ourselves” - Edmund Hillary Contents Summary 3 Zusammenfassung 5 Chapter 1 | Introduction 7 1.1 Symbiosis 7 1.2 Chemosynthetic symbiosis 9 1.2.1 Diversity of chemosynthetic environments 11 1.2.2 Diversity of chemosynthetic symbioses 17 1.3 Symbiont transmission 19 1.3.1 Impact of transmission mode on symbiont genome evolution 21 1.3.2 Impact of transmission mode on symbiont heterogeneity 23 1.4 Bathymodiolin symbiosis 24 1.4.1 Metabolism of SOX and MOX symbionts 27 1.4.2 The SOX symbiont relatives from the SUP05 clade 31 1.4.3 Microdiversity in Bathymodiolus SOX symbionts 35 1.5 Strain diversity and endosymbiosis 36 1.5.1 Diversity in mutualism 36 1.5.2 Intra-specifc diversity of symbionts in nature 39 Aims of this thesis 42 List of publications 47 Chapter 2 | Diversity matters 65 Chapter 3 | Genome structure in the SUP05 clade 139 Chapter 4 | Symbiont evolution 191 Chapter 5 | Preliminary results, concluding remarks and future directions 227 5.1 Microdiversity in Bathymodiolus symbionts 228 5.1.1 How diverse is the MOX symbiont 231 5.1.2 What makes B. brooksi unusual? 234 0 Contents 5.2 Functional heterogeneity in regulatory mechanisms 240 5.3 Mediators of genomic plasticity 243 5.3.1 Potential functions of CRISPR-Cas in the SOX symbionts 244 5.4 Why be diverse? 247 5.4.1 The evolution of ‘being ready’ 252 5.5 Future directions 254 5.5.1 Sharing the compartment 254 5.5.2 From genotype to phenotype 256 5.5.3 Catch me if you can 257 5.6 Concluding remarks 258 Acknowledgements 267 Contribution to manuscripts 271 Eidesstattliche Erklärung 273 “The important thing is not to stop questioning. Curiosity has its own reason for existence. One cannot help but be in awe when he contemplates the mysteries of eternity, of life, of the marvelous structure of reality. It is enough if one tries merely to comprehend a little of this mystery each day.” —"Old Man's Advice to Youth: 'Never Lose a Holy Curiosity.'" LIFE Magazine (2 May 1955) p. 64”, Albert Einstein 1 2 Summary Summary Bacterial populations in the environment are often complex and characterized by high genetic diversity. The factors that determine microbial community composition, spatial organization, co-existence and genome evolution are still not well understood. Accumulating evidence shows that even closely related strains of the same species can difer strongly in their functions. However, it is often unknown whether these closely related lineages can co-exist. Especially in intimate symbioses between bacteria and animal hosts, the impact of strain-level diversity is largely unexplored. Evolutionary theory predicts that mutualistic symbioses, based on reciprocal exchange of costly goods, should be destabilized by genetic diversity among closely-related symbiont strains. Yet, to date it is unknown if this prediction holds true in environmental symbioses. This is because the majority of bacteria has not been cultured under standard laboratory conditions, and due to the methodological challenges of teasing apart highly similar genomes from a complex bacterial community. In this thesis I therefore aimed to deepen our current understanding of mutualistic symbioses, and the potential role that symbiont strain diversity and evolution may play in it. To do so, I preformed high- resolution genomic analyses of the endosymbionts of Bathymodiolus mussels. These mussels dominate hydrothermal vents and cold seeps in the deep sea, and form a chemosynthetic symbiosis with gammaproteobacterial sulfur-oxidizing (SOX) or methane-oxidizing (MOX) symbionts, or both. The symbionts reside intracellularly in gill epithelial cells of the host and are horizontally transmitted between host generations. The Bathymodiolus symbiosis is an ideal system to study intra-specifc symbiont diversity, because of the small number of symbiont phylotypes that colonize each host individual. Their low community diversity allowed the detection of strain diversity within the symbiont populations, yet, standard binning approaches currently cannot resolve highly similar strain genomes from metagenomes. I therefore developed a custom workfow to resolve strain-level diversity in natural symbiont populations from high-resolution metagenomic sequencing data. My analyses revealed an extensive nucleotide diversity in the SOX symbionts, showing up to 11 single-nucleotide polymorphisms (SNPs) per kilobase pair (kbp; chapter II). Using this polymorphism data, up to 16 strains could be detected within single host individuals. To assess whether these strains also difered in their functional repertoires, I used read coverage information to identify regions of low coverage, representing diferences in gene content among symbiont strains. Surprisingly, this analysis unveiled an extensive genomic plasticity among co-existing strains, afecting the energy metabolism, phage defense, lipopolysaccharide synthesis, phosphate uptake and regulation. Most of these functions were transcribed, indicating that the genotypic diversity also afected the symbiont phenotypes. The study included diferent Bathymodiolus species from a range of geographic locations, which made us conclude that high strain diversity of the SOX endosymbiont is pervasive among Bathymodiolus mussels. The SOX symbionts are members of the SUP05 clade, a widespread bacterial group that impacts marine geochemical cycles of sulfur and nitrogen. Lineages of this clade have been reported to occur both as free-living cells, and as symbionts of mussels, clams and sponges. Whether the diferent lifestyles are associated with specifc sets of genes and whether Bathymodiolus symbionts thus share unique genetic features distinct from the other lifestyles and lineages in the SUP05 clade remained unclear. An extensive comparative genomic analysis (chapter III) revealed that the link between lifestyle and gene content similarity was weak, indicating that convergent evolution has led to diferent genetic solutions with the same outcome (e.g. the colonization of Bathymodiolus mussels). Further, our analyses revealed that the SUP05 clade displays extensive gene content variation and metabolic plasticity on all phylogenetic levels, from genus to single strains, possibly driven by horizontal gene transfer. This suggests that evolvability, referring to the potential of a population to evolve adaptive solutions to unknown future conditions, might be a trait that is selected for in the SUP05 clade, contributing to the global success of this bacterial group. 3 Summary The impact of natural selection on symbiont genome evolution is strongly afected by the type of association and transmission mode. In vertically transmitted symbionts the efective population size (Ne) is small, which diminishes selection pressure and results in symbiont genome evolution predominantly driven by genetic drift. In horizontally transmitted symbionts, such as the SOX symbionts, Ne is often unknown and the impact of genetic drift and natural selection is less obvious. Therefore, I used allele frequencies derived from polymorphism data to detect signatures of selection in the symbiont genomes (chapter IV). In order to gain a comprehensive picture of diversifying selection in the SOX symbiont, this study encompasses 88 metagenomes, spanning 15 chemosynthetic sampling sites from all over the world, and including 9 distinct host species. This analysis allowed the identifcation of genes afected by diversifying selection between symbiont populations, which included core traits, such as genes involved in sulfur oxidation. Our results therefore indicate that natural selection is efective in the SOX symbionts, and that environmental conditions and the interaction with the host may be important drivers in diversifying selection. Analyses of allele frequencies in co-occurring host individuals further revealed a large symbiont population overlap (chapter II and IV), suggesting a continuous or extended re- sampling of symbionts either derived from a free-living population or from symbiont release by other host individuals. Intriguingly, the population overlap and degree of heterogeneity between the host species B. brooksi and B. thermophilus was smaller, possibly resulting from a reduced rate of symbiont exchange compared to the other investigated host species (chapter IV). Factors afecting symbiont exchange between hosts may be the age and density of host individuals. The depth and high-resolution of the sequencing approach in this thesis together with the extensive sampling efort allowed me to uncover previously hidden strain diversity in the SOX symbionts of Bathymodiolus. My fndings challenge and extent current evolutionary theories and point out the value of in-depth analysis of environmental bacterial communities to deepen our understanding of evolution, microbial interaction and symbiosis. 4 Zusammenfassung Zusammenfassung Natürliche Bakterienpopulationen sind oft komplex