Haloarchaea are extremophiles, generally thriving at high temperatures and salt concentrations, thus, with limited access to oxygen. As a strategy to maintain a respiratory metabolism, many halophilic archaea are capable of denitrification. Among them are members of the genus Haloferax, which are abundant in saline/hypersaline environments. Based on the haloarchaeal genomes analysed, the genes involved in denitrification are grouped into three gene clusters (nar, nir-nor, nos) coding for denitrification enzymes NarGHI, NirK, qNor and NosZ. In case of incomplete denitrifiers, some of the genes or clusters are absent. Amon all haloarchaea analysed, three reported denitrifiers, H. mediterranei, H. denitrificans and H. volcanii were characterized with respect to their denitrification phenotype using a semi-automatic incubation system. Out of the species tested, only H. mediterranei was able to consistently reduce all available N-oxyanions to N2, while the other two released significant amounts of NO and N2 O, which affect tropospheric and stratospheric chemistries respectively. Also, H. mediterranei showed a well-orchestrated system of gene expression during denitrification, being Nar and Nos, both transcriptionally activated by hypoxia (and probably nitrate), while Nir and Nor expression require the presence of nitric oxide (and possibly nitrite) as well as Nos. The prevalence and magnitude of hypersaline ecosystems are on the rise due to climate change and anthropogenic activity. Thus, the biology of halophilic denitrifiers is inherently interesting, due to their contribution to the global nitrogen cycle, and potential application in bioremediation.

Our understanding of microbial evolution is largely dependent on available genomic data of diverse organisms. Yet, genome-sequencing efforts have mostly ignored the diverse uncultivable majority in favor of cultivable and sociologically relevant organisms. In this thesis, I have applied and developed cultivation independent methods to explore microbial diversity and obtain genomic data in an unbiased manner. The obtained genomes were then used to study the evolution of mitochondria, Rickettsiales and Haloarchaea. Metagenomic binning of oceanic samples recovered draft genomes for thirteen novel Alphaproteobacteria-related lineages. Phylogenomics analyses utilizing the improved taxon sample suggested that mitochondria are not related to Rickettsiales but rather evolved from a proteobacterial lineage closely related to all sampled alphaproteobacteria. Single-cell genomics and metagenomics of lake and oceanic samples, respectively, identified previously unobserved Rickettsiales-related lineages. They branched early relative to characterized Rickettsiales and encoded flagellar genes, a feature once thought absent in this order. Flagella are most likely an ancestral feature, and were independently lost during Rickettsiales diversification. In addition, preliminary analyses suggest that ATP/ADP translocase, the marker for energy parasitism, was acquired after the acquisition of type IV secretion systems during the emergence of the Rickettsiales. Further exploration of the oceanic samples yielded the first draft genomes of Marine Group IV archaea, the closest known relatives of the Haloarchaea. The halophilic and generally aerobic Haloarchaea are thought to have evolved from an anaerobic methanogenic ancestor. The MG-IV genomes allowed us to study this enigmatic evolutionary transition. Preliminary ancestral reconstruction analyses suggest a gradual loss of methanogenesis and adaptation to an aerobic lifestyle, respectively. The thesis further presents a new amplicon sequencing method that captures near full-length 16S and 23S rRNA genes of environmental prokaryotes. The method exploits PacBio's long read technology and the frequent proximity of these genes in prokaryotic genomes. Compared to traditional partial 16S amplicon sequencing, our method classifies environmental lineages that are distantly related to reference taxa more confidently. In conclusion, this thesis provides new insights into the origins of mitochondria, Rickettsiales and Haloarchaea and illustrates the power of cultivation independent methods with respect to the study of microbial evolution.