Joran Martijn

The evolution of obligate host-association of bacterial symbionts and pathogens remains poorly understood. The Rickettsiales represent an order of obligate alphaproteobacterial endosymbionts and parasites that infect a wide variety of eukaryotic hosts, including humans, livestock, insects and protists. Induced by their host-associated lifestyle, Rickettsiales genomes have undergone reductive evolution, leading to small, AT-rich genomes with limited metabolic capacities. We describe several genomes of deep-branching, environmental alphaproteobacteria that branch basal to previously sampled Rickettsiales, and whose genome content are reminiscent of free-living and biofilm-associated lifestyles. Ancestral genome content reconstruction across the Rickettsiales tree revealed that the free-living to host-association transition of this group occurred more recently than previously anticipated, and likely involved the repurposing of a type IV secretion system. One-Sentence Summary Deep-branching Rickettsiales provide insights into the evolution of obligate host-associated lifestyle

Summary: Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost‐effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read‐curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full‐length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high‐throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full‐length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost‐effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.

The bacterial phylum Chlamydiae, which is so far comprised of obligate symbionts of eukaryotic hosts, are well-known as human and animal pathogens ^1-3 . However, the Chlamydiae also include so-called environmental lineages ^4-6 that primarily infect microbial eukaryotes ⁷ . Studying environmental chlamydiae, whose genomes display extended metabolic capabilities compared to their pathogenic relatives ^8-10 has provided first insights into the evolution of the pathogenic and obligate intracellular lifestyle that is characteristic for this phylum. Here, we report an unprecedented relative abundance and diversity of novel lineages of the Chlamydiae phylum, representing previously undetected, yet potentially important, community members in deep marine sediments. We discovered that chlamydial lineages dominate the microbial communities in the Arctic Mid-Ocean Ridge ¹¹ , which revealed the dominance of chlamydial lineages at anoxic depths, reaching relative abundances of up to 43% of the bacterial community, and a maximum diversity of 163 different species-level taxonomic unit. Using genome-resolved metagenomics, we reconstructed 24 draft chlamydial genomes, thereby dramatically expanding known interspecies genomic diversity in this phylum. Phylogenomic and comparative analyses revealed several deep-branching Chlamydiae clades, including a sister clade of the pathogenic Chlamydiaceae. Altogether, our study provides new insights into the diversity, evolution and environmental distribution of the Chlamydiae.

Mitochondria are ATP-generating organelles, the endosymbiotic origin of which was a key event in the evolution of eukaryotic cells 1 . Despite strong phylogenetic evidence that mitochondria had an alphaproteobacterial ancestry 2 , efforts to pinpoint their closest relatives among sampled alphaproteobacteria have generated conflicting results, complicating detailed inferences about the identity and nature of the mitochondrial ancestor. While most studies support the idea that mitochondria evolved from an ancestor related to Rickettsiales3-9, an order that includes several host-associated pathogenic and endosymbiotic lineages10,11, others have suggested that mitochondria evolved from a free-living group12-14. Here we re-evaluate the phylogenetic placement of mitochondria. We used genome-resolved binning of oceanic metagenome datasets and increased the genomic sampling of Alphaproteobacteria with twelve divergent clades, and one clade representing a sister group to all Alphaproteobacteria. Subsequent phylogenomic analyses that specifically address long branch attraction and compositional bias artefacts suggest that mitochondria did not evolve from Rickettsiales or any other currently recognized alphaproteobacterial lineage. Rather, our analyses indicate that mitochondria evolved from a proteobacterial lineage that branched off before the divergence of all sampled alphaproteobacteria. In light of this new result, previous hypotheses on the nature of the mitochondrial ancestor6,15,16 should be re-evaluated.

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions of environmental samples and to discover previously unknown lineages. Its unique structure of interspersed conserved and variable regions is an excellent target for PCR and allows for classification of reads at all taxonomic levels. However, the relatively few phylogenetically informative sites prevent confident phylogenetic placements of novel lineages that are deep branching relative to reference taxa. This problem is exacerbated when only short 16S rRNA gene fragments are sequenced. To resolve their placement, it is common practice to gather more informative sites by combining multiple conserved genes into concatenated datasets. This however requires genomic data which may be obtained through relatively expensive metagenome sequencing and computationally demanding analyses. Here we develop a protocol that amplifies a large part of 16S and 23S rRNA genes within the rRNA operon, including the ITS region, and sequences the amplicons with PacBio long-read technology. We tested our method with a synthetic mock community and developed a read curation pipeline that reduces the overall error rate to 0.18%. Applying our method on four diverse environmental samples, we were able to capture near full-length rRNA operon amplicons from a large diversity of prokaryotes. Phylogenetic trees constructed with these sequences showed an increase in statistical support compared to trees inferred with shorter, Illumina-like sequences using only the 16S rRNA gene (250 bp). Our method is a cost-effective solution to generate high quality, near full-length 16S and 23S rRNA gene sequences from environmental prokaryotes.

Summary: The R ickettsiae comprise intracellular bacterial symbionts and pathogens infecting diverse eukaryotes. Here, we provide a detailed characterization of ‘ C andidatus J idaibacter acanthamoeba’, a rickettsial symbiont of A canthamoeba . The bacterium establishes the infection in its amoeba host within 2 h where it replicates within vacuoles. Higher bacterial loads and accelerated spread of infection at elevated temperatures were observed. The infection had a negative impact on host growth rate, although no increased levels of host cell lysis were seen. Phylogenomic analysis identified this bacterium as member of the M idichloriaceae . Its 2.4 Mb genome represents the largest among R ickettsiales and is characterized by a moderate degree of pseudogenization and a high coding density. We found an unusually large number of genes encoding proteins with eukaryotic‐like domains such as ankyrins, leucine‐rich repeats and tetratricopeptide repeats, which likely function in host interaction. There are a total of three divergent, independently acquired type IV secretion systems, and 35 flagellar genes representing the most complete set found in an obligate intracellular A lphaproteobacterium . The deeply branching phylogenetic position of ‘ C andidatus J idaibacter acanthamoeba’ together with its ancient features place it closely to the rickettsial ancestor and helps to better understand the transition from a free‐living to an intracellular lifestyle.

The origin of the eukaryotic cell remains one of the most contentious puzzles in modern biology. Recent studies have provided support for the emergence of the eukaryotic host cell from within the archaeal domain of life, but the identity and nature of the putative archaeal ancestor remain a subject of debate. Here we describe the discovery of ‘Lokiarchaeota’, a novel candidate archaeal phylum, which forms a monophyletic group with eukaryotes in phylogenomic analyses, and whose genomes encode an expanded repertoire of eukaryotic signature proteins that are suggestive of sophisticated membrane remodelling capabilities. Our results provide strong support for hypotheses in which the eukaryotic host evolved from a bona fide archaeon, and demonstrate that many components that underpin eukaryote-specific features were already present in that ancestor. This provided the host with a rich genomic ‘starter-kit’ to support the increase in the cellular and genomic complexity that is characteristic of eukaryotes.

The bacterial family Rickettsiaceae includes a group of well-known etiological agents of many human and vertebrate diseases, including epidemic typhus-causing pathogen Rickettsia prowazekii. Owing to their medical relevance, rickettsiae have attracted a great deal of attention and their host-pathogen interactions have been thoroughly investigated. All known members display obligate intracellular lifestyles, and the best-studied genera, Rickettsia and Orientia, include species that are hosted by terrestrial arthropods. Their obligate intracellular lifestyle and host adaptation is reflected in the small size of their genomes, a general feature shared with all other families of the Rickettsiales. Yet, despite that the Rickettsiaceae and other Rickettsiales families have been extensively studied for decades, many details of the origin and evolution of their obligate host-association remain elusive. Here we report the discovery and single-cell sequencing of ‘Candidatus Arcanobacter lacustris’, a rare environmental alphaproteobacterium that was sampled from Damariscotta Lake that represents a deeply rooting sister lineage of the Rickettsiaceae. Intriguingly, phylogenomic and comparative analysis of the partial ‘Candidatus Arcanobacter lacustris’ genome revealed the presence chemotaxis genes and vertically inherited flagellar genes, a novelty in sequenced Rickettsiaceae, as well as several host-associated features. This finding suggests that the ancestor of the Rickettsiaceae might have had a facultative intracellular lifestyle. Our study underlines the efficacy of single-cell genomics for studying microbial diversity and evolution in general, and for rare microbial cells in particular.

The Archaea represent the so-called Third Domain of life, which has evolved in parallel with the Bacteria and which is implicated to have played a pivotal role in the emergence of the eukaryotic domain of life. Recent progress in genomic sequencing technologies and cultivation-independent methods has started to unearth a plethora of data of novel, uncultivated archaeal lineages. Here, we review how the availability of such genomic data has revealed several important insights into the diversity, ecological relevance, metabolic capacity, and the origin and evolution of the archaeal domain of life.