Reconciling Asgardarchaeota Phylogenetic Proximity to Eukaryotes and Planctomycetes Cellular Features in the Evolution of Life.

The relationship between the three domains of life-Archaea, Bacteria, and Eukarya-is one of Biology's greatest mysteries. Current favored models imply two ancestral domains, Bacteria and Archaea, with eukaryotes originating within Archaea. This type of models has been supported by the recent description of the Asgardarchaeota, the closest prokaryotic relatives of eukaryotes. However, there are many problems associated with any scenarios implying that eukaryotes originated from within the Archaea, including genome mosaicism, phylogenies, the cellular organization of the Archaea, and their ancestral character. By contrast, all models of eukaryogenesis fail to consider two relevant discoveries: the detection of membrane coat proteins, and of phagocytosis-related processes in Planctomycetes, which are among the bacteria with the most developed endomembrane system. Consideration of these often overlooked features and others found in Planctomycetes and related bacteria suggest an evolutionary model based on a single ancestral domain. In this model, the proximity of Asgard and eukaryotes is not rejected but instead, Asgard are considered as diverging away from a common ancestor instead of on the way toward the eukaryotic ancestor. This model based on a single ancestral domain solves most of the ambiguities associated with the ones based on two ancestral domains. The single-domain model is better suited to explain the origin and evolution of all three domains of life, blurring the distinctions between them. Support for this model as well as the opportunities that it presents not only for reinterpreting previous results, but also for planning future experiments, are explored.

Thalassoglobus polymorphus sp. nov., a novel Planctomycete isolated close to a public beach of Mallorca Island.

Access to axenic cultures is crucial to extend the knowledge of the biology, lifestyle or metabolic capabilities of bacteria from different phyla. The phylum Planctomycetes is an excellent example since its members display an unusual cell biology and complex lifestyles. As a contribution to the current collection of axenic planctomycete cultures, here we describe strain Mal48T isolated from phytoplankton material sampled at the coast of S'Arenal close to Palma de Mallorca (Spain). The isolated strain shows optimal growth at pH 7.0-7.5 and 30 °C and exhibits typical features of Planctomycetes. Cells of the strain are spherical to pear-shaped, divide by polar budding with daughter cells showing the same shape as the mother cell, tend to aggregate, display a stalk and produce matrix or fimbriae. Strain Mal48T showed 95.8% 16S rRNA gene sequence similarity with the recently described Thalassoglobus neptunius KOR42T. The genome sequence of the novel isolate has a size of 6,357,355 bp with a G+C content of 50.3%. A total of 4874 protein-coding genes, 41 tRNA genes and 2 copies of the 16S rRNA gene are encoded in the genome. Based on phylogenetic, morphological and physiological analyses, we conclude that strain Mal48T (= DSM 100737T = LMG 29019T) should be classified as the type strain of a new species in the genus Thalassoglobus, for which the name Thalassoglobus polymorphus sp. nov. is proposed.

Maioricimonas rarisocia gen. nov., sp. nov., a novel planctomycete isolated from marine sediments close to Mallorca Island.

Planctomycetes are ubiquitous bacteria with environmental and biotechnological relevance. Axenic cultures of planctomycetal strains are the basis to analyse their unusual biology and largely uncharacterised metabolism in more detail. Here, we describe strain Mal4T isolated from marine sediments close to Palma de Mallorca, Spain. Strain Mal4T displays common planctomycetal features, such as division by polar budding and the presence of fimbriae and crateriform structures on the cell surface. Cell growth was observed at ranges of 10-39 °C (optimum at 31 °C) and pH 6.5-9.0 (optimum at 7.5). The novel strain shows as pear-shaped cells of 2.0 ± 0.2 × 1.4 ± 0.1 µm and is one of the rare examples of orange colony-forming Planctomycetes. Its genome has a size of 7.7 Mb with a G+C content of 63.4%. Phylogenetically, we conclude that strain Mal4T (= DSM 100296T = LMG 29133T) is the type strain representing the type species of a novel genus, for which we propose the name Maioricimonas rarisocia gen. nov., sp. nov.

Assembly of a complete genome sequence for Gemmata obscuriglobus reveals a novel prokaryotic rRNA operon gene architecture.

Gemmata obscuriglobus is a Gram-negative bacterium with several intriguing biological features. Here, we present a complete, de novo whole genome assembly for G. obscuriglobus which consists of a single, circular 9 Mb chromosome, with no plasmids detected. The genome was annotated using the NCBI Prokaryotic Genome Annotation pipeline to generate common gene annotations. Analysis of the rRNA genes revealed three interesting features for a bacterium. First, linked G. obscuriglobus rrn operons have a unique gene order, 23S-5S-16S, compared to typical prokaryotic rrn operons (16S-23S-5S). Second, G. obscuriglobus rrn operons can either be linked or unlinked (a 16S gene is in a separate genomic location from a 23S and 5S gene pair). Third, all of the 23S genes (5 in total) have unique polymorphisms. Genome analysis of a different Gemmata species (SH-PL17), revealed a similar 23S-5S-16S gene order in all of its linked rrn operons and the presence of an unlinked operon. Together, our findings show that unique and rare features in Gemmata rrn operons among prokaryotes provide a means to better define the evolutionary relatedness of Gemmata species and the divergence time for different Gemmata species. Additionally, these rrn operon differences provide important insights into the rrn operon architecture of common ancestors of the planctomycetes.

Genomic analysis reveals the potential for hydrocarbon degradation of Rhodopirellula sp. MGV isolated from a polluted Brazilian mangrove.

Planctomycetes are bacteria found in several environments, such as mangroves. In the coastline of the State of Sao Paulo (Brazilian Southeast), mangroves occur in different stages of environmental contamination, promoted by the proximity to the city and industrial activities. One of these mangroves (located in the city of Bertioga) is characterized by the high impact due to past petroleum and ongoing urban contamination. We isolated five bacteria affiliated to Planctomycetes from this mangrove and further subjected them to phenotypical and genetic analysis. The tolerance for salinity was demonstrated by the cultivation under distinct concentrations of NaCl. The ability of this bacterium to use diverse carbon sources was revealed by the use of 30 C-sources from a total of 31 tests. We found the isolate Rhodopirellula sp. MGV very closely affiliated to species of the genus Rhodopirellula, harboring a genome with 7.16 Mbp and 55.3% of GC. The annotation of the 77 contigs resulted in 6.284 CDS, with a remarkable occurrence of sequences associated with aromatic carbon metabolism. In conclusion, we present the isolation and characterization of a Planctomycetes from mangroves, suggesting its participation in the degradation of hydrocarbons present in the contaminated mangroves studied.

Marine Sediments Illuminate Chlamydiae Diversity and Evolution.

The bacterial phylum Chlamydiae is so far composed of obligate symbionts of eukaryotic hosts. Well known for Chlamydiaceae, pathogens of humans and other animals, Chlamydiae also include so-called environmental lineages that primarily infect microbial eukaryotes. Environmental surveys indicate that Chlamydiae are found in a wider range of environments than anticipated previously. However, the vast majority of this chlamydial diversity has been underexplored, biasing our current understanding of their biology, ecological importance, and evolution. Here, we report that previously undetected and active chlamydial lineages dominate microbial communities in deep anoxic marine sediments taken from the Arctic Mid-Ocean Ridge. Reaching relative abundances of up to 43% of the bacterial community, and a maximum diversity of 163 different species-level taxonomic units, these Chlamydiae represent important community members. Using genome-resolved metagenomics, we reconstructed 24 draft chlamydial genomes, expanding by over a third the known genomic diversity in this phylum. Phylogenomic analyses revealed several novel clades across the phylum, including a previously unknown sister lineage of the Chlamydiaceae, providing new insights into the origin of pathogenicity in this family. We were unable to identify putative eukaryotic hosts for these marine sediment chlamydiae, despite identifying genomic features that may be indicative of host-association. The high abundance and genomic diversity of Chlamydiae in these anoxic marine sediments indicate that some members could play an important, and thus far overlooked, ecological role in such environments and may indicate alternate lifestyle strategies.

Global Biogeographic Distribution Patterns of Thermoacidophilic Verrucomicrobia Methanotrophs Suggest Allopatric Evolution.

Thermoacidophilic methane-oxidizing Verrucomicrobia of the candidate genus Methylacidiphilum represent a bacterial taxon adapted to highly acidic (pH 1-4) and moderate temperature (∼65°C) methane-containing geothermal environments. Their apparent ubiquity in acidic terrestrial volcanic areas makes them ideal model organisms to study prokaryotic biogeography. Three Methylacidiphilum species isolated from distantly-separated geothermal regions in Russia, New Zealand, and Italy were previously described. We have explored the intra-taxon phylogenetic patterns of these organisms based on comparative genome analyses and phenotypic comparisons with six new Verrucomicrobia methanotroph isolates from other globally-separated acidic geothermal locations. Comparison of rRNA and particulate methane monooxygenase (pmoCAB) operon sequences indicates a close phylogenetic relationship among the new isolates as well as with the previously characterized strains. All share similar cell morphology including the presence of extensive intracellular inclusion bodies and lack of intracellular membrane systems, which are typical for proteobacterial methanotrophs. However, genome sequence comparisons and concatenated MLST-based phylogenetic analyses separate the new isolates into three distinct species-level groups. Three recently processed isolates from the Azores (each from geographically-separate hot springs within the region) and a single isolate from Iceland are highly similar, sharing more than 88% in silico genome homology with each other as well as with the previous isolate, Methylacidiphilum fumariolicum strain SolV, from Italy. These appear to constitute a distinct European/Atlantic clade. However, two of the new isolates - one from the Yellowstone National Park (United States) and another from The Philippines - constitute separate and novel Methylacidiphilum species. There is no clear correlation between fatty acid profiles and geographic distance between origins, or any phylogenetic relationship. Serological analysis using antiserum raised against M. kamchatkense strain Kam1 revealed large differences in the degree of cross-reactivity with no correlation with other factors. However, the genetic distance between the strains does correlate to the distance between their geographic origins and suggests a global biogeographic pattern shaped by an isolation-by-distance mechanism. These results further confirm terrestrial geothermal springs as isolated islands featuring allopatric prokaryotic speciation.

Ancient, highly conserved proteins from a LUCA with complex cell biology provide evidence in support of the nuclear compartment commonality (NuCom) hypothesis.

The nuclear compartment commonality (NuCom) hypothesis posits a complex last common ancestor (LUCA) with membranous compartments including a nuclear membrane. Such a LUCA then evolved to produce two nucleated lineages of the tree of life: the Planctomycetes-Verrucomicrobia-Chlamydia superphylum (PVC) within the Bacteria, and the Eukarya. We propose that a group of ancient essential protokaryotic signature proteins (PSPs) originating in LUCA were incorporated into ancestors of PVC Bacteria and Eukarya. Tubulins, ubiquitin system enzymes and sterol-synthesizing enzymes are consistent with early origins of these features shared between the PVC superphylum and Eukarya.

Evolutionary Cell Biology of Division Mode in the Bacterial Planctomycetes-Verrucomicrobia- Chlamydiae Superphylum.

Bacteria from the Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC) superphylum are exceptions to the otherwise dominant mode of division by binary fission, which is based on the interaction between the FtsZ protein and the peptidoglycan (PG) biosynthesis machinery. Some PVC bacteria are deprived of the FtsZ protein and were also thought to lack PG. How these bacteria divide is still one of the major mysteries of microbiology. The presence of PG has recently been revealed in Planctomycetes and Chlamydiae, and proteins related to PG synthesis have been shown to be implicated in the division process in Chlamydiae, providing important insights into PVC mechanisms of division. Here, we review the historical lack of observation of PG in PVC bacteria, its recent detection in two phyla and its involvement in chlamydial cell division. Based on the detection of PG-related proteins in PVC proteomes, we consider the possible evolution of the diverse division mechanisms in these bacteria. We conclude by summarizing what is known and what remains to be understood about the evolutionary cell biology of PVC division modes.

Characterization of Outer Membrane Proteome of Akkermansia muciniphila Reveals Sets of Novel Proteins Exposed to the Human Intestine.

Akkermansia muciniphila is a common member of the human gut microbiota and belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum. Decreased levels of A. muciniphila have been associated with many diseases, and thus it is considered to be a beneficial resident of the intestinal mucus layer. Surface-exposed molecules produced by this organism likely play important roles in colonization and communication with other microbes and the host, but the protein composition of the outer membrane (OM) has not been characterized thus far. Herein we set out to identify and characterize A. muciniphila proteins using an integrated approach of proteomics and computational analysis. Sarkosyl extraction and sucrose density-gradient centrifugation methods were used to enrich and fractionate the OM proteome of A. muciniphila. Proteins from these fractions were identified by LC-MS/MS and candidates for OM proteins derived from the experimental approach were subjected to computational screening to verify their location in the cell. In total we identified 79 putative OM and membrane-associated extracellular proteins, and 23 of those were found to differ in abundance between cells of A. muciniphila grown on the natural substrate, mucin, and those grown on the non-mucus sugar, glucose. The identified OM proteins included highly abundant proteins involved in secretion and transport, as well as proteins predicted to take part in formation of the pili-like structures observed in A. muciniphila. The most abundant OM protein was a 95-kD protein, termed PilQ, annotated as a type IV pili secretin and predicted to be involved in the production of pili in A. muciniphila. To verify its location we purified the His-Tag labeled N-terminal domain of PilQ and generated rabbit polyclonal antibodies. Immunoelectron microscopy of thin sections immunolabeled with these antibodies demonstrated the OM localization of PilQ, testifying for its predicted function as a type IV pili secretin in A. muciniphila. As pili structures are known to be involved in the modulation of host immune responses, this provides support for the involvement of OM proteins in the host interaction of A. muciniphila. In conclusion, the characterization of A. muciniphila OM proteome provides valuable information that can be used for further functional and immunological studies.

Development of Genetic Tools for the Manipulation of the Planctomycetes.

Bacteria belonging to the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum are of interest for biotechnology, evolutionary cell biology, ecology, and human health. Some PVC species lack a number of typical bacterial features while others possess characteristics that are usually more associated to eukaryotes or archaea. For example, the Planctomycetes phylum is atypical for the absence of the FtsZ protein and for the presence of a developed endomembrane system. Studies of the cellular and molecular biology of these infrequent characteristics are currently limited due to the lack of genetic tools for most of the species. So far, genetic manipulation in Planctomycetes has been described in Planctopirus limnophila only. Here, we show a simple approach that allows mutagenesis by homologous recombination in three different planctomycetes species (i.e., Gemmata obscuriglobus, Gimesia maris, and Blastopirellula marina), in addition to P. limnophila, thus extending the repertoire of genetically modifiable organisms in this superphylum. Although the Planctomycetes show high resistance to most antibiotics, we have used kanamycin resistance genes in G. obscuriglobus, P. limnophila, and G. maris, and tetracycline resistance genes in B. marina, as markers for mutant selection. In all cases, plasmids were introduced in the strains by mating or electroporation, and the genetic modification was verified by Southern Blotting analysis. In addition, we show that the green fluorescent protein (gfp) is expressed in all four backgrounds from an Escherichia coli promoter. The genetic manipulation achievement in four phylogenetically diverse planctomycetes will enable molecular studies in these strains, and opens the door to developing genetic approaches not only in other planctomycetes but also other species of the superphylum, such as the Lentisphaerae.

A Zebrafish Model for Chlamydia Infection with the Obligate Intracellular Pathogen Waddlia chondrophila.

Obligate intracellular chlamydial bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum are important pathogens of terrestrial and marine vertebrates, yet many features of their pathogenesis and host specificity are still unknown. This is particularly true for families such as the Waddliacea which, in addition to epithelia, cellular targets for nearly all Chlamydia, can infect and replicate in macrophages, an important arm of the innate immune system or in their free-living amoebal counterparts. An ideal pathogen model system should include both host and pathogen, which led us to develop the first larval zebrafish model for chlamydial infections with Waddlia chondrophila. By varying the means and sites of application, epithelial cells of the swim bladder, endothelial cells of the vasculature and phagocytosing cells of the innate immune system became preferred targets for infection in zebrafish larvae. Through the use of transgenic zebrafish, we could observe recruitment of neutrophils to the infection site and demonstrate for the first time that W. chondrophila is taken up and replicates in these phagocytic cells and not only in macrophages. Furthermore, we present evidence that myeloid differentiation factor 88 (MyD88) mediated signaling plays a role in the innate immune reaction to W. chondrophila, eventually by Toll-like receptor (TLRs) recognition. Infected larvae with depleted levels of MyD88 showed a higher infection load and a lower survival rate compared to control fish. This work presents a new and potentially powerful non-mammalian experimental model to study the pathology of chlamydial virulence in vivo and opens up new possibilities for investigation of other members of the PVC superphylum.

Evaluating the Evolutionary Origins of Unexpected Character Distributions within the Bacterial Planctomycetes-Verrucomicrobia-Chlamydiae Superphylum.

Recently, several characters that are absent from most bacteria, but which are found in many eukaryotes or archaea, have been identified within the bacterial Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum. Hypotheses of the evolutionary history of such characters are commonly based on the inference of phylogenies of gene or protein families associated with the traits, estimated from multiple sequence alignments (MSAs). So far, studies of this kind have focused on the distribution of (i) two genes involved in the synthesis of sterol, (ii) tubulin genes, and (iii) c1 transfer genes. In many cases, these analyses have concluded that horizontal gene transfer (HGT) is likely to have played a role in shaping the taxonomic distribution of these gene families. In this article, we describe several issues with the inference of HGT from such analyses, in particular concerning the considerable uncertainty associated with our estimation of both gene family phylogenies (especially those containing ancient lineage divergences) and the Tree of Life (ToL), and the need for wider use and further development of explicit probabilistic models to compare hypotheses of vertical and horizontal genetic transmission. We suggest that data which is often taken as evidence for the occurrence of ancient HGT events may not be as convincing as is commonly described, and consideration of alternative theories is recommended. While focusing on analyses including PVCs, this discussion is also relevant for inferences of HGT involving other groups of organisms.

Molecular Signatures for the PVC Clade (Planctomycetes, Verrucomicrobia, Chlamydiae, and Lentisphaerae) of Bacteria Provide Insights into Their Evolutionary Relationships.

The PVC superphylum is an amalgamation of species from the phyla Planctomycetes, Verrucomicrobia, and Chlamydiae, along with the Lentisphaerae, Poribacteria, and two other candidate divisions. The diverse species of this superphylum lack any significant marker that differentiates them from other bacteria. Recently, genome sequences for 37 species covering all of the main PVC groups of bacteria have become available. We have used these sequences to construct a phylogenetic tree based upon concatenated sequences for 16 proteins and identify molecular signatures in protein sequences that are specific for the species from these phyla or those providing molecular links among them. Of the useful molecular markers identified in the present work, six conserved signature indels (CSIs) in the proteins Cyt c oxidase, UvrD helicase, urease, and a helicase-domain containing protein are specific for the species from the Verrucomicrobia phylum; three other CSIs in an ABC transporter protein, cobyrinic acid ac-diamide synthase, and SpoVG protein are specific for the Planctomycetes species. Additionally, a 3 aa insert in the RpoB protein is uniquely present in all sequenced Chlamydiae, Verrucomicrobia, and Lentisphaerae species, providing evidence for the shared ancestry of the species from these three phyla. Lastly, we have also identified a conserved protein of unknown function that is exclusively found in all sequenced species from the phyla Chlamydiae, Verrucomicrobia, Lentisphaerae, and Planctomycetes suggesting a specific linkage among them. The absence of this protein in Poribacteria, which branches separately from other members of the PVC clade, indicates that it is not specifically related to the PVC clade of bacteria. The molecular markers described here in addition to clarifying the evolutionary relationships among the PVC clade of bacteria also provide novel tools for their identification and for genetic and biochemical studies on these organisms.