The cell envelope of Thermotogae suggests a mechanism for outer membrane biogenesis.

The presence of a cell membrane is one of the major structural components defining life. Recent phylogenomic analyses have supported the hypothesis that the last universal common ancestor (LUCA) was likely a diderm. Yet, the mechanisms that guided outer membrane (OM) biogenesis remain unknown. Thermotogae is an early-branching phylum with a unique OM, the toga. Here, we use cryo-electron tomography to characterize the in situ cell envelope architecture of Thermotoga maritima and show that the toga is made of extended sheaths of ß-barrel trimers supporting small (~200 nm) membrane patches. Lipidomic analyses identified the same major lipid species in the inner membrane (IM) and toga, including the rare to bacteria membrane-spanning ether-bound diabolic acids (DAs). Proteomic analyses revealed that the toga was composed of multiple SLH-domain containing Ompα and novel ß-barrel proteins, and homology searches detected variable conservations of these proteins across the phylum. These results highlight that, in contrast to the SlpA/OmpM superfamily of proteins, Thermotoga possess a highly diverse bipartite OM-tethering system. We discuss the implications of our findings with respect to other early-branching phyla and propose that a toga-like intermediate may have facilitated monoderm-to-diderm cell envelope transitions.

Vestiges of Adaptation to the Mesophilic Environment in the Genome of Tepiditoga spiralis gen. nov., sp. nov.

A novel anaerobic heterotrophic strain, designated strain sy52T, was isolated from a hydrothermal chimney at Suiyo Seamount in the Pacific Ocean. A 16S rRNA gene analysis revealed that the strain belonged to the family Petrotogaceae in the phylum Thermotogae. The strain was mesophilic with optimum growth at 48°C and the phylum primarily comprised hyperthermophiles and thermophiles. Strain sy52T possessed unique genomic characteristics, such as an extremely low G+C content and 6 copies of rRNA operons. Genomic analyses of strain sy52T revealed that amino acid usage in the predicted proteins resulted from adjustments to mesophilic environments. Genomic features also indicated independent adaptions to the mesophilic environment of strain sy52T and Mesotoga species, which belong to the mesophilic lineage in the phylum Thermotogae. Based on phenotypic and phylogenetic evidence, strain sy52T is considered to represent a novel genus and species in the family Petrotogaceae with the proposed name Tepiditoga spiralis gen. nov., sp. nov.

Towards a congruent reclassification and nomenclature of the thermophilic species of the genus Pseudothermotoga within the order Thermotogales.

The phylum Thermotogae gathers thermophilic, hyperthermophic, mesophilic, and thermo-acidophilic anaerobic bacteria that are mostly originated from geothermally heated environments. The metabolic and phenotypic properties harbored by the Thermotogae species questions the evolutionary events driving the emergence of this early branch of the universal tree of life. Recent reshaping of the Thermotogae taxonomy has led to the description of a new genus, Pseudothermotoga, a sister group of the genus Thermotoga within the order Thermotogales. Comparative genomics of both Pseudothermotoga and Thermotoga spp., including 16S-rRNA-based phylogenetic, pan-genomic analysis as well as signature indel conservation, provided evidence that Thermotoga caldifontis and Thermotoga profunda species should be reclassified within the genus Pseudothermotoga and renamed as Pseudothermotoga caldifontis comb. nov. (type strain=AZM44c09T) and Pseudothermotoga profunda comb. nov. (type strain=AZM34c06T), respectively. In addition, based upon whole-genome relatedness indices and DNA-DNA Hybridization results, the reclassification of Pseudothermotoga lettingae and Pseudothermotoga subterranea as latter heterotypic synonyms of Pseudothermotoga elfii is proposed. Finally, potential genetic elements resulting from the distinct evolutionary story of the Thermotoga and Pseudothermotoga clades are discussed.

Genomic insights into temperature-dependent transcriptional responses of Kosmotoga olearia, a deep-biosphere bacterium that can grow from 20 to 79 °C.

Temperature is one of the defining parameters of an ecological niche. Most organisms thrive within a temperature range that rarely exceeds ~30 °C, but the deep subsurface bacterium Kosmotoga olearia can grow over a temperature range of 59 °C (20-79 °C). To identify genes correlated with this flexible phenotype, we compared transcriptomes of K. olearia cultures grown at its optimal 65 °C to those at 30, 40, and 77 °C. The temperature treatments affected expression of 573 of 2224 K. olearia genes. Notably, this transcriptional response elicits re-modeling of the cellular membrane and changes in metabolism, with increased expression of genes involved in energy and carbohydrate metabolism at high temperatures and up-regulation of amino acid metabolism at lower temperatures. At sub-optimal temperatures, many transcriptional changes were similar to those observed in mesophilic bacteria at physiologically low temperatures, including up-regulation of typical cold stress genes and ribosomal proteins. Comparative genomic analysis of additional Thermotogae genomes indicates that one of K. olearia's strategies for low-temperature growth is increased copy number of some typical cold response genes through duplication and/or lateral acquisition. At 77 °C one-third of the up-regulated genes are of hypothetical function, indicating that many features of high-temperature growth are unknown.

Methodological flaws introduce strong bias into molecular analysis of microbial populations.

AIMS: In this study, we report how different cell disruption methods, PCR primers and in silico analyses can seriously bias results from microbial population studies, with consequences for the credibility and reproducibility of the findings. Our results emphasize the pitfalls of commonly used experimental methods that can seriously weaken the interpretation of results. METHODS AND RESULTS: Four different cell lysis methods, three commonly used primer pairs and various computer-based analyses were applied to investigate the microbial diversity of a fermentation sample composed of chicken dung. The fault-prone, but still frequently used, amplified rRNA gene restriction analysis was chosen to identify common weaknesses. In contrast to other studies, we focused on the complete analytical process, from cell disruption to in silico analysis, and identified potential error rates. This identified a wide disagreement of results between applied experimental approaches leading to very different community structures depending on the chosen approach. CONCLUSIONS: The interpretation of microbial diversity data remains a challenge. In order to accurately investigate the taxonomic diversity and structure of prokaryotic communities, we suggest a multi-level approach combining DNA-based and DNA-independent techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: The identified weaknesses of commonly used methods to study microbial diversity can be overcome by a multi-level approach, which produces more reliable data about the fate and behaviour of microbial communities of engineered habitats such as biogas plants, so that the best performance can be ensured.

Identification and genome reconstruction of abundant distinct taxa in microbiomes from one thermophilic and three mesophilic production-scale biogas plants.

BACKGROUND: Biofuel production from conversion of biomass is indispensable in the portfolio of renewable energies. Complex microbial communities are involved in the anaerobic digestion process of plant material, agricultural residual products and food wastes. Analysis of the genetic potential and microbiology of communities degrading biomass to biofuels is considered to be the key to develop process optimisation strategies. Hence, due to the still incomplete taxonomic and functional characterisation of corresponding communities, new and unknown species are of special interest. RESULTS: Three mesophilic and one thermophilic production-scale biogas plants (BGPs) were taxonomically profiled using high-throughput 16S rRNA gene amplicon sequencing. All BGPs shared a core microbiome with the thermophilic BGP featuring the lowest diversity. However, the phyla Cloacimonetes and Spirochaetes were unique to BGPs 2 and 3, Fusobacteria were only found in BGP3 and members of the phylum Thermotogae were present only in the thermophilic BGP4. Taxonomic analyses revealed that these distinctive taxa mostly represent so far unknown species. The only exception is the dominant Thermotogae OTU featuring 16S rRNA gene sequence identity to Defluviitoga tunisiensis L3, a sequenced and characterised strain. To further investigate the genetic potential of the biogas communities, corresponding metagenomes were sequenced in a deepness of 347.5 Gbp in total. A combined assembly comprised 80.3 % of all reads and resulted in the prediction of 1.59 million genes on assembled contigs. Genome binning yielded genome bins comprising the prevalent distinctive phyla Cloacimonetes, Spirochaetes, Fusobacteria and Thermotogae. Comparative genome analyses between the most dominant Thermotogae bin and the very closely related Defluviitoga tunisiensis L3 genome originating from the same BGP revealed high genetic similarity. This finding confirmed applicability and reliability of the binning approach. The four highly covered genome bins of the other three distinct phyla showed low or very low genetic similarities to their closest phylogenetic relatives, and therefore indicated their novelty. CONCLUSIONS: In this study, the 16S rRNA gene sequencing approach and a combined metagenome assembly and binning approach were used for the first time on different production-scale biogas plants and revealed insights into the genetic potential and functional role of so far unknown species.

Substrate adaptabilities of Thermotogae mannan binding proteins as a function of their evolutionary histories.

The Thermotogae possess a large number of ATP-binding cassette (ABC) transporters, including two mannan binding proteins, ManD and CelE (previously called ManE). We show that a gene encoding an ancestor of these was acquired by the Thermotogae from the archaea followed by gene duplication. To address the functional evolution of these proteins as a consequence of their evolutionary histories, we measured the binding affinities of ManD and CelE orthologs from representative Thermotogae. Both proteins bind cellobiose, cellotriose, cellotetraose, ß-1,4-mannotriose, and ß-1,4-mannotetraose. The CelE orthologs additionally bind ß-1,4-mannobiose, laminaribiose, laminaritriose and sophorose while the ManD orthologs additionally only weakly bind ß-1,4-mannobiose. The CelE orthologs have higher unfolding temperatures than the ManD orthologs. An examination of codon sites under positive selection revealed that many of these encode residues located near or in the binding site, suggesting that the proteins experienced selective pressures in regions that might have changed their functions. The gene arrangement, phylogeny, binding properties, and putative regulatory networks suggest that the ancestral mannan binding protein was a CelE ortholog which gave rise to the ManD orthologs. This study provides a window on how one class of proteins adapted to new functions and temperatures to fit the physiologies of their new hosts.

Genomic characterization of Defluviitoga tunisiensis L3, a key hydrolytic bacterium in a thermophilic biogas plant and its abundance as determined by metagenome fragment recruitment.

The genome sequence of Defluviitoga tunisiensis L3 originating from a thermophilic biogas-production plant was established and recently published as Genome Announcement by our group. The circular chromosome of D. tunisiensis L3 has a size of 2,053,097bp and a mean GC content of 31.38%. To analyze the D. tunisiensis L3 genome sequence in more detail, a phylogenetic analysis of completely sequenced Thermotogae strains based on shared core genes was performed. It appeared that Petrotoga mobilis DSM 10674(T), originally isolated from a North Sea oil-production well, is the closest relative of D. tunisiensis L3. Comparative genome analyses of P. mobilis DSM 10674(T) and D. tunisiensis L3 showed moderate similarities regarding occurrence of orthologous genes. Both genomes share a common set of 1351 core genes. Reconstruction of metabolic pathways important for the biogas production process revealed that the D. tunisiensis L3 genome encodes a large set of genes predicted to facilitate utilization of a variety of complex polysaccharides including cellulose, chitin and xylan. Ethanol, acetate, hydrogen (H2) and carbon dioxide (CO2) were found as possible end-products of the fermentation process. The latter three metabolites are considered to represent substrates for methanogenic Archaea, the key organisms in the final step of the anaerobic digestion process. To determine the degree of relatedness between D. tunisiensis L3 and dominant biogas community members within the thermophilic biogas-production plant, metagenome sequences obtained from the corresponding microbial community were mapped onto the L3 genome sequence. This fragment recruitment revealed that the D. tunisiensis L3 genome is almost completely covered with metagenome sequences featuring high matching accuracy. This result indicates that strains highly related or even identical to the reference strain D. tunisiensis L3 play a dominant role within the community of the thermophilic biogas-production plant.

Novel, Deep-Branching Heterotrophic Bacterial Populations Recovered from Thermal Spring Metagenomes.

Thermal spring ecosystems are a valuable resource for the discovery of novel hyperthermophilic Bacteria and Archaea, and harbor deeply-branching lineages that provide insight regarding the nature of early microbial life. We characterized bacterial populations in two circumneutral (pH ~8) Yellowstone National Park thermal (T ~80°C) spring filamentous "streamer" communities using random metagenomic DNA sequence to investigate the metabolic potential of these novel populations. Four de novo assemblies representing three abundant, deeply-branching bacterial phylotypes were recovered. Analysis of conserved phylogenetic marker genes indicated that two of the phylotypes represent separate groups of an uncharacterized phylum (for which we propose the candidate phylum name "Pyropristinus"). The third new phylotype falls within the proposed Calescamantes phylum. Metabolic reconstructions of the "Pyropristinus" and Calescamantes populations showed that these organisms appear to be chemoorganoheterotrophs and have the genomic potential for aerobic respiration and oxidative phosphorylation via archaeal-like V-type, and bacterial F-type ATPases, respectively. A survey of similar phylotypes (>97% nt identity) within 16S rRNA gene datasets suggest that the newly described organisms are restricted to terrestrial thermal springs ranging from 70 to 90°C and pH values of ~7-9. The characterization of these lineages is important for understanding the diversity of deeply-branching bacterial phyla, and their functional role in high-temperature circumneutral "streamer" communities.

Complete genome sequence of the strain Defluviitoga tunisiensis L3, isolated from a thermophilic, production-scale biogas plant.

An anaerobic, thermophilic bacterium belonging to the phylum Thermotogae was isolated from a rural, thermophilic biogas plant (54°C) producing methane-rich biogas from maize silage, barley, cattle and pig manure. Here we report the first complete genome sequence of the Defluviitoga tunisiensis strain L3, an isolate from the family Thermotogaceae. The strain L3 encodes several genes predicted to be involved in utilization of a large diversity of complex carbohydrates including cellobiose and xylan for the production of acetate, hydrogen (H2) and carbon dioxide (CO2). The genome sequence of D. tunisiensis L3 provides the basis for biotechnological exploitation of genetic determinants playing an important role in thermophilic fermentation processes utilizing renewable primary products.

Being Aquifex aeolicus: Untangling a hyperthermophile's checkered past.

Lateral gene transfer (LGT) is an important factor contributing to the evolution of prokaryotic genomes. The Aquificae are a hyperthermophilic bacterial group whose genes show affiliations to many other lineages, including the hyperthermophilic Thermotogae, the Proteobacteria, and the Archaea. Previous phylogenomic analyses focused on Aquifex aeolicus identified Thermotogae and Aquificae either as successive early branches or sisters in a rooted bacterial phylogeny, but many phylogenies and cellular traits have suggested a stronger affiliation with the Epsilonproteobacteria. Different scenarios for the evolution of the Aquificae yield different phylogenetic predictions. Here, we outline these scenarios and consider the fit of the available data, including three sequenced Aquificae genomes, to different sets of predictions. Evidence from phylogenetic profiles and trees suggests that the Epsilonproteobacteria have the strongest affinities with the three Aquificae analyzed. However, this pattern is shown by only a minority of encoded proteins, and the Archaea, many lineages of thermophilic bacteria, and members of genus Clostridium and class Deltaproteobacteria also show strong connections to the Aquificae. The phylogenetic affiliations of different functional subsystems showed strong biases: Most but not all genes implicated in the core translational apparatus tended to group Aquificae with Thermotogae, whereas a wide range of metabolic and cellular processes strongly supported the link between Aquificae and Epsilonproteobacteria. Depending on which sets of genes are privileged, either Thermotogae or Epsilonproteobacteria is the most plausible adjacent lineage to the Aquificae. Both scenarios require massive sharing of genes to explain the history of this enigmatic group, whose history is further complicated by specific affinities of different members of Aquificae to different partner lineages.

Reconstruction of ancestral 16S rRNA reveals mutation bias in the evolution of optimal growth temperature in the Thermotogae phylum.

Optimal growth temperature is a complex trait involving many cellular components, and its physiology is not yet fully understood. Evolution of continuous characters, such as optimal growth temperature, is often modeled as a one-dimensional random walk, but such a model may be an oversimplification given the complex processes underlying the evolution of continuous characters. Recent articles have used ancestral sequence reconstruction to infer the optimal growth temperature of ancient organisms from the guanine and cytosine content of the stem regions of ribosomal RNA, allowing inferences about the evolution of optimal growth temperature. Here, we investigate the optimal growth temperature of the bacterial phylum Thermotogae. Ancestral sequence reconstruction using a nonhomogeneous model was used to reconstruct the stem guanine and cytosine content of 16S rRNA sequences. We compare this sequence reconstruction method with other ancestral character reconstruction methods, and show that sequence reconstruction generates smaller confidence intervals and different ancestral values than other reconstruction methods. Unbiased random walk simulation indicates that the lower temperature members of the Thermotogales have been under directional selection; however, when a simulation is performed that takes possible mutations into account, it is the high temperature lineages that are, in fact, under directional selection. We find that the evolution of Thermotogales optimal growth temperatures is best fit by a biased random walk model. These findings suggest that it may be easier to evolve from a high optimal growth temperature to a lower one than vice versa.

Protein based molecular markers provide reliable means to understand prokaryotic phylogeny and support Darwinian mode of evolution.

The analyses of genome sequences have led to the proposal that lateral gene transfers (LGTs) among prokaryotes are so widespread that they disguise the interrelationships among these organisms. This has led to questioning of whether the Darwinian model of evolution is applicable to prokaryotic organisms. In this review, we discuss the usefulness of taxon-specific molecular markers such as conserved signature indels (CSIs) and conserved signature proteins (CSPs) for understanding the evolutionary relationships among prokaryotes and to assess the influence of LGTs on prokaryotic evolution. The analyses of genomic sequences have identified large numbers of CSIs and CSPs that are unique properties of different groups of prokaryotes ranging from phylum to genus levels. The species distribution patterns of these molecular signatures strongly support a tree-like vertical inheritance of the genes containing these molecular signatures that is consistent with phylogenetic trees. Recent detailed studies in this regard on the Thermotogae and Archaea, which are reviewed here, have identified large numbers of CSIs and CSPs that are specific for the species from these two taxa and a number of their major clades. The genetic changes responsible for these CSIs (and CSPs) initially likely occurred in the common ancestors of these taxa and then vertically transferred to various descendants. Although some CSIs and CSPs in unrelated groups of prokaryotes were identified, their small numbers and random occurrence has no apparent influence on the consistent tree-like branching pattern emerging from other markers. These results provide evidence that although LGT is an important evolutionary force, it does not mask the tree-like branching pattern of prokaryotes or understanding of their evolutionary relationships. The identified CSIs and CSPs also provide novel and highly specific means for identification of different groups of microbes and for taxonomical and biochemical studies.