Metagenomics research has recently thrived due to DNA sequencing technologies improvement, driving the emergence of new analysis tools and the growth of taxonomic databases. However, there is no all-purpose strategy that can guarantee the best result for a given project and there are several combinations of software, parameters and databases that can be tested. Therefore, we performed an impartial comparison, using statistical measures of classification for eight bioinformatic tools and four taxonomic databases, defining a benchmark framework to evaluate each tool in a standardized context. Using in silico simulated data for 16S rRNA amplicons and whole metagenome shotgun data, we compared the results from different software and database combinations to detect biases related to algorithms or database annotation. Using our benchmark framework, researchers can define cut-off values to evaluate the expected error rate and coverage for their results, regardless the score used by each software. A quick guide to select the best tool, all datasets and scripts to reproduce our results and benchmark any new method are available at https://github.com/Ales-ibt/Metagenomic-benchmark. Finally, we stress out the importance of gold standards, database curation and manual inspection of taxonomic profiling results, for a better and more accurate microbial diversity description.

Summary Many parasitic bacteria live in the cytoplasm of multicellular animals, but only a few are known to regularly invade their nuclei. In this study, we describe the novel bacterial parasite “Candidatus Endonucleobacter bathymodioli” that invades the nuclei of deep‐sea bathymodiolin mussels from hydrothermal vents and cold seeps. Bathymodiolin mussels are well known for their symbiotic associations with sulfur‐ and methane‐oxidizing bacteria. In contrast, the parasitic bacteria of vent and seep animals have received little attention despite their potential importance for deep‐sea ecosystems. We first discovered the intranuclear parasite “Ca. E. bathymodioli” in Bathymodiolus puteoserpentis from the Logatchev hydrothermal vent field on the Mid‐Atlantic Ridge. Using primers and probes specific to “Ca. E. bathymodioli” we found this intranuclear parasite in at least six other bathymodiolin species from vents and seeps around the world. Fluorescence in situ hybridization and transmission electron microscopy analyses of the developmental cycle of “Ca. E. bathymodioli” showed that the infection of a nucleus begins with a single rod‐shaped bacterium which grows to an unseptated filament of up to 20 μm length and then divides repeatedly until the nucleus is filled with up to 80 000 bacteria. The greatly swollen nucleus destroys its host cell and the bacteria are released after the nuclear membrane bursts. Intriguingly, the only nuclei that were never infected by “Ca. E. bathymodioli” were those of the gill bacteriocytes. These cells contain the symbiotic sulfur‐ and methane‐oxidizing bacteria, suggesting that the mussel symbionts can protect their host nuclei against the parasite. Phylogenetic analyses showed that the “Ca. E. bathymodioli” belongs to a monophyletic clade of Gammaproteobacteria associated with marine metazoans as diverse as sponges, corals, bivalves, gastropods, echinoderms, ascidians and fish. We hypothesize that many of the sequences from this clade originated from intranuclear bacteria, and that these are widespread in marine invertebrates.

Bathymodiolus mussels dominate animal communities at many hydrothermal vents and cold seeps. Essential to the mussels' ecological and evolutionary success is their association with symbiotic methane- and sulfur-oxidizing gammaproteobacteria, which provide them with nutrition. In addition to these well-known gammaproteobacterial endosymbionts, we found epsilonproteobacterial sequences in metatranscriptomes, metagenomes and 16S rRNA clone libraries as well as by polymerase chain reaction screening of Bathymodiolus species sampled from vents and seeps around the world. These epsilonproteobacterial sequences were closely related, indicating that the association is highly specific. The Bathymodiolus-associated epsilonproteobacterial 16S rRNA sequences were at most 87.6% identical to the closest cultured relative, and 91.2% identical to the closest sequences in public databases. This clade therefore represents a novel family within the Epsilonproteobacteria. Fluorescence in situ hybridization and transmission electron microscopy showed that the bacteria are filamentous epibionts associated with the gill epithelia in two Bathymodiolus species. In animals that host highly specific symbioses with one or a few types of endosymbionts, other less-abundant members of the microbiota can be easily overlooked. Our work highlights how widespread and specific associations with less-abundant microbes can be. Possibly, these microbes play an important role in the survival and health of their animal hosts.

Chemosynthetic life was recently discovered at Chapopote, an asphalt hydrocarbon seep in the southern Gulf of Mexico. Preliminary morphological analyses indicated that one tubeworm and two mussel species colonize Chapopote. Our molecular analyses identified the tubeworm as Escarpia sp., and the mussels as Bathymodiolus heckerae and B. brooksi. Comparative 16S rRNA analysis and FISH showed that all three species harbour intracellular sulfur-oxidizing symbionts highly similar or identical to those found in the same host species from northern Gulf of Mexico (nGoM). The mussels also harbour methane-oxidizing symbionts, and these shared highly similar to identical 16S rRNA sequences to their nGoM conspecifics. We discovered a novel symbiont in B. heckerae, which is closely related to hydrocarbon-degrading bacteria of the genus Cycloclasticus. In B. heckerae, we found key genes for the use of aromatic compounds, and its stable carbon isotope values were consistently higher than B. brooksi, indicating that the novel symbiont might use isotopically heavy aromatic hydrocarbons from the asphalt seep. This discovery is particularly intriguing because until now only methane and reduced sulfur compounds have been shown to power cold-seep chemosynthetic symbioses. The abundant hydrocarbons available at Chapopote would provide these mussel symbioses with a rich source of nutrition.

Marine sediments are an example of one of the most complex microbial habitats. These bacterial communities play an important role in several biogeochemical cycles in the marine ecosystem. In particular, the Gulf of Mexico has a ubiquitous concentration of hydrocarbons in its sediments, representing a very interesting niche to explore. Additionally, the Mexican government has opened its oil industry, offering several exploration and production blocks in shallow and deep water in the southwestern Gulf of Mexico (swGoM), from which there are no public results of conducted studies. Given the higher risk of large-scale oil spills, the design of contingency plans and mitigation activities before oil exploitation is of growing concern. Therefore, a bacterial taxonomic baseline profile is crucial to understanding the impact of any eventual oil spill. Here, we show a genus level taxonomic profile to elucidate the bacterial baseline, pointing out richness and relative abundance, as well as relationships with 79 abiotic parameters, in an area encompassing ∼150,000 km2, including a region where the exploitation of new oil wells has already been authorized. Our results describe for the first time the bacterial landscape of the swGoM, establishing a bacterial baseline “core” of 450 genera for marine sediments in this region. We can also differentiate bacterial populations from shallow and deep zones of the swGoM based on their community structure. Shallow sediments have been chronically exposed to aromatic hydrocarbons, unlike deep zones. Our results reveal that the bacterial community structure is particularly enriched with hydrocarbon-degrading bacteria in the shallow zone, where a greater aromatic hydrocarbon concentration was determined. Differences in the bacterial communities in the swGoM were also observed through a comprehensive comparative analysis relative to various marine sediment sequencing projects, including sampled sites from the Deep Water Horizon oil spill. This study in the swGoM provides clues to the bacterial population adaptation to the ubiquitous presence of hydrocarbons and reveals organisms such as Thioprofundum bacteria with potential applications in ecological surveillance. This resource will allow us to differentiate between natural conditions and alterations generated by oil extraction activities, which, in turn, enables us to assess the environmental impact of such activities.

The Gulf of Mexico (GoM) is a particular environment that is continuously exposed to hydrocarbon compounds that may influence the microbial community composition. We carried out a metagenomic assessment of the bacterial community to get an overall view of this geographical zone. We analyzed both taxonomic and metabolic markers profiles to explain how the indigenous GoM microorganims participate in the biogeochemical cycling. Two geographically distant regions in the GoM, one in the north-west (NW) and one in the south-east (SE) of the GoM were analyzed and showed differences in their microbial composition and metabolic potential. These differences provide evidence the delicate equilibrium that sustains microbial communities and biogeochemical cycles. Based on the taxonomy and gene groups, the NW are more oxic sediments than SE ones, which have anaerobic conditions. Both water and sediments show the expected sulfur, nitrogen, and hydrocarbon metabolism genes, with particularly high diversity of the hydrocarbon-degrading ones. Accordingly, many of the assigned genera were associated with hydrocarbon degradation processes, Nitrospira and Sva0081 were the most abundant in sediments, while Vibrio , Alteromonas , and Alcanivorax were mostly detected in water samples. This basal-state analysis presents the GoM as a potential source of aerobic and anaerobic hydrocarbon degradation genes important for the ecological dynamics of hydrocarbons and the potential use for water and sediment bioremediation processes.

The significance of experiments that claim to simulate the properties of prebiotic small peptides and polypeptides as models of the polymers that may have preceded proteins is critically addressed. As discussed here, most of these experiments are based only on a small number of a larger set of amino acids that may have been present in the prebiotic environment, supported by both experimental simulations and the repertoire of organic compounds reported in carbonaceous chondrites. Model experiments with small peptides may offer some insights into the processes that contributed to generate the chemical environment leading to the emergence of informational oligomers, but not to the origin of proteins. The large body of circumstantial evidence indicating that catalytic RNA played a key role in the origin of protein synthesis during the early stages of cellular evolution implies that the emergence of the genetic code and of protein biosynthesis are no longer synonymous with the origin of life. Hence, reports on the abiotic synthesis of small catalytic peptides under potential prebiotic conditions do not provide information on the origin of triplet encoded protein biosynthesis, but in some cases may serve as models to understand the properties of the earliest proteins.

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