Metazoans of redoxcline sediments in Mediterranean deep-sea hypersaline anoxic basins

Background: The deep-sea hypersaline anoxic basins (DHABs) of the Mediterranean (water depth similar to 3500 m) are some of the most extreme oceanic habitats known. Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB haloclines and brines, loriciferans are the only metazoan reported to inhabit the anoxic DHAB brines. Our goal was to further investigate metazoan communities in DHAB haloclines and brines. Results: We report observations from sediments of three DHAB (Urania, Discovery, L'Atalante) haloclines, comparing these to observations from sediments underlying normoxic waters of typical Mediterranean salinity. Due to technical difficulties, sampling of the brines was not possible. Morphotype analysis indicates nematodes are the most abundant taxon; crustaceans, loriciferans and bryozoans were also noted. Among nematodes, Daptonema was the most abundant genus; three morphotypes were noted with a degree of endemicity. The majority of rRNA sequences were from planktonic taxa, suggesting that at least some individual metazoans were preserved and inactive. Nematode abundance data, in some cases determined from direct counts of sediments incubated in situ with CellTracker (TM) Green, was patchy but generally indicates the highest abundances in either normoxic control samples or in upper halocline samples; nematodes were absent or very rare in lower halocline samples. Ultrastructural analysis indicates the nematodes in L'Atalante normoxic control sediments were fit, while specimens from L'Atalante upper halocline were healthy or had only recently died and those from the lower halocline had no identifiable organelles. Loriciferans, which were only rarely encountered, were found in both normoxic control samples as well as in Discovery and L'Atalante haloclines. It is not clear how a metazoan taxon could remain viable under this wide range of conditions. Conclusions: We document a community of living nematodes in normoxic, normal saline deep-sea Mediterranean sediments and in the upper halocline portions of the DHABs. Occurrences of nematodes in mid-halocline and lower halocline samples did not provide compelling evidence of a living community in those zones. The possibility of a viable metazoan community in brines of DHABs is not supported by our data at this time

Hiding in plain sight: the globally distributed bacterial candidate phylum PAUC34f

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Chen, M. L., Becraft, E. D., Pachiadaki, M., Brown, J. M., Jarett, J. K., Gasol, J. M., Ravin, N. V., Moser, D. P., Nunoura, T., Herndl, G. J., Woyke, T., & Stepanauskas, R. Hiding in plain sight: the globally distributed bacterial candidate phylum PAUC34f. Frontiers in Microbiology, 11, (2020): 376, doi: 10.3389/fmicb.2020.00376.Bacterial candidate phylum PAUC34f was originally discovered in marine sponges and is widely considered to be composed of sponge symbionts. Here, we report 21 single amplified genomes (SAGs) of PAUC34f from a variety of environments, including the dark ocean, lake sediments, and a terrestrial aquifer. The diverse origins of the SAGs and the results of metagenome fragment recruitment suggest that some PAUC34f lineages represent relatively abundant, free-living cells in environments other than sponge microbiomes, including the deep ocean. Both phylogenetic and biogeographic patterns, as well as genome content analyses suggest that PAUC34f associations with hosts evolved independently multiple times, while free-living lineages of PAUC34f are distinct and relatively abundant in a wide range of environments.This work was funded by the United States National Science Foundation grants 1460861 (REU site at Bigelow Laboratory for Ocean Sciences), 1441717, 1335810, and 1232982 to RS, and the Simons Foundation (Life Sciences Project Award ID 510023) to RS. NR was supported by the Ministry of Science and Higher Education of Russia. GH was supported by the Austrian Science Fund (FWF) project ARTEMIS (P28781-B21) and the European Research Council under the European Community’s Seventh Framework Program (FP7/2007-2013)/ERC (Grant Agreement No. 268595). JG was supported by Spanish project RTI2018-101025-B-I00. TW and JJ were funded by the U.S. Department of Energy, Joint Genome Institute, a DOE Office of Science User Facility supported under Contract No. DE-AC02-05CH11231

Comparison of Niskin vs. in situ approaches for analysis of gene expression in deep Mediterranean Sea water samples

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Deep Sea Research Part II: Topical Studies in Oceanography 129 (2016): 213-222, doi:10.1016/j.dsr2.2014.10.020.Obtaining an accurate picture of microbial processes occurring in situ is essential for our understanding of marine biogeochemical cycles of global importance. Water samples are typically collected at depth and returned to the sea surface for processing and downstream experiments. Metatranscriptome analysis is one powerful approach for investigating metabolic activities of microorganisms in their habitat and which can be informative for determining responses of microbiota to disturbances such as the Deepwater Horizon oil spill. For studies of microbial processes occurring in the deep sea, however, sample handling, pressure, and other changes during sample recovery can subject microorganisms to physiological changes that alter the expression profile of labile messenger RNA. Here we report a comparison of gene expression profiles for whole microbial communities in a bathypelagic water column sample collected in the Eastern Mediterranean Sea using Niskin bottle sample collection and a new water column sampler for studies of marine microbial ecology, the Microbial Sampler – In Situ Incubation Device (MS-SID). For some taxa, gene expression profiles from samples collected and preserved 33 in situ were significantly different from potentially more stressful Niskin sampling and 34 preservation on deck. Some categories of transcribed genes also appear to be affected by sample 35 handling more than others. This suggests that for future studies of marine microbial ecology, 36 particularly targeting deep sea samples, an in situ sample collection and preservation approach 37 should be considered.This research was funded by NSF OCE-1061774 to VE and CT, NSF DBI-0424599 to CT and NSF OCE-0849578 to VE and colleague J. Bernhard. Cruise participation was partially supported by Deutsche Forschungsgemeinschaft (DFG) grant STO414/10-1 to T. Stoeck

Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Labonte, J. M., Pachiadaki, M., Fergusson, E., McNichol, J., Grosche, A., Gulmann, L. K., Vetriani, C., Sievert, S. M., & Stepanauskas, R. Single cell genomics-based analysis of gene content and expression of prophages in a diffuse-flow deep-sea hydrothermal system. Frontiers in Microbiology, 10, (2019): 1262, doi:10.3389/fmicb.2019.01262.Phage–host interactions likely play a major role in the composition and functioning of many microbiomes, yet remain poorly understood. Here, we employed single cell genomics to investigate phage–host interactions in a diffuse-flow, low-temperature hydrothermal vent that may be reflective of a broadly distributed biosphere in the subseafloor. We identified putative prophages in 13 of 126 sequenced single amplified genomes (SAGs), with no evidence for lytic infections, which is in stark contrast to findings in the surface ocean. Most were distantly related to known prophages, while their hosts included bacterial phyla Campylobacterota, Bacteroidetes, Chlorobi, Proteobacteria, Lentisphaerae, Spirochaetes, and Thermotogae. Our results suggest the predominance of lysogeny over lytic interaction in diffuse-flow, deep-sea hydrothermal vents, despite the high activity of the dominant Campylobacteria that would favor lytic infections. We show that some of the identified lysogens have co-evolved with their host over geological time scales and that their genes are transcribed in the environment. Functional annotations of lysogeny-related genes suggest involvement in horizontal gene transfer enabling host’s protection against toxic metals and antibacterial compounds.This work was supported by the U.S. National Science Foundation’s Dimensions of Biodiversity Program [OCE-1136488 (to RS), OCE-1136727 (to SMS) and OCE-1136451 (to CV)], as well as DEB-1441717 and OCE-1335810 (to RS), and the DOE JGI CSP project 1477

Benthic protists and fungi of Mediterranean deep hypsersaline anoxic basin redoxcline sediments

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 5 (2014): 605, doi:10.3389/fmicb.2014.00605.Some of the most extreme marine habitats known are the Mediterranean deep hypersaline anoxic basins (DHABs; water depth ∼3500 m). Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB water-column haloclines and brines, the existence and activity of benthic DHAB protists have rarely been explored. Here, we report findings regarding protists and fungi recovered from sediments of three DHAB (Discovery, Urania, L’ Atalante) haloclines, and compare these to communities from sediments underlying normoxic waters of typical Mediterranean salinity. Halocline sediments, where the redoxcline impinges the seafloor, were studied from all three DHABs. Microscopic cell counts suggested that halocline sediments supported denser protist populations than those in adjacent control sediments. Pyrosequencing analysis based on ribosomal RNA detected eukaryotic ribotypes in the halocline sediments from each of the three DHABs, most of which were fungi. Sequences affiliated with Ustilaginomycotina Basidiomycota were the most abundant eukaryotic signatures detected. Benthic communities in these DHABs appeared to differ, as expected, due to differing brine chemistries. Microscopy indicated that only a low proportion of protists appeared to bear associated putative symbionts. In a considerable number of cases, when prokaryotes were associated with a protist, DAPI staining did not reveal presence of any nuclei, suggesting that at least some protists were carcasses inhabited by prokaryotic scavengers.K. Kormas was partially supported by the University of Thessaly through a sabbatical in 2013. Supported by NSF grants OCE-0849578 to Virginia P. Edgcomb and Joan M. Bernhard and OCE-1061391 to Joan M. Bernhard and Virginia P. Edgcomb

Hiding in plain sight: The globally distributed bacterial candidate phylum PAUC34f

Bacterial candidate phylum PAUC34f was originally discovered in marine sponges and is widely considered to be composed of sponge symbionts. Here, we report 21 single amplified genomes (SAGs) of PAUC34f from a variety of environments, including the dark ocean, lake sediments, and a terrestrial aquifer. The diverse origins of the SAGs and the results of metagenome fragment recruitment suggest that some PAUC34f lineages represent relatively abundant, free-living cells in environments other than sponge microbiomes, including the deep ocean. Both phylogenetic and biogeographic patterns, as well as genome content analyses suggest that PAUC34f associations with hosts evolved independently multiple times, while free-living lineages of PAUC34f are distinct and relatively abundant in a wide range of environments. © Copyright © 2020 Chen, Becraft, Pachiadaki, Brown, Jarett, Gasol, Ravin, Moser, Nunoura, Herndl, Woyke and Stepanauskas

Evaluation and optimization of PCR primers for selective and quantitative detection of marine ANME subclusters involved in sulfate-dependent anaerobic methane oxidation

Since the discovery that anaerobic methanotrophic archaea (ANME) are involved in the anaerobic oxidation of methane coupled to sulfate reduction in marine sediments, different primers and probes specifically targeting the 16S rRNA gene of these archaea have been developed. Microbial investigation of the different ANME subtypes (ANME-1; ANME-2a, b, and c; and ANME-3) was mainly done in sediments where specific subtypes of ANME were highly enriched and methanogenic cell numbers were low. In different sediments with higher archaeal diversity and abundance, it is important that primers and probes targeting different ANME subtypes are very specific and do not detect other ANME subtypes or methanogens that are also present. In this study, primers and probes that were regularly used in AOM studies were tested in silico on coverage and specificity. Most of the previously developed primers and probes were not specific for the ANME subtypes, thereby not reflecting the actual ANME population in complex samples. Selected primers that showed good coverage and high specificity for the subclades ANME-1, ANME-2a/b, and ANME-2c were thoroughly validated using quantitative polymerase chain reaction (qPCR). From these qPCR tests, only certain combinations seemed suitable for selective amplification. After optimization of these primer sets, we obtained valid primer combinations for the selective detection and quantification of ANME-1, ANME-2a/b, and ANME-2c in samples where different ANME subtypes and possibly methanogens could be present. As a result of this work, we propose a standard workflow to facilitate selection of suitable primers for qPCR experiments on novel environmental samples.This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009). Research of PHATand AJMS is supported by the SIAM Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO).info:eu-repo/semantics/publishedVersio

Integrated analysis of bacterial and microeukaryotic communities from differentially active mud volcanoes in the Gulf of Cadiz

The present study assesses the diversity and composition of sediment bacterial and microeukaryotic communities from deep-sea mud volcanoes (MVs) associated with strike-slip faults in the South-West Iberian Margin (SWIM). We used a 16S/18S rRNA gene based pyrosequencing approach to characterize and correlate the sediment bacterial and microeukaryotic communities from MVs with differing gas seep regimes and from an additional site with no apparent seeping activity. In general, our results showed significant compositional changes of bacterial and microeukaryotic communities in sampling sites with different seepage regimes. Sediment bacterial communities were enriched with Methylococcales (putative methanotrophs) but had lower abundances of Rhodospirillales, Nitrospirales and SAR202 in the more active MVs. Within microeukaryotic communities, members of the Lobosa (lobose amoebae) were enriched in more active MVs. We also showed a strong correlation between Methylococcales populations and lobose amoeba in active MVs. This study provides baseline information on the diversity and composition of bacterial and microeukaryotic communities in deep-sea MVs associated with strike-slip faults

A genomic catalog of Earth’s microbiomes

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth’s continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes

Interconnectivity vs. isolation of prokaryotic communities in European deep-sea mud volcanoes

During the past two decades, European cold seep ecosystems have attracted the scientific interest and to date there are several studies which have investigated the community structure and biodiversity of individual sites. In order to gain a better insight into the biology, biodiversity, and biogeography of seep-associated microbial communities along Europe's continental margins, a comparative approach was applied in the present work. By exploiting the publicly available data on 16S rRNA gene sequences retrieved from sediments of the Hakon Mosby mud volcano, Gulf of Cadiz and the eastern Mediterranean mud volcanoes/pockmarks (Anaximander area and Nile Fan), we investigated the prokaryotic biological components connecting these geographically isolated systems. The construction of interaction networks for both archaeal and bacterial shared operational taxonomic units (OTUs) among the different sites, revealed the presence of persistent OTUs, which can be considered as "key-players". One archaeal OTU (HQ588641) belonging to the ANME-3 group and one delta-Proteobacteria (HQ588562) were found in all five investigated areas. Other Archaea OTUs shared between four sites or less, belonged to the ANME-2c, -2a, MBG-D, -B and Thaumarchaeota. All other shared Bacteria belonged to the delta- and gamma-Proteobacteria, with the exception of one JS1 affiliate OTU. The distribution of the majority of the shared OTUs seems to be restricted in cold seeps, mud volcanoes and other marine methane-rich environments. Although the investigated sites were connected through a small number of OTUs, these microorganisms hold central ecophysiological roles in these sediments, namely methane-and sulfur-mediated mineralization