Diversity of extremely halophilic cultivable prokaryotes in Mediterranean, Atlantic and Pacific solar salterns: Evidence that unexplored sites constitute sources of cultivable novelty

The culturable fraction of aerobic, heterotrophic and extremely halophilic microbiota retrieved from sediment and brine samples of eight sampling sites in the Mediterranean, Canary Islands and Chile was studied by means of a tandem approach combining large-scale cultivation, MALDI-TOF MS targeting whole cell biomass, and phylogenetic reconstruction based on 16S rRNA gene analysis. The approach allowed the identification of more than 4200 strains and a comparison between different sampling sites. The results indicated that the method constituted an excellent tool for the discovery of taxonomic novelty. Four new genera and nine new species could be identified within the archaeal family Halobacteriaceae, as well as one new bacterial species, and a representative of Salinibacter ruber phylotype II, a group that had been refractory to isolation for the last fifteen years. Altogether, the results indicated that in order to provide better yields for the retrieval of novel taxa from the environment, performance of non-redundant environment sampling is recommended together with the screening of large sets of strains.The current study was funded with the scientific support given by the Spanish Ministry of Economy through the projectsCGL2012-39627-C03-01 and CGL2012-39627-C03-03, which were also supported with European Regional Development Fund (FEDER) funds, and the preparatory phase of the Microbial Resource Research Infrastructure (MIRRI) funded by the EU (grant number 312251). In addition, the funding from competitive research groups (Microbiology) of the Government of the Balearic Islands (also co-supported with FEDER funds), is also acknowledged. TVP acknowledges the predoctoral fellowship of the Ministerio de Economía y Competitividad of the Spanish Government for the FPI fellowship (Nr BES-2013-064420) supporting his research activities

Solar salterns as model systems to study the units of bacterial diversity that matter for ecosystem functioning

Microbial communities often harbor overwhelming species and gene diversity, making it challenging to determine the important units to study this diversity. We argue that the reduced, and thus tractable, microbial diversity of manmade salterns provides an ideal system to advance this cornerstone issue. We review recent time-series genomic and metagenomic studies of the saltern-dominating bacterial and archaeal taxa to show that these taxa form persistent, sequence-discrete, species-like populations. While these populations harbor extensive intra-population gene diversity, even within a single saltern site, only a small minority of these genes appear to be functionally important during environmental perturbations. We outline an approach to detect and track such populations and their ecologically important genes that should be broadly applicable.The US National Science Foundation, the Spanish Ministry of Science, Innovation and Universities which were supported with European Regional Development Fund (FEDER) funds.http://www.journals.elsevier.com/current-opinion-in-biotechnologyhj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Ancient saltern metagenomics: tracking changes in microbes and their viruses from the underground to the surface

Microbial communities in hypersaline underground waters derive from ancient organisms trapped within the evaporitic salt crystals and are part of the poorly known subterranean biosphere. Here, we characterized the viral and prokaryotic assemblages present in the hypersaline springs that dissolve Triassic-Keuper evaporite rocks and feed the Anana Salt Valley (Araba/Alava, Basque Country, Spain). Four underground water samples (around 23% total salinity) with different levels of exposure to the open air were analysed by means of microscopy and metagenomics. Cells and viruses in the spring water had lower concentrations than what are normally found in hypersaline environments and seemed to be mostly inactive. Upon exposure to the open air, there was an increase in activity of both cells and viruses as well as a selection of phylotypes. The underground water was inhabited by a rich community harbouring a diverse set of genes coding for retinal binding proteins. A total of 35 viral contigs from 15 to 104 kb, representing partial or total viral genomes, were assembled and their evolutionary changes through the spring system were followed by SNP analysis and metagenomic island tracking. Overall, both the viral and the prokaryotic assemblages changed quickly upon exposure to the open air conditions.We would like to thank Anana Salt Valley Foundation, and Andoni Erkiaga Agirre, its director at the time of sampling, for their kind help. Thanks to Leire Arana, Edorta Loma and Kika Colom for their help with sampling and to Eduardo Gonzalez-Pastor for telling us about the Anana Salt Valley. We thank Heather Maughan for the professional English editing and the critical reading of the manuscript and Esther Rubio-Portillo for her help with statistical analyses. This work was funded by the Spanish Ministry of Science, Innovation and Universities grant MICROMATES (PGC2018-096956-B-C41 and C44, to J.A./F.S. and R.R.-M.), which was also supported with European Regional Development Fund (FEDER) funds, and by the Generalitat Valenciana grant PROMETEO/2017/129. Documen

Food determines ephemerous and non-stable gut microbiome communities in juvenile wild and farmed Mediterranean fish

Novel insights were provided by contrasting the composition of wild and farmed fish gut microbiomes because the latter had essentially different environmental conditions from those in the wild. This was reflected in the gut microbiome of the wild Sparus aurata and Xyrichtys novacula studied here, which showed highly diverse microbial community structures, dominated by Proteobacteria, mostly related to an aerobic or microaerophilic metabolism, but with some common shared major species, such as Ralstonia sp. On the other hand, farmed non-fasted S. aurata individuals had a microbial structure that mirrored the microbial composition of their food source, which was most likely anaerobic, since several members of the genus Lactobacillus, probably revived from the feed and enriched in the gut, dominated the communities. The most striking observation was that after a short fasting period (86 h), farmed gilthead seabream almost lost their whole gut microbiome, and the resident community associated with the mucosa had a very much reduced diversity that was highly dominated by a single potentially aerobic species Micrococcus sp., closely related to M. flavus. The results pointed to the fact that, at least for the juvenile S. aurata studied, most of the microbes in the gut were transient and highly dependent on the feed source, and that only after fasting for at least 2 days could the resident microbiome in the intestinal mucosa be determined. Since an important role of this transient microbiome in relation to fish metabolism could not be discarded, the methodological approach needs to be well designed in order not to bias the results. The results have important implications for fish gut studies that could explain the diversity and occasional contradictory results published in relation to the stability of marine fish gut microbiomes, and might provide important information for feed formulation in the aquaculture industry.info:eu-repo/semantics/publishedVersio

The low diverse gastric microbiome of the jellyfish Cotylorhiza tuberculata is dominated by four novel taxa

Cotylorhiza tuberculata is an important scyphozoan jellyfish producing population blooms in the Mediterranean probably due to pelagic ecosystem's decay. Its gastric cavity can serve as a simple model of microbial–animal digestive associations, yet poorly characterized. Using state-of-the-art metagenomic population binning and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we show that only four novel clonal phylotypes were consistently associated with multiple jellyfish adults. Two affiliated close to Spiroplasma and Mycoplasma genera, one to chlamydial ‘Candidatus Syngnamydia’, and one to bacteroidetal Tenacibaculum, and were at least one order of magnitude more abundant than any other bacteria detected. Metabolic modelling predicted an aerobic heterotrophic lifestyle for the chlamydia, which were found intracellularly in Onychodromopsis-like ciliates. The Spiroplasma-like organism was predicted to be an anaerobic fermenter associated to some jellyfish cells, whereas the Tenacibaculum-like as free-living aerobic heterotroph, densely colonizing the mesogleal axis inside the gastric filaments. The association between the jellyfish and its reduced microbiome was close and temporally stable, and possibly related to food digestion and protection from pathogens. Based on the genomic and microscopic data, we propose three candidate taxa: ‘Candidatus Syngnamydia medusae’, ‘Candidatus Medusoplasma mediterranei’ and ‘Candidatus Tenacibaculum medusae’.This research from RRM’s group was partially supported by the Spanish Ministry of Economy projects CGL2012-39627-C03-03 and CLG2015_66686-C3-1-P, which were also supported with European Regional Development Fund (FEDER) funds. KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1241046). RRM acknowledges the economic support of grant PR2015-00008 included in the program Salvador de Madariaga of the Ministry of Education, Culture and Sports in order to undertake a research stay at the MPI-MM in Bremen. TVP acknowledges the predoctoral fellowship of the Ministerio de Economía y Competitividad of the Spanish Government for the FPI fellowship (Nr BES-2013-064420) supporting his research activities

Microbial Motility at the Bottom of North America: Digital Holographic Microscopy and Genomic Motility Signatures in Badwater Spring, Death Valley National Park

Motility is widely distributed across the tree of life and can be recognized by microscopy regardless of phylogenetic affiliation, biochemical composition, or mechanism. Microscopy has thus been proposed as a potential tool for detection of biosignatures for extraterrestrial life; however, traditional light microscopy is poorly suited for this purpose, as it requires sample preparation, involves fragile moving parts, and has a limited volume of view. In this study, we deployed a field-portable digital holographic microscope (DHM) to explore microbial motility in Badwater Spring, a saline spring in Death Valley National Park, and complemented DHM imaging with 16S rRNA gene amplicon sequencing and shotgun metagenomics. The DHM identified diverse morphologies and distinguished run-reverse-flick and run-reverse types of flagellar motility. PICRUSt2- and literature-based predictions based on 16S rRNA gene amplicons were used to predict motility genotypes/phenotypes for 36.0-60.1% of identified taxa, with the predicted motile taxa being dominated by members of Burkholderiaceae and Spirochaetota. A shotgun metagenome confirmed the abundance of genes encoding flagellar motility, and a Ralstonia metagenome-assembled genome encoded a full flagellar gene cluster. This study demonstrates the potential of DHM for planetary life detection, presents the first microbial census of Badwater Spring and brine pool, and confirms the abundance of mobile microbial taxa in an extreme environment.R.R.-M. research was supported by the Spanish Ministry of Science, Innovation and Universities projects PGC2018-096956-B-C41 and PRX18/00048, both also supported with European Regional Development Fund (FEDER) funds.Peer reviewe

Predominance of deterministic microbial community dynamics in salterns exposed to different light intensities

While the dynamics of microbial community assembly driven by environmental perturbations have been extensively studied, our understanding is far from complete, particularly for light‐induced perturbations. Extremely halophilic communities thriving in coastal solar salterns are mainly influenced by two environmental factors—salt concentrations and high sunlight irradiation. By experimentally manipulating light intensity through the application of shading, we showed that light acts as a deterministic factor that ultimately drives the establishment of recurrent microbial communities under near‐saturation salt concentrations. In particular, the stable and highly change‐resistant communities that established under high‐light intensities were dominated (>90% of metagenomic reads) by Haloquadratum spp. and Salinibacter spp. On the other hand, under 37‐fold lower light intensity, different, less stable and change‐resistant communities were established, mainly dominated by yet unclassified haloarchaea and relatively diverse photosynthetic microorganisms. These communities harboured, in general, much lower carotenoid pigment content than their high‐irradiation counterparts. Both assemblage types appeared to be highly resilient, re‐establishing when favourable conditions returned after perturbation (i.e. high‐irradiation for the former communities and low‐irradiation for the latter ones). Overall, our results revealed that stochastic processes were of limited significance to explain these patterns.This study was funded by the Spanish Ministry of Economy projects CGL2012-39627-C03-03 CLG2015_66686-C3-1-P and PGC2018-096956-B-C41 (to R.R.M.), CGL2015_66686-C3-3-P (to J.A.) and CGL2015_66686-C3-2-P (to J.E.G.P.), which were also supported with European Regional Development Fund (FEDER) funds. R.A. was funded by the Max Planck Society. KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1831582). T.V.P. received a pre-doctoral fellowship (No. BES-2013-064420) from the Spanish Government Ministry for Finance and Competition. R.R.M. acknowledges the financial support of the sabbatical stay at Georgia Tech supported by the Grant PRX18/00048 of the Ministry of Sciences, Innovation and Universities

Genomic comparison between members of the Salinibacteraceae family, and description of a new species of Salinibacter (Salinibacter altiplanensis sp. nov.) isolated from high altitude hypersaline environments of the Argentinian Altiplano

The application of tandem MALDI-TOF MS screening with 16S rRNA gene sequencing of selected isolates has been demonstrated to be an excellent approach for retrieving novelty from large-scale culturing. The application of such methodologies in different hypersaline samples allowed the isolation of the culture-recalcitrant Salinibacter ruber second phylotype (EHB-2) for the first time, as well as a new species recently isolated from the Argentinian Altiplano hypersaline lakes. In this study, the genome sequences of the different species of the phylum Rhodothermaeota were compared and the genetic repertoire along the evolutionary gradient was analyzed together with each intraspecific variability. Altogether, the results indicated an open pan-genome for the family Salinibacteraceae, as well as the codification of relevant traits such as diverse rhodopsin genes, CRISPR-Cas systems and spacers, and one T6SS secretion system that could give ecological advantages to an EHB-2 isolate. For the new Salinibacter species, we propose the name Salinibacter altiplanensis sp. nov. (the designated type strain is AN15T = CECT 9105T = IBRC-M 11031T).This study was funded by the Spanish Ministry of Economy projects CGL2012-39627-C03-03 and CLG2015-66686-C3-1-P (to RRM), CLG2015-66686-C3-3-P (to JA), which were also both supported with European Regional Development Fund (FEDER) funds. RA was financed by the Max Planck Society. KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1241046). TVP acknowledges a pre-doctoral fellowship (Nr BES-2013-064420) from the Ministerio de Economía y Competitividad of the Spanish Government. The authors also acknowledge the economic support and unconditional scientific interest of Lipotrue SL and Deep Blue Sea SL

Food determines ephemerous and non-stable gut microbiome communities in juvenile wild and farmed Mediterranean fish

Novel insights were provided by contrasting the composition of wild and farmed fish gut microbiomes because the latter had essentially different environmental conditions from those in the wild. This was reflected in the gut microbiome of the wild Sparus aurata and Xyrichtys novacula studied here, which showed highly diverse microbial community structures, dominated by Proteobacteria, mostly related to an aerobic or microaerophilic metabolism, but with some common shared major species, such as Ralstonia sp. On the other hand, farmed non-fasted S. aurata individuals had a microbial structure that mirrored the microbial composition of their food source, which was most likely anaerobic, since several members of the genus Lactobacillus, probably revived from the feed and enriched in the gut, dominated the communities. The most striking observation was that after a short fasting period (86 h), farmed gilthead seabream almost lost their whole gut microbiome, and the resident community associated with the mucosa had a very much reduced diversity that was highly dominated by a single potentially aerobic species Micrococcus sp., closely related to M. flavus. The results pointed to the fact that, at least for the juvenile S. aurata studied, most of the microbes in the gut were transient and highly dependent on the feed source, and that only after fasting for at least 2 days could the resident microbiome in the intestinal mucosa be determined. Since an important role of this transient microbiome in relation to fish metabolism could not be discarded, the methodological approach needs to be well designed in order not to bias the results. The results have important implications for fish gut studies that could explain the diversity and occasional contradictory results published in relation to the stability of marine fish gut microbiomes, and might provide important information for feed formulation in the aquaculture industry.This study was funded by the Spanish Ministry of Science and Innovation projects CTM2017-91490-EXP, PGC2018-096956-B-C41, RTC-2017-6405-1 and PID2021-126114NB-C42, which were also supported by the European Regional Development Fund (FEDER). RRM acknowledges financial support from a sabbatical stay at Helmholz Zentrum München by grant PRX21/00043, which was also from the Spanish Ministry of Science, Innovation and Universities. TV acknowledges the “Margarita Salas” postdoctoral grant, funded by the Spanish Ministry of Universities, within the framework of the Recovery, Transformation and Resilience Plan funded by the European Union (NextGenerationEU), with the participation of the University of the Balearic Islands (UIB). AP was supported by an FPI pre-doctoral fellowship (ref. FPI/2269/2019) from the Balearic Islands Government General Direction of Innovation and Research. AR was supported by a pre-doctoral grant (PRE2019-091259) linked to the ADIPOQUIZ project (RTI2018-095653-R-I00), funded by the Spanish Ministry of Science and Innovation.The research was carried out within the framework of the activities of the Spanish Government through the “Maria de Maeztu Centre of Excellence” accreditation to IMEDEA (CSIC-UIB) (CEX2021-001198).With funding from the Spanish government through the ‘María de Maeztu Unit of Excelence’ accreditation (CEX2021-001198).Peer reviewe

Description of two cultivated and two uncultivated new Salinibacter species, one named following the rules of the bacteriological code: Salinibacter grassmerensis sp. nov.; and three named following the rules of the SeqCode: Salinibacter pepae sp. nov., Salinibacter abyssi sp. nov., and Salinibacter pampae sp. nov.

Current -omics methods allow the collection of a large amount of information that helps in describing the microbial diversity in nature. Here, and as a result of a culturomic approach that rendered the collection of thousands of isolates from 5 different hypersaline sites (in Spain, USA and New Zealand), we obtained 21 strains that represent two new Salinibacter species. For these species we propose the names Salinibacter pepae sp. nov. and Salinibacter grassmerensis sp. nov. (showing average nucleotide identity (ANI) values < 95.09% and 87.08% with Sal. ruber M31T, respectively). Metabolomics revealed species-specific discriminative profiles. Sal. ruber strains were distinguished by a higher percentage of polyunsaturated fatty acids and specific N-functionalized fatty acids; and Sal. altiplanensis was distinguished by an increased number of glycosylated molecules. Based on sequence characteristics and inferred phenotype of metagenome-assembled genomes (MAGs), we describe two new members of the genus Salinibacter. These species dominated in different sites and always coexisted with Sal. ruber and Sal. pepae. Based on the MAGs from three Argentinian lakes in the Pampa region of Argentina and the MAG of the Romanian lake Fără Fund, we describe the species Salinibacter pampae sp. nov. and Salinibacter abyssi sp. nov. respectively (showing ANI values 90.94% and 91.48% with Sal. ruber M31T, respectively). Sal. grassmerensis sp. nov. name was formed according to the rules of the International Code for Nomenclature of Prokaryotes (ICNP), and Sal. pepae, Sal. pampae sp. nov. and Sal. abyssi sp. nov. are proposed following the rules of the newly published Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode). This work constitutes an example on how classification under ICNP and SeqCode can coexist, and how the official naming a cultivated organism for which the deposit in public repositories is difficult finds an intermediate solution.This study was funded by the Spanish Ministry of Science, Innovation and Universities projects PGC2018-096956-B-C41, RTC-2017-6405-1 and PID2021-126114NB-C42, which were also supported by the European Regional Development Fund (FEDER). RRM acknowledges the financial support of the sabbatical stay at Georgia Tech and HelmholzZentrum München by the grants PRX18/00048 and PRX21/00043 respectively also from the Spanish Ministry of Science, Innovation and Universities. This research was carried out within the framework of the activities of the Spanish Government through the “Maria de Maeztu Centre of Excellence” accreditation to IMEDEA (CSIC-UIB) (CEX2021-001198). KTK’s research was supported, in part, by the U.S. National Science Foundation (Award No. 1831582 and No. 2129823). IMG. AC and HLB were financially supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI – UEFISCDI, project number PN-III-P4-ID-PCE-2020-1559, within PNCDI III. HLB acknowledges Ocna Sibiului City Hall (Sibiu County, Romania) for granting the access to Fără Fund Lake and A. Baricz and D.F. Bogdan for technical support during sampling and sample preparation. MBS thanks Dominion Salt for their assistance in sample Lake Grassmere. MELL acknowledges the financial support of the Argentinian National Scientific and Technical Research Council (Grant CONICET-NSFC 2017 N° IF-2018-10102222-APN-GDCT-CONICET) and the National Geographic Society (Grant # NGS 357R-18). BPH was supported by NASA (award 80NSSC18M0027). TV acknowledges the “Margarita Salas” postdoctoral grant, funded by the Spanish Ministry of Universities, within the framework of Recovery, Transformation and Resilience Plan, and funded by the European Union (NextGenerationEU), with the participation of the University of Balearic Islands (UIB)