Discovery of the streamlined haloarchaeon Halorutilus salinus, comprising a new order widespread in hypersaline environments across the world

Discovery of the Streamlined Haloarchaeon Halorutilus salinus, Comprising a New Order Widespread in Hypersaline Environments across the World Ana Durán-Viseras,a,b Cristina Sánchez-Porro,aTomeu Viver,cKonstantinos T. Konstantinidis,bAntonio Ventosaa a Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain b School of Civil & Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA c Marine Microbiology Group, Department of Animal and Microbial Biodiversity, Mediterranean Institute for Advanced Studies (IMEDEA, CSIC-UIB), Esporles, Spain ABSTRACT The class Halobacteria isoneofthemostdiversegroupswithinthe Euryarchaeota phylum, whose members are ubiquitously distributed in hypersaline environments, where they often constitute the major population. Here, we report the discovery and isolation of a new halophilic archaeon, strain F3-133 T exhibiting #86.3% 16S rRNA gene identity to any previously cultivated archaeon, and, thus, representing a new order. Analysis of available 16S rRNA gene amplicon and metagenomic data sets showed that the new isolate represents an abundant group in intermediate-to-high salinity ecosystems and is widely distributed across the world. The isolate presents a streamlined genome, which probably accounts for its ecological success in nature and its fastidious growth in culture. The predominant osmoprotection mechanism appears to be the typical salt-in strategy used by other haloarchaea. Furthermore, the genome contains the complete gene set for nucleotide monophosphate degradation pathway through archaeal RuBisCO, being within the first halophilic archaea representatives reported to code this enzyme. Genomic comparisons with previously described representatives of the phylum Euryarchaeota were consistent with the 16S rRNA gene data in supporting that our isolate represents a novel order within the class Halobacteria for which we propose the names Halorutilales ord. nov., Halorutilaceae fam. nov., Halorutilus gen. nov. and Halorutilus salinus sp. nov. IMPORTANCE The discovery of the new halophilic archaeon, Halorutilus salinus, representing a novel order, family, genus, and species within the class Halobacteria and phylum Euryarchaeota clearly enables insights into the microbial dark matter, expanding the current taxonomical knowledge of this group of archaea. The indepth comparative genomic analysis performed on this new taxon revealed one of the first known examples of an Halobacteria representative coding the archaeal RuBisCO gene and with a streamlined genome, being ecologically successful in nature and explaining its previous non-isolation. Altogether, this research brings light into the understanding of the physiology of the Halobacteria class members, their ecological distribution, and capacity to thrive in hypersaline environments. KEYWORDS hypersaline environments, Halorutilales ord. nov., comparative genomics, streamlining, RuBisCO, microbial ecology, osmoadaptation Hypersaline ecosystems are extreme environments characterized by high salt concentrations, much greater than that of seawater and often close to salt saturation (1, 2). The interest of the scientific community on hypersaline environments have been hugely increased as possible analogues to life on other planets such as Mars (3–5), as well as by its biotechnological potential in the biomedicine and biotechnology fields (6–8). Editor Xiao-Hua Zhang, Ocean University of China Copyright © 2023 Durán-Viseras et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Antonio Ventosa, [email protected], or Konstantinos T. Konstantinidis, [email protected]. The authors declare no conflict of interest. Received 30 November 2022 Accepted 21 February 2023 Published 21 March 2023 March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 1 RESEARCH ARTICLE Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. These ecosystems, whether natural or manmade, are characterized by severe stresses for microbial organisms, due not only to high salt concentration, but to other factors, such as low nutrient and oxygen availability, alkalinity, high UV irradiance, and the possible presence of toxic compounds and heavy metals (2, 9). These conditions have selected for low class/family diversity, generally consisting of two major lineages i.e., the archaeal Halobacteria class and the bacterial family of Salinibacteraceae,class Rhodothermia (10, 11), but with relatively high species richness (microdiversity) within each class (12, 13). Members of the class Halobacteria are among the best adapted organisms to deal with the high salt concentration encountered in those habitats (14, 15), and are widely distributed in hypersaline systems, from marine salterns to salt lakes, coastal sabkhas, salt mines, hypersaline soda lakes, hypersaline soils, and salt-fermented seafood (15–17). The class Halobacteria of the phylum Euryarchaeota isoneofthemostdiversegroups within the domain Archaea that, at the time of this writing, is constituted by 71 genera and 308 species (18, 19). Currently, the class Halobacteria is divided into 3 orders (Halobacteriales, Haloferacales,andNatrialbales), that each comprises 3 (Haloarculaceae,Halobacteriaceae,and Halococcaceae), 2 (Haloferacaceae and Halorubraceae)and1(Natrialbaceae) families, respectively (7, 20, 21). Although traditional cultivation techniques have led to the isolation and description of hundreds of new groups of halophilic bacteria and archaea (19, 22–30), a great number of recent culture-independent studies from hypersaline habitats have revealed the prominence of several yet-uncultivated lineages in these systems (31–34). Most of these uncultivated groups represent dominant populations in hypersaline environments (31, 32, 35, 36), and are presumably slow growing and fastidious to isolate microorganisms. Thus, persistent efforts and improvements of the cultivation methodologies are still needed to bring these uncultivated lineages to culture (37). Within this context the term “culturomics”was coined to denote the application of high-throughput culturing efforts for the identification of a higher microbial culturable diversity (38). Our recent exploration of the microbial diversity in Isla Cristina salterns (Huelva, Spain) following the “culturomics”approach (39, 40), has yielded the isolation of several novel haloarchaeal taxa, such as the new genera Halosegnis (40) and Haloglomus (26),aswellas several new species of the genera Halonotius (41, 42) and Natronomonas (43). Indeed, subsequent ecological studies highlighted the wide geographical distribution of these groups and their substantial abundance in different hypersaline systems (26, 40, 42, 44–47). As part of the same isolation campaign in the multi-pond, manmade Isla Cristina saltern, a novel haloarchaeon, designated as strain F3-133 T , was isolated in pure culture. Based on the genome-aggregate average amino acid identity (AAI; #51.7%) and 16S rRNA gene (#86.3% identity), strain F3-133 T was shown to be distantly related to any previously described haloarchaea. Here, we report the characteristic of its genome sequence, its environmental distribution based on available 16S rRNA gene amplicon and metagenomic data sets, as well as the ecophysiological strategies that underly its ecological success. RESULTS AND DISCUSSION Isolation of strain F3-133 T by culturomics. The culturomics approach was applied with the aim of isolating and characterizing the functional roles of the abundant saltern taxa lacking currently cultured representatives. Based on previously successful methodological approaches, for the isolation of other elusive microorganisms (26, 40), several growth media and conditions were tested. The water samples collected from the different saltern ponds of Isla Cristina (37° 129N7°199W) were serially diluted and plated on the different culture media. Plates were subsequently incubated under different conditions (aerobic, microaerophilic, or anaerobic, in dark or light) and for longtime periods (up to 3 months). During this period, colonies appearing on plates were carefully observed, and those of fastest growth and large morphology were labeled in order to be further discarded. Afterwards, an arduous high-throughput isolation screening of those unlabeled colonies with slow growth and tiny morphology was conducted Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 2 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. resulting in more than 2,000 newly-isolated-single-colonies. These isolates were identified by 16S rRNA gene amplification. Isolates likely to represent new groups of microorganisms based on 16S rRNA gene identity, were selected for further characterization. Among them, 1 tiny, red-pigmented colony (,0.5 mm) with extremely slow growth, designated strain F3-133 T , exhibited a remarkably low relatedness with any previously cultivated haloarchaea. Strain F3-133 T was isolated from a culture medium containing pyruvate, which indeed has been considered as a key compound for the isolation of fastidious microorganisms (48), such as the halophilic archaeon Haloquadratum waslbyi,orthestreamlined halophilic and marine bacteria Spiribacter salinus and Pelagibacter ubique,among others. Due to its very slow and limited growth under laboratory conditions, several attempts were required before the successful subculturing of strain F3-133 T .Thepurityof the strain was subsequently checked by the colony morphology in the plate, microscopy and 16S rRNA gene amplification. Once its purity was confirmed, its genome was sequenced for further analyses. Genome-based phylogeny and taxonomy. Complete 16S rRNA gene sequence (1470 nt) comparison of strain F3-133 T against available sequences in public databases revealed relative low relatedness (#86.3% identity) with any previously cultivated microorganism. Strain F3-133 T proved to be most closely related to Halobacteria class representatives. Based on recently recommended values (e.g., 83 to 86% and 86 to 89% for the class-level and order-level classification, respectively [49, 50]), such 16S rRNA gene identity values indicate that strain F3-133 T represents a new order or even a new class within the phylum Euryarchaeota. In order to further clarify the taxonomic position of this strain, a concatenated alignment of 62 single-copy genes shared between strain F3-133 T and representative members of different euryarchaeotal classes was used for an approximate maximum-likelihood tree reconstruction (Fig. 1A). Based on these results, strain F3-133 T represents an independent clade, a sister taxon to the class Halobacteria, with 100% bootstrap support (Fig. 1A). Consistent with this phylogeny-based picture, strain F3-133 T exhibited the highest AAI values (50 to 51%) to representatives of the class Halobacteria (Fig. 2A), with AAI values among Halobacteria members being substantially higher (58 to 62%) (Fig. 2A). Further comparison of strain F3-133 T with all type species from all orders and families of the class Halobacteria with available genomes supported its placement as a distinct phylogenomic branch within the class (Fig. 1B), which was also consistent with the AAI values among the same genomes (Fig. 2B). Collectively, it is clear enough that the evidence from 16S rRNA gene, universal protein-coding gene phylogenies and AAI analysis consistently FIG 1 Approximate maximum-likelihood phylogenetic tree based on the concatenated amino acid sequence alignment of 62 (A) and 94 (B) single-copy genes. Shown are strain F3-133 T and the type strains of the type species of each type genus within all families of the class Halobacteria and representative members from the other classes of the phylum Euryarchaeota (A), and strain F3-133 T and the type strains of all type species within the class Halobacteria (B). The DPANN representatives Candidatus Nanosalinarum J07AB56 and Candidatus Nanosalina J07AB43 were used as outgroups. Genome accession numbers are shown in parentheses. Bootstrap values $70% are shown at branch points. Scale bars denotes 0.1 (A) and 1.0 (B) changes per nucleotide position. Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 3 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. FIG 1 (Continued) Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 4 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. and clearly support the proposal of strain F3-133 T as a novel order within the class Halobacteria, currently represented by a single family, genus, and species. Furthermore, 16S rRNA gene phylogenetic analysis of strain F3-133 T and closely related environmental sequences from publicly available clone library data sets revealed that there might be other new species or genera within the proposed new family and order to be FIG 2 (A) Heatmap of AAI relatedness among strain F3-133 T and the type strains of all type genera of the class Halobacteria and representative members of the other classes of the phylum Euryarchaeota. (B) Heatmap of AAI relatedness among strain F3-133 T and the type strains of all type species within the class Halobacteria with available genomes. Genome accession numbers are specified on Fig. 1B and Table S4. Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 5 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. described in the future (Fig. S1). The genomic sequences of representatives of these groups will provide better resolution of the class status and a more thorough taxonomic description of its extant diversity in the future. Ecological distribution. To gain insight into the ecological distribution and relative abundance of strain F3-133 T in hypersaline environments from around the world, a large number of available metagenomic and 16S rRNA gene amplicons data sets were screened (Tables S1 and S2). Among ;200 metagenomes assessed (Table S2), only three provided significant read recruitment plots against the F3-133 T genome above the limit of detection of the metagenomic sequencing effort applied (Fig. 3). Specifically, F3-133 T -like populations were present in the environment of origin, the brine of a pond of Isla Cristina saltern (42% total salts), as well as in a solar saltern in Velddrif (South Africa; 15% total salts) and a natural saline lake in the Chilean Altiplano (34% total salts) (Fig. 3), with a relative abundance of 0.1%, 0.03% and 0.02% of the total community, respectively. These results may indicate that strain F3-133 T increases its population abundance from non-detectable levels (rare biosphere) to an abundant member of the community under its optimal environmental conditions. Therefore, these results not only revealed that strain F3-133 T constitutes an abundant member of the microbial community in hypersaline environments widely distributed across the globe, but also reflected its versatility, being able to inhabit intermediate-to-high salinity systems. Moreover, the high number of reads recruiting below 95% identity in all three data sets suggested that there are likely additional, closely related, yet-to-be described species that are abundant (in order to be detected in the corresponding metagenomes) in these environments. Further, screening of 16S rRNA gene amplicon or clone library data sets for close matches to the 16S rRNA gene sequence of F3-133 T revealed a remarkable number of sequences (1024 amplicons and 3 clones) related at the species (.98.6% identity), genus (.95% identity), and family (.92% identity) levels in samples from inland and coastal hypersaline environments in Australia, Romania, Tunisia, or USA (Fig. 4A). The salinities of those environments ranged from 5.1% total salts (Lake Strawbridge sediment) to 34% total salts (Bajool saltern) (Table S1), which are indeed comparable to the salinities of the metagenomes in which strain F3-133 T was found to be abundant (as specified above), further supporting the widespread presence of strain F3-133 T and related species in a broad range of salinity niches. With respect to 16S rRNA gene amplicon data set matches at genus level, the highest relative abundances were found in the sediments of Strawbridge Lake (Australia) (up to 0.9% of the total community) (Fig. 4B). However, the number of matches at species level in this environment were much fewer (Fig. 4B), reaching the highest relative abundance (0.3% of the total) in the meromictic hypersaline lake of Fara Fund (Romania). This fact, together with the metagenomic-based results mentioned above, enforce the hypothesis that aquatic niches are the preferred habitat for this group of microorganisms. Overall, these results further corroborated the worldwide distribution of F3-133 T and related species in a wide range of ecological niches associated with natural and manmade salterns, and suggested that more isolation efforts are needed to explore the extant diversity of this group of archaea. General genome characteristics. ThemaingenomecharacteristicsofstrainF3-133 T are provided in Table S3. One of the most remarkable features is the relatively small genome size of this new taxon (2.1 Mb) compared to other characterized halophilic archaeal species. To the best our knowledge, this is within the smallest genome sizes reported for any described member of the class Halobacteria to date (together with Halodesulfurarchaeum formicicum HSR6 T ,Halanaeroarchaeum sulfurireducens HSR2 T ,andHalobacterium salinarum NRC-1 T chromosome), with the available Halobacteria genomes to average ;4Mb (Table S4). Small genomes in Archaea and Bacteria representatives are frequently associated with parasitic or symbiotic lifestyles, although free-living members in environments with low nutrient availability, such as aquatic ecosystems, can also exhibit small genomic sizes in comparison to nutrient-rich terrestrial ecosystems (51, 52). Moreover, smaller genome size species are often associated with an auxotrophic lifestyle (51, 53). These Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 6 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. FIG 3 Read recruitment plot of metagenomic reads from (A) Isla Cristina solar saltern (PRJNA890281), (B) Velddrif solar saltern (ERR5981341), and (C) Chilean Altiplano lake against the genomic sequence (Continued on next page) Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 7 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. characteristics may also explain the preference of strain F3-133 T and relatives to aquatic environments (as discussed above), as well as its narrow nutritional requirements (strain F3-133 T can be only cultured in a defined medium with pyruvate, and is unable to use other compounds as carbon and energy sources) that presumably led to its fastidious growth during our culturomics isolation efforts. Further, autotrophy genes were also present in the F3-133 T genome, indicating that it can be truly free-living. Moreover, a single rRNA operon was identified in the strain F3-133 T genome, consistent with an oligotrophic lifestyle (54). The presence of a single rRNA operon may also be related to the slow growth phylotype (54) exhibited by strain F3-133 T ,whichalsomakes its cultivation under laboratory conditions challenging. Notably, the genome of strain F3- 133 T is relatively low in DNA G1C content (59.8 mol%) in comparison with other halophilic archaea, which usually have G1C contents well above 60 mol% (Table S4), with the only known exceptions being Haloquadratum walsbyi (47.8 mol%), Halocatena pleomorpha SPPAMP-1 T (57.1 mol%), Halonotius pteroides CECT 7525 T (59.5 mol%), and representatives of the Candidatus phylum Nanohaloarchaea (;40 mol%) (55). The genome of strain F3-133 T revealed a very low percentage of non-coding sequences and an outstanding short median and average intergenic spacers (Fig. S2) in comparison to any previously cultivated Halobacteria. Altogether, these unique features indicate a streamlined genomic strategy for this strain (51, 56). Specifically, under some circumstances, selection may lead to a genomic complexity reduction in organisms that have large effective population sizes and resting cell stages or periods without significance growth, resulting in ecological successful strategies in nature, as for Pelagibacter ubique (57–59). Advances in multi-omics techniques revealed that the number of streamlined genomes in nature has been previously underestimated due to their fastidious growth under laboratory conditions (60). Besides, genome reduction is frequently associated with unusual nutritional requirements that necessitate specific cultivation approaches (56). In fact, strain F3-133 T was isolated using a culture medium similar to the ones used for growing other streamlined organisms. Indeed, the streamlined genome of strain F3-133 T probably accounts, at least in part, for the remarkable abundance and the worldwide-distribution of this new taxon in different hypersaline environments, but also to the previously unsuccessful attempts to isolate it. Lastly, small genome organisms are frequently related with relatively unchanging (stable) ecological niches, in contrast with fluctuating niches for which sigma factors play a crucial role (56). No sigma factors were identified in the isolate genome, corroborating its likely preferred distribution in aquatic ecosystems. Central metabolism. Based on the bioinformatically annotated genome, strain F3- 133 T encodes genes involved in gluconeogenesis, tricarboxylic acid cycle, and glyoxylate cycle (central carbohydrate metabolism; Fig. 5). However, as previously reported for other haloarchaea (40, 42, 61, 62), the gene coding for 6-phosphofructokinase involved in the lower part of the classical Embden–Meyerhof–Parnas (EM) pathway of glycolysis is absent in strain F3-133 T , which could be alternatively replaced by the oxidative pentose phosphate pathway (Fig. 5) (63). Strain F3-133 T also possesses the pyruvate ferredoxin oxidoreductase genes (porA and porB) involved in the oxidation of pyruvate to acetyl-CoA (Fig. 5). With respect to nitrogen metabolism, the genes encoding for nitrite reductases, i.e., nirA, nirB, and nirK, involved in assimilatory and dissimilatory nitrate reduction and denitrification, respectively, were all identified in the genome (Fig. 5). However, the remaining genes involved in these pathways, such as narB,narGHI,norBC,ornosZ, FIG 3 Legend (Continued) of strain F3-133 T . Within each plot: bottom middle panel represents the reads recruited, placed by position in the genome (X-axis) and percent of identity (Y-axis). Upper middle panel shows the sequencing depth per genome region in logarithmic scale. Bars at the bottom represent regions without mapping reads (sequencing depth of zero). Bottom right panel represents the number of nucleotide bases in logarithmic scale at each unit of nucleotide identity (yaxis) in the bottom middle panel. Upper right panel exhibits the sequencing depth histogram across the genome. Left panel shows the color scale for the number of stacked reads per 2-dimensional bin in the bottom middle panel. The dark blue or light blue colors in the background of panels or the color lines correspond to matches with identity above and below 95% nucleotide identity cutoff. Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 8 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. were not found, indicating that F3-133 T is probably doing only specific steps (modular) of denitrification. Strain F3-133 T also exhibited the high-affinity ammonium transporter (Amt) for ammonia uptake and the ABC transporter for the nitrogen derived compound spermidine (Fig. 5). The responsible genes for ammonia assimilation, glutamine synthetase, and glutamate synthase were also found in the genome (Fig. 5). Complete biosynthesis pathways of several amino acids (i.e., arginine, histidine, isoleucine, lysine, ornithine, proline, serine, threonine, and valine) were likewise identified. Consistent with its physiological characterization, genes encoding archaellum were encountered in the genome (Fig. 5). Inorganic phosphate might be incorporated to the cell via an ABC transporter (pstSCAB). Rhodopsin-like sequences such as a sensory rhodopsin FIG 4 (A) Global distribution of strain F3-133 T and related organisms in hypersaline environments based on 16S rRNA gene amplicon and clone library data sets. Circles denote the geographic location of the related 16S rRNA gene sequences found in different amplicon data sets, while squares represent the sequences from clone library data sets. The star symbol indicates the place where strain F3-133 T was isolated. (B) Relative abundance of 16S rRNA gene sequences related to F3-133 T relative to all amplicon sequences in the corresponding data sets (at the geographic location highlighted with circles in [A]) assigned to the (same) family, genus, and species as strain F3-333 T based on identity threshold as described in the Materials and Methods section. Note that in some samples same-species sequences dominated the relatives found (e.g., Fara Fund Lake, Romania) while in some other samples the related sequences were mostly assigned to different species and genera than F3-133 T (e.g., Strawbridge Lake, Australia). RNA indicates transcriptomic data, while by default genomic data is used. Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 9 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. Kayacik, Kaldirim and Yavsan Salterns (Turkey) assessed by 16S rRNA amplicon sequencing. Microorganisms 9:1525. https://doi.org/10.3390/ microorganisms9071525. 45. Jookar Kashi F, Owlia P, Amoozegar MA, Kazemi B. 2021. Halophilic prokaryotes in Urmia Salt Lake, a hypersaline environment in Iran. Curr Microbiol 78:3230–3238. https://doi.org/10.1007/s00284-021-02583-w. 46. Martínez JM, Escudero C, Rodríguez N, Rubin S, Amils R. 2021. Subsurface and surface halophile communities of the chaotropic Salar de Uyuni. Environ Microbiol 23:3987–4001. https://doi.org/10.1111/1462-2920.15411. 47. Mani K, Taib N, Hugoni M, Bronner G, Bragança JM, Debroas D. 2020. Transient dynamics of archaea and bacteria in sediments and brine across a salinity gradient in a solar saltern of Goa, India. Front Microbiol 11:1891. https://doi.org/10.3389/fmicb.2020.01891. 48. Oren A. 2015. Pyruvate: a key nutrient in hypersaline environments? Microorganisms 3:407–416. https://doi.org/10.3390/microorganisms3030407. 49. Konstantinidis KT, Rosselló-Móra R, Amann R. 2017. Uncultivated microbes in need of their own taxonomy. ISME J 11:2399–2406. https://doi.org/10 .1038/ismej.2017.113. 50. Rosselló-Móra R, Amann R. 2015. Past and future species definitions for Bacteria and Archaea. Syst Appl Microbiol 38:209–216. https://doi.org/10 .1016/j.syapm.2015.02.001. 51. Rodríguez-Gijón A, Nuy JK, Mehrshad M, Buck M, Schulz F, Woyke T, Garcia SL. 2022. A genomic perspective across earth’s microbiomes reveals that genome size in Archaea and Bacteria is linked to ecosystem type and trophic strategy. Front Microbiol 12:1–9. https://doi.org/10.3389/fmicb.2021.761869. 52. Kellner S, Spang A, Offre P, Szöllo†si GJ, Petitjean C, Williams TA. 2018. Genome size evolution in the Archaea. Emerg Top Life Sci 2:595–605. https:// doi.org/10.1042/ETLS20180021. 53. Grote J, Thrash JC, Huggett MJ, Landry ZC, Carini P, Giovannoni SJ, Rappé MS. 2012. Streamlining and core genome conservation among highly divergent members of the SAR11 clade. mBio 3:e00252–e00312. https://doi .org/10.1128/mBio.00252-12. 54. Klappenbach JA, Dunbar JM, Schmidt TM. 2000. rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol 66: 1328–1333. https://doi.org/10.1128/AEM.66.4.1328-1333.2000. 55. Hamm JN, Erdmann S, Eloe-Fadrosh EA, Angeloni A, Zhong L, Brownlee C, Williams TJ, Barton K, Carswell S, Smith MA, Brazendale S, Hancock AM, Allen MA, Raftery MJ, Cavicchioli R. 2019. Unexpected host dependency of antarctic Nanohaloarchaeota. Proc Natl Acad Sci U S A 116:14661–14670. https://doi.org/10.1073/pnas.1905179116. 56. Giovannoni SJ, Thrash JC, Temperton B. 2014. Implications of streamlining theory for microbial ecology. ISME J 8:1553–1565. https://doi.org/10 .1038/ismej.2014.60. 57. Dufresne A, Garczarek L, Partensky F. 2005. Accelerated evolution associated with genome reduction in a free-living prokaryote. Genome Biol 6:R14. https://doi.org/10.1186/gb-2005-6-2-r14. 58. Lynch M, Conery JS. 2003. The origins of genome complexity. Science 302:1401–1404. https://doi.org/10.1126/science.1089370. 59. Giovannoni SJ, Tripp HJ, Givan S, Podar M, Vergin KL, Baptista D, Bibbs L, EadsJ,RichardsonTH,NoordewierM,RappéMS,ShortJM,CarringtonJC, Mathur EJ. 2005. Genetics: genome streamlining in a cosmopolitan oceanic bacterium. Science 309:1242–1245. https://doi.org/10.1126/science.1114057. 60. Button DK, Robertson BR. 2001. Determination of DNA content of aquatic bacteria by flow cytometry. Appl Environ Microbiol 67:1636–1645. https:// doi.org/10.1128/AEM.67.4.1636-1645.2001. 61. Falb M, Müller K, Königsmaier L, Oberwinkler T, Horn P, von Gronau S, Gonzalez O, Pfeiffer F, Bornberg-Bauer E, Oesterhelt D. 2008. Metabolism of halophilic archaea. Extremophiles 12:177–196. https://doi.org/10.1007/s00792 -008-0138-x. 62. Anderson I, Scheuner C, Göker M, Mavromatis K, Hooper SD, Porat I, Klenk H-P, Ivanova N, Kyrpides N. 2011. Novel insights into the diversity of catabolic metabolism from ten haloarchaeal genomes. PLoS One 6:e20237. https://doi.org/10.1371/journal.pone.0020237. 63. Verhees CH, Kengen SWM, Tuininga JE, Schut GJ, Adams MWW, de Vos WM, van der Oost J. 2003. The unique features of glycolytic pathways in Archaea. Biochem J 375:231–246. https://doi.org/10.1042/BJ20021472. 64. Sato T, Atomi H, Imanaka T. 2007. Archaeal type III RuBisCOs function in a pathway for AMP metabolism. Science 315:1003–1006. https://doi.org/10 .1126/science.1135999. 65. Schönheit P, Buckel W, Martin WF. 2016. On the origin of heterotrophy. Trends Microbiol 24:12–25. https://doi.org/10.1016/j.tim.2015.10.003. 66. Aono R, Sato T, Yano A, Yoshida S, Nishitani Y, Miki K, Imanaka T, Atomi H. 2012. Enzymatic characterization of amp phosphorylase and ribose-1,5- bisphosphate isomerase functioning in an archaeal amp metabolic pathway. J Bacteriol 194:6847–6855. https://doi.org/10.1128/JB.01335-12. 67. Bulzu PA, Andrei AS, Salcher MM, Mehrshad M, Inoue K, Kandori H, Beja O, Ghai R, Banciu HL. 2019. Casting light on Asgardarchaeota metabolism in a sunlit microoxic niche. Nat Microbiol 4:1129–1137. https://doi.org/10 .1038/s41564-019-0404-y. 68. Castelle CJ, Banfield JF. 2018. Major new microbial groups expand diversity and alter our understanding of the tree of life. Cell 172:1181–1197. https://doi.org/10.1016/j.cell.2018.02.016. 69. Sorokin D, Makarova KS, Abbas B, Ferrer M, Golyshin PN, Galinski EA, Ciordia S, Mena MC, Merkel AY, Wolf YI, Van Loosdrecht MCM, Koonin EV. 2017. Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis. Nat Microbiol 2:17081. https://doi.org/10.1038/nmicrobiol.2017.81. 70. Sato T, Utashima S, Yoshii Y, Hirata K, Kanda S, Onoda Y, Qiang Jin J, Xiao S, Minami R, Fukushima H, Noguchi A, Manabe Y, Fukase K, Atomi H. 2022. A non-carboxylating pentose bisphosphate pathway in halophilic archaea. Commun Biol 5:1–13. https://doi.org/10.1038/s42003-022-04247-2. 71. Oren A. 2011. Thermodynamic limits to microbial life at high salt concentrations. Environ Microbiol 13:1908–1923. https://doi.org/10.1111/j.1462 -2920.2010.02365.x. 72. Gunde-Cimerman N, Oren A, PlemenitašA. 2018. Strategies of adaptation of microorganisms of the three domains of life to high salt concentrations. FEMS Microbiol Rev 42:353–375. https://doi.org/10.1093/ femsre/fuy009. 73. Booth IR, Blount P. 2012. The MscS and MscL families of mechanosensitive channels act as microbial emergency release valves. J Bacteriol 194: 4802–4809. https://doi.org/10.1128/JB.00576-12. 74. Oren A, Ventosa A, Grant WD. 1997. Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47:233–238. https://doi.org/10.1099/00207713-47-1-233. 75. Oren A, Arahal DR, Ventosa A. 2009. Emended descriptions of genera of the family Halobacteriaceae. Int J Syst Evol Microbiol 59:637–642. https:// doi.org/10.1099/ijs.0.008904-0. 76. DeLong EF. 1992. Archaea in coastal marine environments. Proc Natl Acad Sci U S A 89:5685–5689. https://doi.org/10.1073/pnas.89.12.5685. 77. Arahal DR, Dewhirst FE, Paster BJ, Volcani BE, Ventosa A. 1996. Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences. Appl Environ Microbiol 62:3779–3786. https://doi.org/10.1128/aem.62.10.3779-3786.1996. 78. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021. 79. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: Assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043–1055. https:// doi.org/10.1101/gr.186072.114. 80. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. https:// doi.org/10.1093/bioinformatics/btt086. 81. Hyatt D, Chen G-L, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. https://doi.org/10.1186/1471-2105 -11-119. 82. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/ btu153. 83. Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428:726–731. https://doi.org/10.1016/j.jmb.2015.11 .006. 84. Kanehisa M, Sato Y, Kawashima M. 2022. KEGG mapping tools for uncovering hidden features in biological data. Protein Sci 31:47–53. https://doi .org/10.1002/pro.4172. 85. Rodriguez-R LM, Konstantinidis KT. 2016. The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ Preprints 4:e1900v1. https://doi.org/10.7287/peerj.preprints.1900v1. 86. Rice P, Longden L, Bleasby A. 2000. EMBOSS: the European molecular biology open software suite. Trends Genet 16:276–277. https://doi.org/10 .1016/s0168-9525(00)02024-2. 87. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J. 2017. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 16 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235. and whole-genome assemblies. Int J Syst Evol Microbiol 67:1613–1617. https://doi.org/10.1099/ijsem.0.001755. 88. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO. 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596. https:// doi.org/10.1093/nar/gks1219. 89. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. https://doi.org/10.1093/molbev/mst010. 90. Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313. https:// doi.org/10.1093/bioinformatics/btu033. 91. Edgar RC. 2004. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5:113. https:// doi.org/10.1186/1471-2105-5-113. 92. Price MN, Dehal PS, Arkin AP. 2010. FastTree 2 –approximately maximum-likelihood trees for large alignments. PLoS One 5:e9490. https://doi .org/10.1371/journal.pone.0009490. 93. Letunic I, Bork P. 2021. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 49:W293–W296. https://doi.org/10.1093/nar/gkab301. 94. Gerhardt K, Ruiz-Perez CA, Rodriguez-R LM, Conrad RE, Konstantinidis KT. 2022. RecruitPlotEasy: an advanced read recruitment plot tool for assessing metagenomic population abundance and genetic diversity. Front Bioinform 1:1–5. https://doi.org/10.3389/fbinf.2021.826701. 95. Lagkouvardos I, Joseph D, Kapfhammer M, Giritli S, Horn M, Haller D, Clavel T. 2016. IMNGS: a comprehensive open resource of processed 16S rRNA microbial profiles for ecology and diversity studies. Sci Rep 6:33721. https://doi.org/10.1038/srep33721. Streamlined New Haloarchaeon Halorutilus salinus mSystems March/April 2023 Volume 8 Issue 2 10.1128/msystems.01198-22 17 Downloaded from https://journals.asm.org/journal/msystems on 09 April 2024 by 150.214.182.235.