Unexpected host dependency of Antarctic Nanohaloarchaeota.

In hypersaline environments, Nanohaloarchaeota (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaeota [DPANN] superphylum) are thought to be free-living microorganisms. We report cultivation of 2 strains of Antarctic Nanohaloarchaeota and show that they require the haloarchaeon Halorubrum lacusprofundi for growth. By performing growth using enrichments and fluorescence-activated cell sorting, we demonstrated successful cultivation of Candidatus Nanohaloarchaeum antarcticus, purification of Ca. Nha. antarcticus away from other species, and growth and verification of Ca. Nha. antarcticus with Hrr. lacusprofundi; these findings are analogous to those required for fulfilling Koch's postulates. We use fluorescent in situ hybridization and transmission electron microscopy to assess cell structures and interactions; metagenomics to characterize enrichment taxa, generate metagenome assembled genomes, and interrogate Antarctic communities; and proteomics to assess metabolic pathways and speculate about the roles of certain proteins. Metagenome analysis indicates the presence of a single species, which is endemic to Antarctic hypersaline systems that support the growth of haloarchaea. The presence of unusually large proteins predicted to function in attachment and invasion of hosts plus the absence of key biosynthetic pathways (e.g., lipids) in metagenome assembled genomes of globally distributed Nanohaloarchaeota indicate that all members of the lineage have evolved as symbionts. Our work expands the range of archaeal symbiotic lifestyles and provides a genetically tractable model system for advancing understanding of the factors controlling microbial symbiotic relationships.

Redshifted and Near-infrared Active Analog Pigments Based upon Archaerhodopsin-3.

Archaerhodopsin-3 (AR3) is a member of the microbial rhodopsin family of hepta-helical transmembrane proteins, containing a covalently bound molecule of all-trans retinal as a chromophore. It displays an absorbance band in the visible region of the solar spectrum (λmax 556 nm) and functions as a light-driven proton pump in the archaeon Halorubrum sodomense. AR3 and its mutants are widely used in neuroscience as optogenetic neural silencers and in particular as fluorescent indicators of transmembrane potential. In this study, we investigated the effect of analogs of the native ligand all-trans retinal A1 on the spectral properties and proton-pumping activity of AR3 and its single mutant AR3 (F229S). While, surprisingly, the 3-methoxyretinal A2 analog did not redshift the absorbance maximum of AR3, the analogs retinal A2 and 3-methylamino-16-nor-1,2,3,4-didehydroretinal (MMAR) did generate active redshifted AR3 pigments. The MMAR analog pigments could even be activated by near-infrared light. Furthermore, the MMAR pigments showed strongly enhanced fluorescence with an emission band in the near-infrared peaking around 815 nm. We anticipate that the AR3 pigments generated in this study have widespread potential for near-infrared exploitation as fluorescent voltage-gated sensors in optogenetics and artificial leafs and as proton pumps in bioenergy-based applications.

Cold adaptation of the Antarctic haloarchaea Halohasta litchfieldiae and Halorubrum lacusprofundi.

Halohasta litchfieldiae represents ∼ 44% and Halorubrum lacusprofundi ∼ 10% of the hypersaline, perennially cold (≥ -20°C) Deep Lake community in Antarctica. We used proteomics and microscopy to define physiological responses of these haloarchaea to growth at high (30°C) and low (10 and 4°C) temperatures. The proteomic data indicate that both species responded to low temperature by modifying their cell envelope including protein N-glycosylation, maintaining osmotic balance and translation initiation, and modifying RNA turnover and tRNA modification. Distinctions between the two species included DNA protection and repair strategies (e.g. roles of UspA and Rad50), and metabolism of glycerol and pyruvate. For Hrr. lacusprofundi, low temperature led to the formation of polyhydroxyalkanoate-like granules, with granule formation occurring by an unknown mechanism. Hrr. lacusprofundi also formed biofilms and synthesized high levels of Hsp20 chaperones. Hht. litchfieldiae was characterized by an active CRISPR system, and elevated levels of the core gene expression machinery, which contrasted markedly to the decreased levels of Hrr. lacusprofundi. These findings greatly expand the understanding of cellular mechanisms of cold adaptation in psychrophilic archaea, and provide insight into how Hht. litchfieldiae gains dominance in Deep Lake.

Morphological and proteomic analysis of biofilms from the Antarctic archaeon, Halorubrum lacusprofundi.

Biofilms enhance rates of gene exchange, access to specific nutrients, and cell survivability. Haloarchaea in Deep Lake, Antarctica, are characterized by high rates of intergenera gene exchange, metabolic specialization that promotes niche adaptation, and are exposed to high levels of UV-irradiation in summer. Halorubrum lacusprofundi from Deep Lake has previously been reported to form biofilms. Here we defined growth conditions that promoted the formation of biofilms and used microscopy and enzymatic digestion of extracellular material to characterize biofilm structures. Extracellular DNA was found to be critical to biofilms, with cell surface proteins and quorum sensing also implicated in biofilm formation. Quantitative proteomics was used to define pathways and cellular processes involved in forming biofilms; these included enhanced purine synthesis and specific cell surface proteins involved in DNA metabolism; post-translational modification of cell surface proteins; specific pathways of carbon metabolism involving acetyl-CoA; and specific responses to oxidative stress. The study provides a new level of understanding about the molecular mechanisms involved in biofilm formation of this important member of the Deep Lake community.

Surface biosignatures of exo-earths: remote detection of extraterrestrial life.

Exoplanet discovery has made remarkable progress, with the first rocky planets having been detected in the central star's liquid water habitable zone. The remote sensing techniques used to characterize such planets for potential habitability and life rely solely on our understanding of life on Earth. The vegetation red edge from terrestrial land plants is often used as a direct signature of life, but it occupies only a small niche in the environmental parameter space that binds life on present-day Earth and has been widespread for only about 460 My. To more fully exploit the diversity of the one example of life known, we measured the spectral characteristics of 137 microorganisms containing a range of pigments, including ones isolated from Earth's most extreme environments. Our database covers the visible and near-infrared to the short-wavelength infrared (0.35-2.5 µm) portions of the electromagnetic spectrum and is made freely available from biosignatures.astro.cornell.edu. Our results show how the reflectance properties are dominated by the absorption of light by pigments in the visible portion and by strong absorptions by the cellular water of hydration in the infrared (up to 2.5 µm) portion of the spectrum. Our spectral library provides a broader and more realistic guide based on Earth life for the search for surface features of extraterrestrial life. The library, when used as inputs for modeling disk-integrated spectra of exoplanets, in preparation for the next generation of space- and ground-based instruments, will increase the chances of detecting life.

Halorubrum laminariae sp. nov., isolated from the brine of salted brown alga Laminaria.

Two halophilic archaeal strains, R60(T) and R61, were isolated from the brine of salted brown alga Laminaria. Cells of the two strains were observed to be rod-shaped, stain Gram-negative and to lyse in distilled water. Strain R60(T) was found to contain gas vacuoles and to produce pink-pigmented colonies, while strain R61 lacked gas vacuoles and produces red-pigmented colonies. Both strains were found to be able to grow at 20-50 °C (optimum 30 °C), at 1.7-4.8 M NaCl (optimum 2.6-3.1 M NaCl), at 0-1.0 M MgCl2 (optimum 0.005-0.1 M MgCl2) and at pH 6.0-9.5 (optimum pH 7.0). The major polar lipids were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and one major glycolipid chromatographically identical to a sulfated mannosyl glucosyl diether produced by Halorubrum members of the Halobacteriaceae. The 16S rRNA gene sequences of the two strains were 99.9 % identical, showing 94.6-98.0 % similarity to those of members of the genus Halorubrum. The EF-2 gene similarity between strains R60(T) and R60 was 100 % and showed 84.6-94.5 % similarity to those of members of the genus Halorubrum. The DNA G+C contents of the two strains were determined to be 63.0 mol %. The DNA-DNA hybridization value between strain R60(T) and strain R61 was 92 % and the two strains showed low DNA-DNA relatedness with the most related members of Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain R60(T) (= CGMCC 1.12689(T) = JCM 30040(T)) and strain R61 (= CGMCC 1.12696) represent a novel species of the genus Halorubrum, for which the name Halorubrum laminariae sp. nov. is proposed.

Draft genome sequence of Halorubrum halophilum B8(T), an extremely halophilic archaeon isolated from salt-fermented seafood.

The extremely halophilic archaeon, Halorubrum halophilum B8(T) (=JCM 18963(T), CECT 8278(T)) was isolated from salt-fermented seafood. We report here the draft genome of H. halophilum B8(T), containing 3,677,984bp with a G+C content of 65.1%. The genome consists of 19 genes predicted to encode esterases.

Characterization of ancient DNA supports long-term survival of Haloarchaea.

Bacteria and archaea isolated from crystals of halite 10(4) to 10(8) years old suggest long-term survival of halophilic microorganisms, but the results are controversial. Independent verification of the authenticity of reputed living prokaryotes in ancient salt is required because of the high potential for environmental and laboratory contamination. Low success rates of prokaryote cultivation from ancient halite, however, hamper direct replication experiments. In such cases, culture-independent approaches that use the polymerase chain reaction (PCR) and sequencing of 16S ribosomal DNA are a robust alternative. Here, we use amplification, cloning, and sequencing of 16S ribosomal DNA to investigate the authenticity of halophilic archaea cultured from subsurface halite, Death Valley, California, 22,000 to 34,000 years old. We recovered 16S ribosomal DNA sequences that are identical, or nearly so (>99%), to two strains, Natronomonas DV462A and Halorubrum DV427, which were previously isolated from the same halite interval. These results provide the best independent support to date for the long-term survival of halophilic archaea in ancient halite. PCR-based approaches are sensitive to small amounts of DNA and could allow investigation of even older halites, 10(6) to 10(8) years old, from which microbial cultures have been reported. Such studies of microbial life in ancient salt are particularly important as we search for microbial signatures in similar deposits on Mars and elsewhere in the Solar System.

Halorubrum salinum sp. nov., isolated from a marine solar saltern.

The halophilic archaeal strain GX71(T) was isolated from the Gangxi marine solar saltern near the Weihai city of Shandong Province, China. Cells of the strain were pleomorphic and lysed in distilled water, stained Gram-negative and formed red-pigmented colonies. Strain GX71(T) was able to grow at 25-45 °C (optimum 30 °C), in the presence of 1.7-4.8 M NaCl (optimum 2.6 M NaCl), with 0.005-0.7 M MgCl2 (optimum 0.05 M MgCl2) and at pH 5.5-9.5 (optimum pH 7.0-7.5). Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 10 % (w/v). The major polar lipids of the strain were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-3) and an unidentified lipid was also detected. The 16S rRNA gene sequence of strain GX71(T) showed 94.0-97.0 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB' gene sequence of strain GX71(T) was 87.3-93.4 % similarity to current members of the genus Halorubrum. The DNA G+C content of GX71(T) was 67.1 mol%. Strain GX71(T) showed low DNA-DNA relatedness with Halorubrum lipolyticum CGMCC 1.5332(T), Halorubrum saccharovorum CGMCC 1.2147(T), Halorubrum kocurii CGMCC 1.7018(T) and Halorubrum arcis CGMCC 1.5343(T), the most closely related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strain GX71(T) represents a novel species of the genus Halorubrum, for which the name Halorubrum salinum sp. nov. is proposed. The type strain is GX71(T) (= CGMCC 1.10458(T) = JCM 17093(T)).

Hyperhalophilic archaeal biofilms: growth kinetics, structure, and antagonistic interaction in continuous culture.

Biofilms by the hyperhalophilic archaea Halorubrum sp. and Halobacterium sp. were analyzed, and for the first time the progression of structural features and the developmental parameters of these sessile populations are described. Optical slicing and digital analysis of sequential micrographs showed that their three dimensional structure was microorganism dependent. Biofilms of Halobacterium sp. developed in clusters that covered about 30% of the supporting surface at the interface level and expanded over about 86 ± 4 µm in thickness, while Halorubrum sp. biofilms covered less than 20% of the surface and reached a thickness of 41 ± 1 µm. The kinetics of growth was lower in biofilms, with generation times of 27 ± 1 and 36 ± 2 h for Halobacterium sp. and Halorubrum sp., respectively, as compared to 8.4 ± 0.3 and 14 ± 1 h in planktonic cultures. Differences between microorganisms were also observed at the cell morphology level. The interaction between the two microorganisms was also evaluated, showing that Halobacterium sp. can outcompete already established Halorubrum sp. biofilms by a mechanism that might include the combined action of tunnelling swimmers and antimicrobial compounds.

Halorubrum rubrum sp. nov., an extremely halophilic archaeon from a Chinese salt lake.

Two halophilic archaeal strains, YC87(T) and YCA11, were isolated from Yuncheng salt lake in Shanxi, China. Cells of the two strains were observed to be pleomorphic rod-shaped, stained Gram-negative and produced red-pigmented colonies. Strain YC87(T) was able to grow at 20-50 °C (optimum 37 °C), at 1.4-4.8 M NaCl (optimum 2.1 M NaCl), at 0.05-1.0 M MgCl2 (optimum 0.3 M MgCl2) and at pH 6.0-9.0 (optimum pH 7.0) while strain YCA11 was able to grow at 20-50 °C (optimum 37 °C), at 2.1-4.8 M NaCl (optimum 3.1 M NaCl), at 0.01-0.7 M MgCl2 (optimum 0.1 M MgCl2) and at pH 6.0-9.0 (optimum pH 7.5). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 8 % (w/v) for strain YC87(T) and 12 % (w/v) for strain YCA11. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and one major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether; another major glycolipid and trace amounts of several unidentified lipids were also detected. The 16S rRNA gene sequences of the two strains were 99.8 % identical, showing 93.2-98.2 % similarity to members of the genus Halorubrum of the family Halobacteriaceae. The rpoB' gene similarity between strains YC87(T) and YCA11 was 99.3 % and showed 87.5-95.2 % similarity to the closest relative members of the genus Halorubrum. The DNA G+C content of strains YC87(T) and YCA11 were determined to be 64.9 and 64.5 mol%, respectively. The DNA-DNA hybridization value between strain YC20(T) and strain YC77 was 87 % and the two strains showed low DNA-DNA relatedness with Halorubrum cibi JCM 15757(T) and Halorubrum aquaticum CGMCC 1.6377(T), the most related members of the genus Halorubrum. The phenotypic, chemotaxonomic and phylogenetic properties suggest that strains YC87(T) and YCA11 represent a novel species of the genus Halorubrum, for which the name Halorubrum rubrum sp. nov. is proposed. The type strain is YC87(T) (=CGMCC 1.12124(T) = JCM 18365(T)).

Halorubrum californiense sp. nov., an extreme archaeal halophile isolated from a crystallizer pond at a solar salt plant in California, USA.

A motile, rod-shaped, pink-pigmented, extremely halophilic archaeon, strain SF3-213(T), was isolated from a crystallizer pond at the Cargill Solar Salt Plant, Newark, California (USA). Analysis of the almost-complete 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genus Halorubrum, with a close relationship to Halorubrum trapanicum NRC 34021(T) (98.6 % similarity), Halorubrum sodomense ATCC 33755(T) (98.3 %) and Halorubrum xinjiangense AS 1.3527(T) (98.2 %). The polar lipids of strain SF3-213(T) were C(20)C(20) derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and a sulfated diglycosyl-diether. Strain SF3-213(T) grew in 2.5-5.0 M NaCl. The temperature and pH ranges for growth were 25-42 degrees C and 6.8-8.5, respectively. Optimal growth occurred at 3.5-4.5 M NaCl, 37 degrees C and pH 7.3. Mg(2+) was required for growth. The DNA G+C content was 69.4 mol%. DNA-DNA hybridization values lower than 70 % were obtained between strain SF3-213(T) and the closely related species of the genus Halorubrum. Based on the data presented in this study, strain SF3-213(T) represents a novel species for which the name Halorubrum californiense sp. nov. is proposed; the type strain is SF3-213(T) (=CECT 7256(T)=DSM 19288(T)=JCM 14715(T)).