Oxyplasma meridianum gen. nov., sp. nov., an extremely acidophilic organotrophic member of the order Thermoplasmatales.

A mesophilic, hyperacidophilic archaeon, strain M1T, was isolated from a rock sample from Vulcano Island, Italy. Cells of this organism were cocci with an average diameter of 1 µm. Some cells possessed filaments. The strain grew in the range of temperatures between 15 and 52 °C and pH 0.5-4.0 with growth optima at 40 °C and pH 1.0. Strain M1T was aerobic and chemoorganotrophic, growing on complex substrates, such as casamino acids, trypticase, tryptone, yeast and beef extracts. No growth at expenses of oxidation of elemental sulphur or reduced sulphur compounds, pyrite, or ferrous sulphate was observed. The core lipids were glycerol dibiphytanyl glycerol tetraether lipids (membrane spanning) with 0 to 4 cyclopentane moieties and archaeol, with trace amounts of hydroxy archaeol. The dominant quinone was MK-7 : 7. The genome size of M1T was 1.67 Mbp with a G+C content of 39.76 mol%, and both characteristics were well within the common range for Thermoplasmatales. The phylogenetic analysis based on 16S rRNA gene sequence placed the strain M1T within the order Thermoplasmatales with sequence identities of 90.9, 90.3 and 90.5% to the closest SSU rRNA gene sequences from organisms with validly published names, Thermoplasma acidophilum, Thermoplasma volcanium and Thermogymnomonas acidicola, respectively. Based on the results of our genomic, phylogenetic, physiological and chemotaxonomic studies, we propose that strain M1T (=DSM 116605T=JCM 36570T) represents a new genus and species, Oxyplasma meridianum gen. nov., sp. nov., within the order Thermoplasmatales.

Limnoglobus roseus gen. nov., sp. nov., a novel freshwater planctomycete with a giant genome from the family Gemmataceae.

The family Gemmataceae accommodates aerobic, chemoorganotrophic planctomycetes, which inhabit various freshwater ecosystems, wetlands and soils. Here, we describe a novel member of this family, strain PX52T, which was isolated from a boreal eutrophic lake in Northern Russia. This isolate formed pink-pigmented colonies and was represented by spherical cells that occurred singly, in pairs or aggregates and multiplied by budding. Daughter cells were highly motile. PX52T was an obligate aerobic chemoorganotroph, which utilized various sugars and some heteropolysaccharides. Growth occurred at pH 5.0-7.5 (optimum pH 6.5) and at temperatures between 10 and 30 °C (optimum 20-25 °C). The major fatty acids were C18 : 1É·7c, C18 : 0 and ßOH-C16:0; the major intact polar lipid was trimethylornithine, and the quinone was MK-6. The complete genome of PX52T was 9.38 Mb in size and contained nearly 8000 potential protein-coding genes. Among those were genes encoding a wide repertoire of carbohydrate-active enzymes (CAZymes) including 33 glycoside hydrolases (GH) and 87 glycosyltransferases (GT) affiliated with 17 and 12 CAZy families, respectively. DNA G+C content was 65.6 mol%. PX52T displayed only 86.0-89.8 % 16S rRNA gene sequence similarity to taxonomically described Gemmataceae planctomycetes and differed from them by a number of phenotypic characteristics and by fatty acid composition. We, therefore, propose to classify it as representing a novel genus and species, Limnoglobus roseus gen. nov., sp. nov. The type strain is strain PX52T (=KCTC 72397T=VKM B-3275T).

Phylogenomic analysis of lipid biosynthetic genes of Archaea shed light on the 'lipid divide'.

The lipid membrane is one of the most characteristic traits distinguishing the three domains of life. Membrane lipids of Bacteria and Eukarya are composed of fatty acids linked to glycerol-3-phosphate (G3P) via ester bonds, while those of Archaea possess isoprene-based alkyl chains linked by ether linkages to glycerol-1-phosphate (G1P), resulting in the opposite stereochemistry of the glycerol phosphate backbone. This 'lipid divide' has raised questions on the evolution of microbial life since eukaryotes are thought to have evolved from the Archaea, requiring a radical change in membrane composition. Here, we searched for homologs of enzymes involved in membrane lipid and fatty acid synthesis in a wide variety of archaeal genomes and performed phylogenomic analyses. We found that two uncultured archaeal groups, i.e. marine euryarchaeota group II/III and 'Lokiarchaeota', recently discovered descendants of the archaeal ancestor leading to eukaryotes, lack the gene to synthesize G1P and, consequently, the capacity to synthesize archaeal membrane lipids. However, our analyses reveal their genetic capacity to synthesize G3P-based 'chimeric lipids' with either two ether-bound isoprenoidal chains or with an ester-bound fatty acid instead of an ether-bound isoprenoid. These archaea may reflect the 'archaea-to-eukaryote' membrane transition stage which have led to the current 'lipid divide'.

Elucidation and identification of amino acid containing membrane lipids using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: Intact polar lipids (IPLs) are the building blocks of cell membranes, and amino acid containing IPLs have been observed to be involved in response to changing environmental conditions in various species of bacteria. High-performance liquid chromatography/mass spectrometry (HPLC/MS) has become the primary method for analysis of IPLs. Many glycerol-free amino acid containing membrane lipids (AA-IPLs), which are structurally different than abundant aminophospholipids, have not been characterized using HPLC/MS. This results in many lipids remaining unrecognized in IPL analysis of microbial cultures and environmental samples, hampering the study of their occurrence and functionality. METHODS: We analyzed the amino acid containing IPLs of a number of bacteria (i.e. Gluconobacter cerinus, Cyclobacterium marinus, Rhodobacter sphaeroides, and Pedobacter heparinus) in order to decipher fragmentation pathways, and explore potential novel lipid structures using HPLC/electrospray ionization ion trap MS (HPLC/ESI-IT-MS) and HPLC/high-resolution MS (HPLC/HRMS). RESULTS: We report differentiation between glutamine and lysine lipids with the same nominal masses, novel MS fragmentation pathways of cytolipin, the lipopeptides cerilipin and flavolipin, head group hydroxylated ornithine lipids, and the novel identification of cerilipin with a hydroxylated fatty acid. CONCLUSIONS: Non-glycerol AA lipids can be readily recognized as their fragmentation follows a clear pattern with initial dehydration or other loss from the head group, followed by fatty acid losses resulting in a diagnostic fragment ion. Higher level MSn and HRMS are valuable tools in characterizing AA lipid head group structural components.

Methanosalsum natronophilum sp. nov., and Methanocalculus alkaliphilus sp. nov., haloalkaliphilic methanogens from hypersaline soda lakes.

Two groups of haloalkaliphilic methanogenic archaea were dominating in enrichments from hypersaline soda lake sediments at pH 10. At moderate salt concentrations with formate or H2 as electron donor, methanogens belonging to the genus Methanocalculus were enriched, while at high salt concentrations with methylated substrates, a group related to Methanosalsum zhilinae was dominating. For both groups, several pure cultures were obtained including the type strains AMF2T for the Methanocalculus group and AME2T for the Methanosalsum group. The Methanocalculus group is characterized by lithoheterotrophic growth with either formate (preferable substrate) or H2 at moderate salinity up to 1.5-2 M total Na+ and obligate alkaliphilic growth with an optimum at pH 9.5. According to phylogenetic analysis, the group also includes closely related strains isolated previously from the low-salt alkaline Lonar Lake. The novel Methanosalsum group is characterized by high salt tolerance (up to 3.5 M total Na+) and obligate alkaliphilic growth with an optimum at pH 9.5. It has a typical methylotrophic substrate profile, utilizing methanol, methylamines and dimethyl sulfide (at low concentrations) as methanogenic substrates. On the basis of physiological and phylogenetic data, it is proposed that the two groups of soda lake methanogenic isolates are assigned into two novel species, Methanocalculus alkaliphilus sp. nov. (type strain AMF2T = DSM 24457T = UNIQEM U859T) and Methanosalsum natronophilum sp. nov. (type strain AME2T = DSM 24634T = NBRC 110091T).

Diversity and distribution of a key sulpholipid biosynthetic gene in marine microbial assemblages.

Sulphoquinovosyldiacylglycerols (SQDG) are polar sulphur-containing membrane lipids, whose presence has been related to a microbial strategy to adapt to phosphate deprivation. In this study, we have targeted the sqdB gene coding the uridine 5'-diphosphate-sulphoquinovose (UDP-SQ) synthase involved in the SQDG biosynthetic pathway to assess potential microbial sources of SQDGs in the marine environment. The phylogeny of the sqdB-coding protein reveals two distinct clusters: one including green algae, higher plants and cyanobacteria, and another one comprising mainly non-photosynthetic bacteria, as well as other cyanobacteria and algal groups. Evolutionary analysis suggests that the appearance of UDP-SQ synthase occurred twice in cyanobacterial evolution, and one of those branches led to the diversification of the protein in members of the phylum Proteobacteria. A search of homologues of sqdB-proteins in marine metagenomes strongly suggested the presence of heterotrophic bacteria potential SQDG producers. Application of newly developed sqdB gene primers in the marine environment revealed a high diversity of sequences affiliated to cyanobacteria and Proteobacteria in microbial mats, while in North Sea surface water, most of the detected sqdB genes were attributed to the cyanobacterium Synechococcus sp. Lipid analysis revealed that specific SQDGs were characteristic of microbial mat depth, suggesting that SQDG lipids are associated with specific producers.

Paludibaculum fermentans gen. nov., sp. nov., a facultative anaerobe capable of dissimilatory iron reduction from subdivision 3 of the Acidobacteria.

A facultatively anaerobic, non-pigmented, non-spore-forming bacterium was isolated from a littoral wetland of a boreal lake located on Valaam Island, northern Russia, and designated strain P105(T). Cells of this isolate were Gram-negative, non-motile rods coated by S-layers with p2 lattice symmetry. Sugars were the preferred growth substrates. Under anoxic conditions, strain P105(T) was capable of fermentation and dissimilatory Fe(III) reduction. End products of fermentation were acetate, propionate and H2. Strain P105(T) was a mildly acidophilic, mesophilic organism, capable of growth at pH 4.0-7.2 (optimum pH 5.5-6.0) and at 4-35 °C (optimum at 20-28 °C). The major fatty acids were iso-C(15 : 0) and C(16 : 1)ω7c; the cells also contained significant amounts of 13,16-dimethyl octacosanedioic acid (isodiabolic acid). The major polar lipids were phosphocholine and phosphoethanolamine; the quinone was MK-8. The G+C content of the DNA was 60.5 mol%. 16S rRNA gene sequence analysis showed that strain P105(T) belongs to subdivision 3 of the Acidobacteria and is only distantly related (90% sequence similarity) to the only currently characterized member of this subdivision, Bryobacter aggregatus. The novel isolate differs from Bryobacter aggregatus in its cell morphology and ability to grow under anoxic conditions and in the presence of iron- and nitrate-reducing capabilities as well as quinone and polar lipid compositions. These differences suggest that strain P105(T) represents a novel genus and species, for which the name Paludibaculum fermentans gen. nov., sp. nov., is proposed. The type strain of Paludibaculum fermentans is P105(T) ( = DSM 26340(T) = VKM B-2878(T)).

Ercella succinigenes gen. nov., sp. nov., an anaerobic succinate-producing bacterium.

A novel anaerobic succinate-producing bacterium, strain ZWB(T), was isolated from sludge collected from a biogas desulfurization bioreactor (Eerbeek, the Netherlands). Cells were non-spore-forming, motile, slightly curved rods (0.4-0.5 µm in diameter and 2-3 µm in length), and stained Gram-negative. The temperature range for growth was 25-40 °C, with an optimum at 37 °C. The pH range for growth was 7.0-9.0, with an optimum at pH 7.5. Strain ZWB(T) was able to ferment glycerol and several carbohydrates mainly to H2, succinate and acetate. Sulfur and fumarate could be used as electron acceptors by strain ZWB(T). The G+C content of the genomic DNA was 37.6 mol%. The most abundant fatty acids were iso-C14 : 0 and iso-C16 : 0 DMA. On the basis of 16S rRNA gene sequence similarity, strain ZWB(T) belongs to the family Ruminococcaceae and it is distantly related to Saccharofermentans acetigenes JCM 14006(T) (92.1%). Based on the physiological features and phylogenetic analysis, strain ZWB(T) represents a novel species of a new genus, for which the name Ercella succinigenes gen. nov., sp. nov. is proposed. The type strain of Ercella succinigenes is ZWB(T) ( = DSM 27333(T) = JCM 19283(T)).

Organic matter degradation in the deep, sulfidic waters of the Black Sea: insights into the ecophysiology of novel anaerobic bacteria.

BACKGROUND: Recent studies have reported the identity and functions of key anaerobes involved in the degradation of organic matter (OM) in deep (> 1000 m) sulfidic marine habitats. However, due to the lack of available isolates, detailed investigation of their physiology has been precluded. In this study, we cultivated and characterized the ecophysiology of a wide range of novel anaerobes potentially involved in OM degradation in deep (2000 m depth) sulfidic waters of the Black Sea. RESULTS: We have successfully cultivated a diverse group of novel anaerobes belonging to various phyla, including Fusobacteriota (strain S5), Bacillota (strains A1T and A2), Spirochaetota (strains M1T, M2, and S2), Bacteroidota (strains B1T, B2, S6, L6, SYP, and M2P), Cloacimonadota (Cloa-SY6), Planctomycetota (Plnct-SY6), Mycoplasmatota (Izemo-BS), Chloroflexota (Chflx-SY6), and Desulfobacterota (strains S3T and S3-i). These microorganisms were able to grow at an elevated hydrostatic pressure of up to 50 MPa. Moreover, this study revealed that different anaerobes were specialized in degrading specific types of OM. Strains affiliated with the phyla Fusobacteriota, Bacillota, Planctomycetota, and Mycoplasmatota were found to be specialized in the degradation of cellulose, cellobiose, chitin, and DNA, respectively, while strains affiliated with Spirochaetota, Bacteroidota, Cloacimonadota, and Chloroflexota preferred to ferment less complex forms of OM. We also identified members of the phylum Desulfobacterota as terminal oxidizers, potentially involved in the consumption of hydrogen produced during fermentation. These results were supported by the identification of genes in the (meta)genomes of the cultivated microbial taxa which encode proteins of specific metabolic pathways. Additionally, we analyzed the composition of membrane lipids of selected taxa, which could be critical for their survival in the harsh environment of the deep sulfidic waters and could potentially be used as biosignatures for these strains in the sulfidic waters of the Black Sea. CONCLUSIONS: This is the first report that demonstrates the cultivation and ecophysiology of such a diverse group of microorganisms from any sulfidic marine habitat. Collectively, this study provides a step forward in our understanding of the microbes thriving in the extreme conditions of the deep sulfidic waters of the Black Sea. Video Abstract.

Linking isoprenoidal GDGT membrane lipid distributions with gene abundances of ammonia-oxidizing Thaumarchaeota and uncultured crenarchaeotal groups in the water column of a tropical lake (Lake Challa, East Africa).

Stratified lakes are important reservoirs of microbial diversity and provide habitats for niche differentiation of Archaea. In this study, we used a lipid biomarker/DNA-based approach to reveal the diversity and abundance of Archaea in the water column of Lake Challa (East Africa). Concentrations of intact polar lipid (IPL) crenarchaeol, a specific biomarker of Thaumarchaeota, were enhanced (1 ng l(-1) ) at the oxycline/nitrocline. The predominance of the more labile IPL hexose-phosphohexose crenarchaeol indicated the presence of an actively living community of Thaumarchaeota. Archaeal 16S rRNA clone libraries revealed the presence of thaumarchaeotal groups 1.1a and 1.1b at and above the oxycline. In the anoxic deep water, amoA gene abundance was an order of magnitude lower than at the oxycline and high abundance (∼90 ng l(-1) ) of an IPL with the acyclic glycerol dialkyl glycerol tetraether (GDGT-0) was evident. The predominance of archaeal 16S rRNA sequences affiliated to the uncultured crenarchaeota groups 1.2 and miscellaneous crenarchaeotic group (MCG) points to an origin of GDGT-0 from uncultured crenarchaeota. This study demonstrates the importance of thermal stratification and nutrient availability in the distribution of archaeal groups in lakes, which is relevant to constrain and validate temperature proxies based on archaeal GDGTs (i.e. TEX86 ).

Thermoanaerobaculum aquaticum gen. nov., sp. nov., the first cultivated member of Acidobacteria subdivision 23, isolated from a hot spring.

A novel bacterium was isolated from a freshwater hot spring, the Hale House Spring, located at Hot Springs National Park, Hot Springs, AR, USA. Cells of strain MP-01(T) stained Gram-negative, were rod-shaped, non-motile, strictly anaerobic and chemo-organotrophic and did not form spores. Growth occurred at 50-65 °C, with an optimum at 60 °C, at pH 6.0-8.0, with an optimum at pH 6.5-7.0, and at NaCl concentrations up to 0.5 % (w/v), with optimum growth in the absence of NaCl. Strain MP-01(T) was capable of fermentative growth on pyruvate or proteinaceous substrates as well as reducing Fe(III) and Mn(IV). Major fatty acids were iso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0 and iso-C17 : 0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine and the major isoprenoid quinone was MK-10. In the polyamine pattern, sym-homospermidine was the predominant compound. The DNA G+C content was 62.7 mol%. Analysis of the 16S rRNA gene sequence of the isolate indicated that strain MP-01(T) represents the first reported cultivated member of subdivision 23 of the Acidobacteria. It is proposed that strain MP-01(T) represents a novel genus and species, for which the name Thermoanaerobaculum aquaticum gen. nov., sp. nov. is proposed. The type strain of Thermoanaerobaculum aquaticum is MP-01(T) ( = DSM 24856(T) = JCM 18256(T)).

Fossilized glycolipids reveal past oceanic N2 fixation by heterocystous cyanobacteria.

N(2)-fixing cyanobacteria play an essential role in sustaining primary productivity in contemporary oceans and freshwater systems. However, the significance of N(2)-fixing cyanobacteria in past nitrogen cycling is difficult to establish as their preservation potential is relatively poor and specific biological markers are presently lacking. Heterocystous N(2)-fixing cyanobacteria synthesize unique long-chain glycolipids in the cell envelope covering the heterocyst cell to protect the oxygen-sensitive nitrogenase enzyme. We found that these heterocyst glycolipids are remarkably well preserved in (ancient) lacustrine and marine sediments, unambiguously indicating the (past) presence of N(2)-fixing heterocystous cyanobacteria. Analysis of Pleistocene sediments of the eastern Mediterranean Sea showed that heterocystous cyanobacteria, likely as epiphytes in symbiosis with planktonic diatoms, were particularly abundant during deposition of sapropels. Eocene Arctic Ocean sediments deposited at a time of large Azolla blooms contained glycolipids typical for heterocystous cyanobacteria presently living in symbiosis with the freshwater fern Azolla, indicating that this symbiosis already existed in that time. Our study thus suggests that heterocystous cyanobacteria played a major role in adding "new" fixed nitrogen to surface waters in past stratified oceans.

A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria.

Nitrospira are barely studied and mostly uncultured nitrite-oxidizing bacteria, which are, according to molecular data, among the most diverse and widespread nitrifiers in natural ecosystems and biological wastewater treatment. Here, environmental genomics was used to reconstruct the complete genome of "Candidatus Nitrospira defluvii" from an activated sludge enrichment culture. On the basis of this first-deciphered Nitrospira genome and of experimental data, we show that Ca. N. defluvii differs dramatically from other known nitrite oxidizers in the key enzyme nitrite oxidoreductase (NXR), in the composition of the respiratory chain, and in the pathway used for autotrophic carbon fixation, suggesting multiple independent evolution of chemolithoautotrophic nitrite oxidation. Adaptations of Ca. N. defluvii to substrate-limited conditions include an unusual periplasmic NXR, which is constitutively expressed, and pathways for the transport, oxidation, and assimilation of simple organic compounds that allow a mixotrophic lifestyle. The reverse tricarboxylic acid cycle as the pathway for CO2 fixation and the lack of most classical defense mechanisms against oxidative stress suggest that Nitrospira evolved from microaerophilic or even anaerobic ancestors. Unexpectedly, comparative genomic analyses indicate functionally significant lateral gene-transfer events between the genus Nitrospira and anaerobic ammonium-oxidizing planctomycetes, which share highly similar forms of NXR and other proteins reflecting that two key processes of the nitrogen cycle are evolutionarily connected.

Intact polar and core glycerol dibiphytanyl glycerol tetraether lipids of group I.1a and I.1b thaumarchaeota in soil.

Ecological studies of thaumarchaeota often apply glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids. However, these components have only been characterized for thaumarchaeota from aquatic environments. Thaumarchaeota have been shown to play an important role in the nitrogen cycle in soil as ammonium oxidizers, and GDGTs are common lipids encountered in soil. We report the core and intact polar lipid (IPL) GDGTs produced by three newly available thaumarchaeota isolated from grassland soil in Austria ("Nitrososphaera viennensis," group I.1b) and enriched from agricultural soils in South Korea ("Candidatus Nitrosoarchaeum koreensis" MY1, group I.1a; and "Candidatus Nitrososphaera" strain JG1, group I.1b). The soil thaumarchaeota all synthesize crenarchaeol as their major core GDGT, in agreement with the fact that crenarchaeol has also been detected in thaumarchaeota from aquatic environments. The crenarchaeol regioisomer apparently is produced in significant quantities only by soil thaumarchaeota of the I.1b subgroup. In addition, GDGTs with 0 to 4 cyclopentane moieties and GDGTs containing an additional hydroxyl group were detected. The IPL head groups of their membrane lipids comprised mainly monohexose, dihexose, trihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose head group bound to crenarchaeol occurred in all soil thaumarchaeota, and this IPL is at present the only lipid that is detected in all thaumarchaeota analyzed so far. This specificity and its lability indicate that it is the most suitable biomarker lipid to trace living thaumarchaeota. This study, in combination with previous studies, also suggests that hydroxylated GDGTs occur in the I.1a, but not in the I.1b, subgroup of the thaumarchaeota.

Arctic hydrology during global warming at the Palaeocene/Eocene thermal maximum.

The Palaeocene/Eocene thermal maximum represents a period of rapid, extreme global warming 55 million years ago, superimposed on an already warm world. This warming is associated with a severe shoaling of the ocean calcite compensation depth and a >2.5 per mil negative carbon isotope excursion in marine and soil carbonates. Together these observations indicate a massive release of 13C-depleted carbon and greenhouse-gas-induced warming. Recently, sediments were recovered from the central Arctic Ocean, providing the first opportunity to evaluate the environmental response at the North Pole at this time. Here we present stable hydrogen and carbon isotope measurements of terrestrial-plant- and aquatic-derived n-alkanes that record changes in hydrology, including surface water salinity and precipitation, and the global carbon cycle. Hydrogen isotope records are interpreted as documenting decreased rainout during moisture transport from lower latitudes and increased moisture delivery to the Arctic at the onset of the Palaeocene/Eocene thermal maximum, consistent with predictions of poleward storm track migrations during global warming. The terrestrial-plant carbon isotope excursion (about -4.5 to -6 per mil) is substantially larger than those of marine carbonates. Previously, this offset was explained by the physiological response of plants to increases in surface humidity. But this mechanism is not an effective explanation in this wet Arctic setting, leading us to hypothesize that the true magnitude of the excursion--and associated carbon input--was greater than originally surmised. Greater carbon release and strong hydrological cycle feedbacks may help explain the maintenance of this unprecedented warmth.

A microbial consortium couples anaerobic methane oxidation to denitrification.

Modern agriculture has accelerated biological methane and nitrogen cycling on a global scale. Freshwater sediments often receive increased downward fluxes of nitrate from agricultural runoff and upward fluxes of methane generated by anaerobic decomposition. In theory, prokaryotes should be capable of using nitrate to oxidize methane anaerobically, but such organisms have neither been observed in nature nor isolated in the laboratory. Microbial oxidation of methane is thus believed to proceed only with oxygen or sulphate. Here we show that the direct, anaerobic oxidation of methane coupled to denitrification of nitrate is possible. A microbial consortium, enriched from anoxic sediments, oxidized methane to carbon dioxide coupled to denitrification in the complete absence of oxygen. This consortium consisted of two microorganisms, a bacterium representing a phylum without any cultured species and an archaeon distantly related to marine methanotrophic Archaea. The detection of relatives of these prokaryotes in different freshwater ecosystems worldwide indicates that the reaction presented here may make a substantial contribution to biological methane and nitrogen cycles.

Natronocalculus amylovorans gen. nov., sp. nov., and Natranaeroarchaeum aerophilus sp. nov., dominant culturable amylolytic natronoarchaea from hypersaline soda lakes in southwestern Siberia.

Several pure cultures of alkaliphilic haloaloarchaea were enriched and isolated from hypersaline soda lakes in southwestern Siberia using amylopectin and fructans as substrates. Phylogenomic analysis placed the isolates into two distinct groups within the class Halobacteria. Four isolates forming group 1 were closely related to a recently described Natranaeroarchaeum sulfidigenes and the other three strains forming group 2 represent a novel genus-level phylogenetic lineage. All isolates are saccharolytic archaea growing with various starch-like alpha-glucans including soluble starch, amylopectin, dextrin, glycogen, pullulane and cyclodextrin. In addition, group 1 can use levan while group 2 - inulin (plant storage beta-fructans). Group 1 strains can also grow anaerobically with either glucose or maltose using elemental sulfur as the electron acceptor. Both groups are moderately alkaliphilic with a pH range for growth from 7.2 to 9.3 (optimum between 8.0-8.8) and low Mg-demanding extreme halophiles growing optimally at 4 M total Na+. The major respiratory menaquinone is MK-8:8 and the core biphytanyl lipids are dominated by archaeol (C20-C20) and a less abundant extended archaeol (C20-C25) with PG and PGP-Me as polar groups. The four isolates of group 1 are suggested to be classified into a new species as Natranaeroarchaeum aerophilus sp. nov. (type strain AArc-St1-1T = JCM 32519T). The three isolates of group 2 are proposed to form a new genus and species for which the name Natronocalculus amylovorans gen. nov., sp. nov. is suggested (type strain AArc-St2T = JCM 32475T).

13,16-Dimethyl octacosanedioic acid (iso-diabolic acid), a common membrane-spanning lipid of Acidobacteria subdivisions 1 and 3.

The distribution of membrane lipids of 17 different strains representing 13 species of subdivisions 1 and 3 of the phylum Acidobacteria, a highly diverse phylum of the Bacteria, were examined by hydrolysis and gas chromatography-mass spectrometry (MS) and by high-performance liquid chromatography-MS of intact polar lipids. Upon both acid and base hydrolyses of total cell material, the uncommon membrane-spanning lipid 13,16-dimethyl octacosanedioic acid (iso-diabolic acid) was released in substantial amounts (22 to 43% of the total fatty acids) from all of the acidobacteria studied. This lipid has previously been encountered only in thermophilic Thermoanaerobacter species but bears a structural resemblance to the alkyl chains of bacterial glycerol dialkyl glycerol tetraethers (GDGTs) that occur ubiquitously in peat and soil and are suspected to be produced by acidobacteria. As reported previously, most species also contained iso-C(15) and C(16:1ω7C) as major fatty acids but the presence of iso-diabolic acid was unnoticed in previous studies, most probably because the complex lipid that contained this moiety was not extractable from the cells; it could only be released by hydrolysis. Direct analysis of intact polar lipids in the Bligh-Dyer extract of three acidobacterial strains, indeed, did not reveal any membrane-spanning lipids containing iso-diabolic acid. In 3 of the 17 strains, ether-bound iso-diabolic acid was detected after hydrolysis of the cells, including one branched GDGT containing iso-diabolic acid-derived alkyl chains. Since the GDGT distribution in soils is much more complex, branched GDGTs in soil likely also originate from other (acido)bacteria capable of biosynthesizing these components.

Core and intact polar glycerol dibiphytanyl glycerol tetraether lipids of ammonia-oxidizing archaea enriched from marine and estuarine sediments.

Glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids are increasingly being used as complements to conventional molecular methods in ecological studies of ammonia-oxidizing archaea (AOA) in the marine environment. However, the few studies that have been done on the detailed lipid structures synthesized by AOA in (enrichment) culture are based on species enriched from nonmarine environments, i.e., a hot spring, an aquarium filter, and a sponge. Here we have analyzed core and intact polar lipid (IPL)-GDGTs synthesized by three newly available AOA enriched directly from marine sediments taken from the San Francisco Bay estuary ("Candidatus Nitrosoarchaeum limnia"), and coastal marine sediments from Svalbard, Norway, and South Korea. Like previously screened AOA, the sedimentary AOA all synthesize crenarchaeol (a GDGT containing a cyclohexane moiety and four cyclopentane moieties) as a major core GDGT, thereby supporting the hypothesis that crenarchaeol is a biomarker lipid for AOA. The IPL headgroups synthesized by sedimentary AOA comprised mainly monohexose, dihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose headgroup bound to crenarchaeol was common to all enrichments and, in fact, the only IPL common to every AOA enrichment analyzed to date. This apparent specificity, in combination with its inferred lability, suggests that it may be the most suitable biomarker lipid to trace living AOA. GDGTs bound to headgroups with a mass of 180 Da of unknown structure appear to be specific to the marine group I.1a AOA: they were synthesized by all three sedimentary AOA and "Candidatus Nitrosopumilus maritimus"; however, they were absent in the group I.1b AOA "Candidatus Nitrososphaera gargensis."

Detection, isolation, and characterization of acidophilic methanotrophs from Sphagnum mosses.

Sphagnum peatlands are important ecosystems in the methane cycle. Methane-oxidizing bacteria in these ecosystems serve as a methane filter and limit methane emissions. Yet little is known about the diversity and identity of the methanotrophs present in and on Sphagnum mosses of peatlands, and only a few isolates are known. The methanotrophic community in Sphagnum mosses, originating from a Dutch peat bog, was investigated using a pmoA microarray. A high biodiversity of both gamma- and alphaproteobacterial methanotrophs was found. With Sphagnum mosses as the inoculum, alpha- and gammaproteobacterial acidophilic methanotrophs were isolated using established and newly designed media. The 16S rRNA, pmoA, pxmA, and mmoX gene sequences showed that the alphaproteobacterial isolates belonged to the Methylocystis and Methylosinus genera. The Methylosinus species isolated are the first acid-tolerant members of this genus. Of the acidophilic gammaproteobacterial strains isolated, strain M5 was affiliated with the Methylomonas genus, and the other strain, M200, may represent a novel genus, most closely related to the genera Methylosoma and Methylovulum. So far, no acidophilic or acid-tolerant methanotrophs in the Gammaproteobacteria class are known. All strains showed the typical features of either type I or II methanotrophs and are, to the best of our knowledge, the first isolated (acidophilic or acid-tolerant) methanotrophs from Sphagnum mosses.