Diversity of Archaea in marine sediments from Skan Bay, Alaska, including cultivated methanogens, and description of Methanogenium boonei sp. nov

Methanogenesis in cold marine sediments is a globally important process leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. We employed a multidisciplinary approach that combined culture-dependent and -independent analyses with geochemical measurements in the sediments of Skan Bay, Alaska (53 degrees N, 167 degrees W), to investigate methanogenesis there. Cultivation-independent analyses of the archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present at Skan Bay and that methanogens constituted a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO(2)-reducing methanogens: AK-3, AK7, and AK-8. The phylogenetic positions and physiological characteristics of these strains are described. We propose a new species, Methanogenium boonei, with strain AK-7 as the type strain.     

Isolation, characterization, and ecology of cold-active, chemolithotrophic, sulfur-oxidizing bacteria from perennially ice-covered Lake Fryxell, Antarctica

Novel strains of obligately chemolithoautotrophic, sulfur-oxidizing bacteria have been isolated from various depths of Lake Fryxell, Antarctica. Physiological, morphological, and phylogenetic analyses showed these strains to be related to mesophilic Thiobacillus species, such as T. thioparus. However, the psychrotolerant Antarctic isolates showed an adaptation to cold temperatures and thus should be active in the nearly freezing waters of the lake. Enumeration by most-probable-number analysis in an oxic, thiosulfate-containing medium revealed that the sulfur-oxidizing chemolithotroph population peaks precisely at the oxycline (9.5 m), although viable cells exist well into the anoxic, sulfidic waters of the lake. The sulfur-oxidizing bacteria described here likely play a key role in the biogeochemical cycling of carbon and sulfur in Lake Fryxell.     

Climatic influence on mesophilic Bacillus cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical, temperate and alpine soil

Bacillus weihenstephanensis strains are psychrotolerant and grow from below 7°C to 38°C. Closely related mesophilic Bacillus cereus strains can grow from above 7°C to 46°C. We classified 1060 B. cereus group isolates from different soil samples with respect to their psychrotolerant and mesophilic genotypes by polymerase chain reaction (PCR) targeting of specific 16S rDNA and cold shock protein A gene signatures. In parallel, growth tests at 7°C were carried out to determine the thermal phenotype. The geographic distribution of psychrotolerant and mesophilic isolates was found to depend significantly on the prevalent annual average temperature. In one tropical, one temperate and two alpine habitats, the proportion of psychrotolerant cspA genotypes was found to be 0%, 45% and 86% and 98%, respectively, with the corresponding annual average temperatures being 28°C, 7°C, 4°C and 1°C. In the tropical habitat, only the mesophilic B. cereus was found, characterized by correspondence of thermal genotype and phenotype. In the alpine habitat, almost only the psychrotolerant B. weihenstephanensis was isolated. In the temperate habitat, mesophilic B. cereus and psychrotolerant B. weihenstephanensis as well as 'intermediate thermal types' occurred, the latter having opposite thermal genotypes and phenotypes or opposing sets of thermal DNA signatures, characterized by the coexistence of mesophilic and psychrotolerant 16S rDNA operon copies within a single isolate. Both sugar utilization and DNA fingerprinting patterns revealed a high, probably non-clonal microsite diversity within the population of the temperate habitat. We interpret our observations in terms of a temperature-dependent selection regime, acting on recombining B. cereus / B. weihenstephanensis populations in soil.     

Methanosarcina lacustris sp. nov., a new psychrotolerant methanogenic archaeon from anoxic lake sediments

A new psychrotolerant methanogenic archaeon strain ZS was isolated from anoxic lake sediments (Switzerland). The cells of the organism were non-motile cocci, 1.5-3.5 microm in diameter. The cells aggregated and formed pseudoparenchyma. The cell wall was Gram-positive. The organism utilized methanol, mono-, di-, trimethylamine and H2/CO2 with methane production. The temperature range for growth was 1-35 degrees C with an optimum at 25 degrees C. The DNA G+C content of the organism was 43.4. mol%. Analysis of the 16S rRNA gene sequence showed that strain ZS was phylogenetically closely related to members of the genus Methanosarcina, but clearly differed from all described species of this genus (95.6-97.6% of sequence similarity). The level of DNA-DNA hybridization of strain ZS with Methanosarcina barkeri and Methanosarcina mazei was 15 and 31%, respectively. Based on the results of physiological and phylogenetic studies strain ZS can be assigned to a new species of the genus Methanasarcina. The name Methanosarcina lacustris sp. nov. is proposed. The type strain is ZS (= DSM 13486T, VKM B-2268).     

Isolation, Characterization, and Ecology of Cold-Active, Chemolithotrophic, Sulfur-Oxidizing Bacteria from Perennially Ice-Covered Lake Fryxell, Antarctica

Novel strains of obligately chemolithoautotrophic, sulfur-oxidizing bacteria have been isolated from various depths of Lake Fryxell, Antarctica. Physiological, morphological, and phylogenetic analyses showed these strains to be related to mesophilic Thiobacillus species, such as T. thioparus. However, the psychrotolerant Antarctic isolates showed an adaptation to cold temperatures and thus should be active in the nearly freezing waters of the lake. Enumeration by most-probable-number analysis in an oxic, thiosulfate-containing medium revealed that the sulfur-oxidizing chemolithotroph population peaks precisely at the oxycline (9.5 m), although viable cells exist well into the anoxic, sulfidic waters of the lake. The sulfur-oxidizing bacteria described here likely play a key role in the biogeochemical cycling of carbon and sulfur in Lake Fryxell.     

Synchronous effects of temperature, hydrostatic pressure, and salinity on growth, phospholipid profiles, and protein patterns of four Halomonas species isolated from deep-sea hydrothermal-vent and sea surface environments

Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30 degrees C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30 degrees C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30 degrees C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30 degrees C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.     

Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice,Spitzbergen

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4°C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses and of Proteobacteria, the Bacillus–Clostridium group, the order Actinomycetales, and the Cytophaga–Flexibacter–Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25°C. Only a few strains were psychrophilic, with an optimal growth at 10–15°C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4°C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, -amylase and -galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10°C, and almost no production was detected at higher temperatures (20–30°C). Catalytic activity was detected even below the freezing point of water (at –5°C), demonstrating the unique properties of these enzymes.     

SEASONAL DIFFERENCES IN THE TEMPERATURE RANGES OF GROWTH OF VIRGINIA ALGAE1

One hundred and fifteen clonal, unialgal strains were isolated and tested for their ability to grow over a range of temperatures from 2 to 40° C. Responses of 63 strains isolated from habitats that were 6° C when sampled and 52 strains isolated from habitats that were 20° C when sampled showed trends toward increasing adaptation to cold or warm temperatures commensurate with their seasonal in situ temperatures. Based on temperature-growth responses alone, 24% of the plankton isolates and 17% of the periphyton isolates could be perennial within the natural habitats. At 5° C, 56% of the warm water plankton isolates and 48% of the warm water periphyton isolates were incapable of growth and, therefore, probably could not be important components of the winter algal community. Likewise at 25° C, 25% of the cold water plankton isolates and 13% of the cold water periphyton isolates were incapable of growth. Thus, temperature alone probably is an important variable regulating seasonal changes in algal community structure. Pollution of these habitats by a thermal enrichment averaging + 5° C year-round could effect a pronounced change in algal species composition because many more taxa could be perennial and more taxa would be incapable of growth during naturally warm periods.     

Characterization of a heme-dependent catalase from Methanobrevibacter arboriphilus.

Recently it was reported that methanogens of the genus Methanobrevibacter exhibit catalase activity. This was surprising, since Methanobrevibacter species belong to the order Methanobacteriales, which are known not to contain cytochromes and to lack the ability to synthesize heme. We report here that Methanobrevibacter arboriphilus strains AZ and DH1 contained catalase activity only when the growth medium was supplemented with hemin. The heme catalase was purified and characterized, and the encoding gene was cloned. The amino acid sequence of the catalase from the methanogens is most similar to that of Methanosarcina barkeri.     

Correlation of 16S ribosomal DNA signature sequences with temperature-dependent growth rates of mesophilic and psychrotolerant strains of the Bacillus cereus group

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance.     

Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. isolated from fast ice from Adelie Land, Antarctica

Nine psychrotolerant bacteria were isolated from fast ice in the middle of Geologie Archipelago, Adelie Land, Antarctica and were categorized into two groups, based on their SDS-PAGE profiles. Representatives from each of the two groups, namely strains DD 48^T and SJ 14^T exhibited phenotypic and chemotaxonomic characteristics confirming to the genus Psychrobacter. The 16S rRNA gene sequence indicated that the two isolates are closely related to each other and to the already reported fifteen species of Psychrobacter. Detailed studies on the phenotypic characteristics, chemotaxonomic properties and phylogenetic analysis of strains DD 48^T and SJ 14^T indicated that they are distinctly different from each other and the reported species of Psychrobacter. At the DNA-DNA hybridisation level, the two species exhibit less than 70% similarity. Thus, strains DD 48^T and SJ 14^T are identified as new species of the genus Psychrobacter for which the names Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. respectively are proposed.     

Aceticlastic and NaCl-requiring methanogen "Methanosaeta pelagica" sp. nov., isolated from marine tidal flat sediment

Among methanogens, only 2 genera, Methanosaeta and Methanosarcina, are known to contribute to methanogenesis from acetate, and Methanosaeta is a specialist that uses acetate specifically. However, Methanosaeta strains so far have mainly been isolated from anaerobic digesters, despite the fact that it is widespread, not only in anaerobic methanogenic reactors and freshwater environments, but also in marine environments, based upon extensive 16S rRNA gene-cloning analyses. In this study, we isolated an aceticlastic methanogen, designated strain 03d30q(T), from a tidal flat sediment. Phylogenetic analyses based on 16S rRNA and mcrA genes revealed that the isolate belongs to the genus Methanosaeta. Unlike the other known Methanosaeta species, this isolate grows at Na(+) concentrations of 0.20 to 0.80 M, with an optimum concentration of 0.28 M. Quantitative estimation using real-time PCR detected the 16S rRNA gene of the genus Methanosaeta in the marine sediment, and relative abundance ranged from 3.9% to 11.8% of the total archaeal 16S rRNA genes. In addition, the number of Methanosaeta organisms increased with increasing depth and was much higher than that of Methanosarcina organisms, suggesting that aceticlastic methanogens contribute to acetate metabolism to a greater extent than previously thought in marine environments, where sulfate-reducing acetate oxidation prevails. This is the first report on marine Methanosaeta species, and based on phylogenetic and characteristic studies, the name "Methanosaeta pelagica" sp. nov. is proposed for this novel species, with type strain 03d30q.     

Vermistella arctica n. sp. Nominates the Genus Vermistella as a Candidate for Taxon with Bipolar Distribution

A new amoebozoan species, Vermistella arctica n. sp., is described from marine habitats in the central part of Svalbard archipelago. This is the first report on Arctic amoebae belonging to the genus Vermistella Moran and Anderson, 2007, the type species of which was described from the opposite pole of the planet. Psychrophily proved in the new strains qualifies the genus Vermistella as a bipolar taxon. Molecular phylogenetic analyses based on 18S rDNA and actin sequences did not show any affinity of the genus Vermistella to Stygamoeba regulata ATCC^® 50892^? strain. A close phylogenetic relationship was found between Vermistella spp. and a sequence originating from an environmental sample from Cariaco basin, the largest marine permanently anoxic system in the world. Possible mechanisms of bipolar distribution are discussed.     

Culture isolation and culture-independent clone libraries reveal new marine Synechococcus ecotypes with distinctive light and N physiologies

Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.     

Analysis of the Composition and Characteristics of Culturable Endophytic Bacteria Within Subnival Plants of the Tianshan Mountains, Northwestern China

This study first described the composition and characteristics of culturable endophytic bacteria isolated from wild alpine-subnival plant species growing under extreme environmental conditions (i.e., on the border of a glacier with frequently fluctuating and freezing temperatures, strong wind, and high ultraviolet radiation). Using a cultivation-dependent approach and 16S rRNA gene amplification techniques, 93 bacterial isolates showing different phenotypic properties were obtained from 20 different subnival plant species, of which gram-positive bacteria (61.5%), psychrotolerant bacteria (67.3%), and pigmented isolates (70.9%) accounted for a large proportion. All these characteristics of endophytes were closely related to the survival environment of their host plants and were in good agreement with microbes occurring in other cold environments. Phylogenetic analysis indicated that the endophytic isolates consisted of five phylogenetic groups comprising α-proteobacteria, γ-proteobacteria, the high G+C content gram-positive bacteria, the low G+C content gram-positive bacteria, and Flavobacterium-Bacteroides-Cytophaga. The largest generic diversity was found in the HGC group, while Clavibacter, Agreia, Rhodococcus, Sphingomonas, and Pseudomonas were the most prevalent genera. Of all isolates, 46.4% showed a high sequence similarity (98-100%) to strains discovered from other cold environments such as glaciers, tundra, and polar seas. Furthermore, 36.4% of the isolates produced Indole-3-acetic acid and 76.3% were able to solubilize mineral phosphate, which revealed that endophytic bacteria with multiple physiological functions were abundant and widespread in subnival plants. These results are essential for understanding the ecological roles of endophytes and as a foundation for further studying the interactions with plants and environment.     

Methanoferrodoxin represents a new class of superoxide reductase containing an iron-sulfur cluster

Protein MM0632 from the methanogenic archaeon Methanosarcina mazei showed strong superoxide reductase activity and rapidly decomposed superoxide radicals to peroxides. The superoxide reductase activity of the heterologously produced enzyme was determined by a cytochrome c assay and in a test system with NADPH, ferredoxin:NADP(+) reductase, and rubredoxin. Furthermore, EPR spectroscopy showed that MM0632 is the first superoxide reductase that possesses an iron-sulfur cluster instead of a second mononuclear iron center. We propose the name methanoferrodoxin for this new class of superoxide reductase with an [Fe(NHis)(4)(SCys)] site as the catalytic center and a [4Fe-4S] cluster as second prosthetic group that is probably involved in electron transfer to the catalytic center. Methanosarcina mazei grows only under anaerobic conditions, but is one of the most aerotolerant methanogens. It is tempting to speculate that methanoferrodoxin contributes to the protection of cells from oxygen radicals formed by flavoproteins during periodic exposure to oxygen in natural environments.     

Analysis of the anaerobic microbial community capable of degrading p-toluene sulphonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.     

Synchronous Effects of Temperature, Hydrostatic Pressure, and Salinity on Growth, Phospholipid Profiles, and Protein Patterns of Four Halomonas Species Isolated from Deep-Sea Hydrothermal- Vent and Sea Surface Environments

Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30°C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30°C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30°C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30°C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.     

Characterization of a Heme-Dependent Catalase from Methanobrevibacter arboriphilus

Recently it was reported that methanogens of the genus Methanobrevibacter exhibit catalase activity. This was surprising, since Methanobrevibacter species belong to the order Methanobacteriales, which are known not to contain cytochromes and to lack the ability to synthesize heme. We report here that Methanobrevibacter arboriphilus strains AZ and DH1 contained catalase activity only when the growth medium was supplemented with hemin. The heme catalase was purified and characterized, and the encoding gene was cloned. The amino acid sequence of the catalase from the methanogens is most similar to that of Methanosarcina barkeri.     

Culture Isolation and Culture-Independent Clone Libraries Reveal New Marine Synechococcus Ecotypes with Distinctive Light and N Physiologies

Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.