Diversity of halophilic archaea from six hypersaline environments in Turkey

The diversity of archaeal strains from six hypersaline environments in Turkey was analyzed by comparing their phenotypic characteristics and 16S rDNA sequences. Thirty-three isolates were characterized in terms of their phenotypic properties including morphological and biochemical characteristics, susceptibility to different antibiotics, and total lipid and plasmid contents, and finally compared by 16S rDNA gene sequences. The results showed that all isolates belong to the family Halobacteriaceae. Phylogenetic analyses using approximately 1,388 bp comparisions of 16S rDNA sequences demonstrated that all isolates clustered closely to species belonging to 9 genera, namely Halorubrum (8 isolates), Natrinema (5 isolates), Haloarcula (4 isolates), Natronococcus (4 isolates), Natrialba (4 isolates), Haloferax (3 isolates), Haloterrigena (3 isolates), Halalkalicoccus (1 isolate), and Halomicrobium (1 isolate). The results revealed a high diversity among the isolated halophilic strains and indicated that some of these strains constitute new taxa of extremely halophilic archaea.     

Hydrocarbon biodegradation in hypersaline environments

When mineral oil, hexadecane, and glutamate were added to natural samples of varying salinity (3.3 to 28.4%) from salt evaporation ponds and Great Salt Lake, Utah, rates of metabolism of these compounds decreased as salinity increased. Rate limitations did not appear to relate to low oxygen levels or to the availability of organic nutrients. Some oxidation of l-[U-C]glutamic acid occurred even at extreme salinities, whereas oxidation of [1-C]hexadecane was too low to be detected. Gas chromatographic examination of hexane-soluble components of tar samples from natural seeps at Rozel Point in Great Salt Lake demonstrated no evidence of biological oxidation of isoprenoid alkanes subject to degradation in normal environments. Some hexane-soluble components of the same tar were altered by incubation in a low-salinity enrichment culture inoculated with garden soil. Attempts to enrich for microorganisms in saline waters able to use mineral oil as a sole source of carbon and energy were successful below, but not above, about 20% salinity. This study strongly suggests a general reduction of metabolic rate at extreme salinities and raises doubt about the biodegradation of hydrocarbons in hypersaline environments.     

Archaea in dry soil environments

Archaea belong to the least well known major group of soil inhabiting microbes as the concept of the very existence of the archaea was introduced only in 1977 and the domain of Archaea established in 1990. The first reports of finding these organisms in soils were published even later. This paper will review the research carried out of the archaea in dry moderate soil environments. It will particularly consider the specific habitats where the archaea live in soils, as well as their associations with other organisms. There is thus far relatively little knowledge about the metabolism of the soil archaea, but the knowledge about their exact habitats and associations as well as their genetic potential point the way to discovering more about the different soil archaeal functions.     

Characterization of halophilic Archaea isolated from different hypersaline ecosystems

Halobacterial representatives were isolated from salted fish, naturally occurring salt pans as well as artificial saline poolsin the Mediterranean area. The isolation techniques experimented proved successful, allowing halophilic archaea to be retrieved iralmost 72% of analysed samples. About 65% of strains could be presumptively ascribed to the speciellaloarcula marismortui by RFLP (Restriction Fragment Length Polymorphism) grouping and 16S rRNA gene sequence comparison. Nevertheless, cluster analysis of RAPD-PCR patterns revealed a wide heterogeneity among isolated strains. Biochemical features of technological interest, such as proteclic, lipolytic and decarboxylase activity, were investigated in order to elucidate the role of archaea during ripening of salted anchovies. Proteolytic activity was only evidenced on the sarcoplasmic fraction extracted from fresh anchovies and for only 4 strains out of 2 No strain revealed either lipolytic capability on cod liver oil or proteolytic activity on fresh anchovies myofibrillar extract. Finally, only one strain exhibited decarboxylase activity, minimizing the potential responsibility of cultivable archaea microflora in the spoilage of salted anchovies.     

Uptake and turnover of acetate in hypersaline environments

Abstract: Acetate uptake and turnover rates were determined for the heterotrophic community in hypersaline environments (saltern crystallizer ponds, the Dead Sea) dominated by halpphilic Archaea. Acetate was formed from glycerol, which is potentially the major available carbon source for natural communities of halophilic Archaea. Values of [ K _t+ S _n] (the sum of the substrate affinity and the substrate concentration present in situ) for acetate measured in saltern crystallizer ponds were around 4.5–11.5 μM, while in the Dead Sea during a Dunaliella bloom values up to 12.8 μM were found. Maximal theoretical rates ( V _max) of acetate uptake in saltern crystallizer ponds were 12–56 nmol l⁻¹ h⁻¹, with estimated turnover times for acetate ( T _t) between 127–730 h at 35°C. V _max values measured in the Dead Sea were between 0.8 and 12.8 nmol l⁻¹ h⁻¹, with turnover times in the range of 320–2190 h. V _max values for acetate were much lower than those for glycerol. Comparisons with pure cultures of halophilic Archaea grown under different conditions showed that the natural communities were not adapted for preferential use of acetate. Both in natural brines and in pure cultures of halophilic Archaea, acetate incorporation rates rapidly decreased above the optimum pH value, probably since acetate enters the cell only in its unionized form. The low affinity for acetate, together with low potential utilization rates result in the long acetate turnover times, which explains the accumulation of acetate observed when low concentrations of glycerol are supplied as a nutrient to natural communities of halophilic Archaea.     

Osmotic adjustment in cyanobacteria from hypersaline environments

The intracellular concentrations of the monovalent inorganic cations K⁺ and Na⁺, low molecular weight carbohydrates and quaternary ammonium compounds have been determined for 4 strains of cyanobacteria (Aphanothece halophytica, Coccochloris elabens, Dactylococcopsis salina and Synechocystis DUN52) originally isolated from hypersaline habitats (i.e. habitats with a salinity greater than that of seawater) over a range of external salt concentration (from 50% to 400% seawater). Intracellular cation levels (Na⁺ and K⁺) were determined to be within the range 80–320 mmol · dm^-3 (cell volume), showing only minor changes in response to salinity. Intracellular carbohydrates were found to comprise a negligible component of the intracellular osmotic potential [at 2–19 mmol · dm^-3 (cell volume)], throughout the salinity range. Quaternary ammonium compounds, however, were recorded in osmotically significant quantities [up to 1,640 mmol · dm^-3 (cell volume)] in these strains, showing major variation in response to salinity. Thus Synechocystis DUN 52 showed an increase in quaternary ammonium compounds in the oder of 1,200 mmol · dm^-3 between 50% and 400% seawater medium, accounting for a significant proportion of the change in external osmotic potential.Examination of intact cells and cell extracts using ¹³C and ¹H nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of the quaternary ammonium compound glycine betaine as the major osmoticum in the 4 strains; no other compounds were detected during NMR assays. These results suggest a common mechanism of osmotic adjustment, involving quaternary ammonium compounds, in cyanobacteria from hypersaline environments.     

Phenol degradation by halophilic bacteria isolated from hypersaline environments

Phenol is a toxic aromatic compound used or produced in many industries and as a result a common component of industrial wastewaters. Phenol containing waste streams are frequently hypersaline and therefore require halophilic microorganisms for efficient biotreatment without dilution. In this study three halophilic bacteria isolated from different saline environments and identified as Halomonas organivorans, Arhodomonas aquaeolei and Modicisalibacter tunisiensis were shown to be able to grow on phenol in hypersaline media containing 100 g/L of total salts at a concentration of 3 mM (280 mg/L), well above the concentration found in most waste streams. Genes encoding the aromatic dioxygenase enzymes catechol 1,2 dioxygenase and protocatechuate 3,4-dioxygenase were present in all strains as determined by PCR amplification using primers specific for highly conserved regions of the genes. The gene for protocatechuate 3,4-dioxygenase was cloned from the isolated H. organivorans and the translated protein was evaluated by comparative protein sequence analysis with protocatechuate 3,4-dioxygenase proteins from other microorganisms. Although the analysis revealed a wide range of sequence divergence among the protocatechuate 3,4-dioxygenase family, all of the conserved domain amino acid structures identified for this enzyme family are identical or conservatively substituted in the H. organivorans enzyme.     

高盐环境可培养细菌噬菌体的研究进展

病毒广泛存在于各类环境中并担负着重要的生态功能,其中包括高盐环境。对高盐环境病毒的研究已成为极端环境微生物研究领域的新热点,目前已被报道的100多株盐病毒中,90多株感染古菌,仅有14株感染细菌。本文综述了目前已知的14株高盐环境细菌噬菌体的形态特征、盐度响应及基因组学的研究进展,并分析了高盐噬菌体的形态多样性、生存策略以及包含在基因组中的进化和起源信息,分析结果表明:高盐噬菌体以有尾噬菌体为主;它们具有广盐性(Euryhaline)的特征,盐度极大地影响其吸附和增殖;它们与非高盐环境噬菌体可能具有共同的起源。高盐噬菌体虽然历经近30年的研究历程,但仅有14株被分离与培养,所以其分离纯化是今后重要工作之一,且结合免培养技术揭示高盐噬菌体的多样性与生态功能是其研究的发展方向。     

Abundance and Diversity of Archaea in Heavy-Metal-Contaminated Soils

The impact of heavy-metal contamination on archaean communities was studied in soils amended with sewage sludge contaminated with heavy metals to varying extents. Fluorescent in situ hybridization showed a decrease in the percentage of Archaea from 1.3% ± 0.3% of 4′,6-diamidino-2-phenylindole-stained cells in untreated soil to below the detection limit in soils amended with heavy metals. A comparison of the archaean communities of the different plots by denaturing gradient gel electrophoresis revealed differences in the structure of the archaean communities in soils with increasing heavy-metal contamination. Analysis of cloned 16S ribosomal DNA showed close similarities to a unique and globally distributed lineage of the kingdom Crenarchaeota that is phylogenetically distinct from currently characterized crenarchaeotal species.     

Availability, uptake and turnover of glycerol in hypersaline environments

Abstract A sensitive assay for glycerol and other polyols was developed, based on periodate oxidation to formaldehyde, followed by a colorimetric assay with 3-methyl-2-benzothiazolone hydrazone. Apparent glycerol concentrations thus measured in saltern crystallizer ponds were around 20–36 μM, while in the Dead Sea, during a Dunaliella bloom, values were up to 27 μM. However, these values probably overestimate the glycerol concentrations present, as shown by labeled glycerol uptake experiments. Values of [K + S_n] (natural concentration + affinity constant) in saltern ponds were as low as 0.76–1.4 μM, with V_max values of 193–303 nmol 1⁻¹h⁻¹, and turnover times between 2.6–7.2 h at 35°C. Similar measurements in the Dead Sea were: [K + S_n] 0.07–1.41 μM, V_max values 160–426 nmol 1⁻¹h⁻¹, and turnover times in the range of 0.45–3.3 h.     

Diversity of Archaea in Brazilian savanna soils

Although the richness of Bacteria and Fungi in Cerrado’ soils has been reported, here we report, for the first time, the archaeal community in Cerrado’s soils. DNA extracted from soil of two distinct vegetation types, a dense subtype of sensu strict (cerrado denso) and riverbank forest (mata de galeria), was used to amplify Archaea-specific 16S rRNA gene. All of the fragments sequenced were classified as Archaea into the phylum Thaumarchaeota, predominantly affiliated to groups I.1b and I.1c. Sequences affiliated to the group I.1a were found only in the soil from riverbank forest. Soils from ‘cerrado denso’ had greater Archaea richness than those from ‘mata de galeria’ based on the richness indexes and on the rarefaction curve. β-Diversity analysis showed significant differences between the sequences from the two soil areas studied because of their different thaumarchaeal group composition. These results provide information about the third domain of life from Cerrado soils.     

Global network of specific virus-host interactions in hypersaline environments

Hypersaline environments are dominated by archaea and bacteria and are almost entirely devoid of eukaryotic organisms. In addition, hypersaline environments contain considerable numbers of viruses. Currently, there is only a limited amount of information about these haloviruses. The ones described in detail mostly resemble head-tail bacteriophages, whereas observations based on direct microscopy of the hypersaline environmental samples highlight the abundance of non-tailed virus-like particles. Here we studied nine spatially distant hypersaline environments for the isolation of new halophilic archaea (61 isolates), halophilic bacteria (24 isolates) and their viruses (49 isolates) using a culture-dependent approach. The obtained virus isolates approximately double the number of currently described archaeal viruses. The new isolates could be divided into three tailed and two non-tailed virus morphotypes, suggesting that both types of viruses are widely distributed and characteristic for haloarchaeal viruses. We determined the sensitivity of the hosts against all isolated viruses. It appeared that the host ranges of numerous viruses extend to hosts in distant locations, supporting the idea that there is a global exchange of microbes and their viruses. It suggests that hypersaline environments worldwide function like a single habitat.     

Formation and breakdown of glycine betaine and trimethylamine in hypersaline environments

Glycine betaine is accumulated as a compatible solute in many photosynthetic and non-photosynthetic bacteria — the last being unable to synthesize the compound - and thus large pools of betaine can be expected to be present in hypersaline environments. A variety of aerobic and anaerobic microorganisms degrade betaine to among other products trimethylamine and methylamine, in a number of different pathways. Curiously, very few of these betaine breakdown processes have yet been identified in hypersaline environments. Trimethylamine can also be formed by bacterial reduction of trimethylamine N-oxide (also by extremely halophilic archaeobacteria). Degradation of trimethylamine in hypersaline environments by halophilic methanogenic bacteria is relatively well documented, and leads to the formation of methane, carbon dioxide and ammonia.     

Cell sorting analysis of geographically separated hypersaline environments

Biogeography of microbial populations remains to be poorly understood, and a novel technique of single cell sorting promises a new level of resolution for microbial diversity studies. Using single cell sorting, we compared saturated NaCl brine environments (32–35 %) of the South Bay Salt Works in Chula Vista in California (USA) and Santa Pola saltern near Alicante (Spain). Although some overlap in community composition was detected, both samples were significantly different and included previously undiscovered 16S rRNA sequences. The community from Chula Vista saltern had a large bacterial fraction, which consisted of diverse Bacteroidetes and Proteobacteria. In contrast, Archaea dominated Santa Pola’s community and its bacterial fraction consisted of the previously known Salinibacter lineages. The recently reported group of halophilic Archaea, Nanohaloarchaea, was detected at both sites. We demonstrate that cell sorting is a useful technique for analysis of halophilic microbial communities, and is capable of identifying yet unknown or divergent lineages. Furthermore, we argue that observed differences in community composition reflect restricted dispersal between sites, a likely mechanism for diversification of halophilic microorganisms.     

西藏米拉山土壤古茵16S rRNA及amoA基因多样性分析

[目的]本研究旨在了解西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况.[方法]采用未培养技术直接从土壤中提取微生物总DNA,分别利用通用引物构建古菌16S rRNA基因和氨氧化古菌amoA基因克隆文库.利用DOTUR软件将古菌和氨氧化古菌序列按照相似性97%的标准分成若干个可操作分类单元(OTUs).[结果]通过构建系统发育树,表明古菌16s rRNA基因克隆文库包括泉古菌门和未分类的古菌两大类,并且所有泉古菌均属于热变形菌纲.氨氧化古菌amoA基因克隆文库中序列均为泉古菌.古菌16s rRNA基因和古菌amoA基因克隆文库分别包括64个OTUs和75个OTUs.[结论]西藏米拉山高寒草甸土壤中古菌多样性比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用.     

Ergosterol biosynthesis in novel melanized fungi from hypersaline environments

Halotolerant and halophilic melanized fungi were recently described in hypersaline waters. A close study of the sterol composition of such fungi, namely Hortaea werneckii, Alternaria alternata, Cladosporium sphaerospermum, Cladosporium sp., and Aureobasidium pullulans revealed the dominance of ergosterol and the presence of 29 intermediates of its biosynthesis pathway. The presence or absence of intermediates from distinct synthesis routes gave insight into the operative synthetic pathways from 4,4,14-trimethylcholesta-8,24-dien-3 beta-ol (lanosterol) to ergosterol in melanized fungi and in Saccharomyces cerevisiae, a reference yeast cultured in parallel. In all studied melanized fungi, initial methylation at C-24 took place before C-14 and C-4 demethylation, involving a different reaction sequence from that observed in S. cerevisiae. Further transformation was observed to occur through various routes. In A. alternata, isomerization at C-7 takes place prior to desaturation at C-5 and C-22, and methylene reduction at C-24. In addition to these pathways in Cladosporium spp., H. werneckii, and A. pullulans, ergosterol may also be synthesized through reduction of the C-24 methylene group before desaturation at C-5 and C-22 or vice versa. Moreover, in all studied melanized fungi except A. alternata, ergosterol biosynthesis may also proceed through C-24 methylene reduction prior to C-4 demethylation. -- Méjanelle, L., J. F. Lòpez, N. Gunde-Cimerman, and J. O. Grimalt. Ergosterol biosynthesis in novel melanized fungi from hypersaline environments. J. Lipid Res. 2001. 42: 352--358.     

Diversity in Chemotaxis Mechanisms among the Bacteria and Archaea

The study of chemotaxis describes the cellular processes that control the movement of organisms toward favorable environments. In bacteria and archaea, motility is controlled by a two-component system involving a histidine kinase that senses the environment and a response regulator, a very common type of signal transduction in prokaryotes. Most insights into the processes involved have come from studies of Escherichia coli over the last three decades. However, in the last 10 years, with the sequencing of many prokaryotic genomes, it has become clear that E. coli represents a streamlined example of bacterial chemotaxis. While general features of excitation remain conserved among bacteria and archaea, specific features, such as adaptational processes and hydrolysis of the intracellular signal CheY-P, are quite diverse. The Bacillus subtilis chemotaxis system is considerably more complex and appears to be similar to the one that existed when the bacteria and archaea separated during evolution, so that understanding this mechanism should provide insight into the variety of mechanisms used today by the broad sweep of chemotactic bacteria and archaea. However, processes even beyond those used in E. coli and B. subtilis have been discovered in other organisms. This review emphasizes those used by B. subtilis and these other organisms but also gives an account of the mechanism in E. coli.