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1.  Phylogenetic Diversity of Archaea and the Archaeal Ammonia Monooxygenase Gene in Uranium Mining-Impacted Locations in Bulgaria 
Archaea  2014;2014:196140.
Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/Nitrososphaera cluster of Crenarchaeota. The composition of the archaeal community is distinct for each site of interest and dependent on environmental characteristics, including pollution levels. Since the members of 1.1b/Nitrososphaera cluster have been implicated in the nitrogen cycle, the archaeal communities from these sites were probed for the presence of the ammonia monooxygenase gene (amoA). Our data indicate that amoA gene sequences are distributed in a similar manner as in Crenarchaeota, suggesting that archaeal nitrification processes in uranium mining-impacted locations are under the control of the same key factors controlling archaeal diversity.
doi:10.1155/2014/196140
PMCID: PMC3966351
2.  Towards a Computational Model of a Methane Producing Archaeum 
Archaea  2014;2014:898453.
Progress towards a complete model of the methanogenic archaeum Methanosarcina acetivorans is reported. We characterized size distribution of the cells using differential interference contrast microscopy, finding them to be ellipsoidal with mean length and width of 2.9 μm and 2.3 μm, respectively, when grown on methanol and 30% smaller when grown on acetate. We used the single molecule pull down (SiMPull) technique to measure average copy number of the Mcr complex and ribosomes. A kinetic model for the methanogenesis pathways based on biochemical studies and recent metabolic reconstructions for several related methanogens is presented. In this model, 26 reactions in the methanogenesis pathways are coupled to a cell mass production reaction that updates enzyme concentrations. RNA expression data (RNA-seq) measured for cell cultures grown on acetate and methanol is used to estimate relative protein production per mole of ATP consumed. The model captures the experimentally observed methane production rates for cells growing on methanol and is most sensitive to the number of methyl-coenzyme-M reductase (Mcr) and methyl-tetrahydromethanopterin:coenzyme-M methyltransferase (Mtr) proteins. A draft transcriptional regulation network based on known interactions is proposed which we intend to integrate with the kinetic model to allow dynamic regulation.
doi:10.1155/2014/898453
PMCID: PMC3960522
3.  Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic Archaeon Pyrococcus furiosus 
Archaea  2014;2014:176863.
The hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have α2β2 structures but the genome contains genes encoding three additional α-subunits. The ten possible combinations of α and β genes were expressed in E. coli and each resulted in stable and active α2β2 isoenzymes. The α-subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. The β-subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.
doi:10.1155/2014/176863
PMCID: PMC3942289  PMID: 24669200
4.  Comparative Analysis of the Methanogen Diversity in Horse and Pony by Using mcrA Gene and Archaeal 16S rRNA Gene Clone Libraries 
Archaea  2014;2014:483574.
Comparative analysis of methanogen compositions in the feces of horse and pony was carried out by constructing the α-subunit of methyl coenzyme-M reductase (mcrA) gene and 16S ribosomal RNA gene (16S rRNA) clone libraries. The mcrA clone library analysis indicated that Methanomicrobiales was predominant in both horse and pony. Furthermore, most of the clones of the 16S rRNA gene library showed that Methanomicrobiales was also predominant in horse and pony, but the LIBSHUFF analysis showed that the horse and pony libraries were significantly different (P < 0.05). Most of operational taxonomic units (OTUs) showed low similarity to the identified methanogens in both the mcrA and the 16S rRNA clone libraries. The results suggest that horse and pony harbor unidentified and novel methanogens in their hindgut. The methanogen population was higher in horse than in pony; however, the anaerobic fungal population was similar in horse and pony. The methanogen diversity was different between two breeds of Equus caballus.
doi:10.1155/2014/483574
PMCID: PMC3941164  PMID: 24678264
5.  Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette 
Archaea  2014;2014:374146.
Pyrrolysine (Pyl), the 22nd proteogenic amino acid, was restricted until recently to few organisms. Its translational use necessitates the presence of enzymes for synthesizing it from lysine, a dedicated amber stop codon suppressor tRNA, and a specific amino-acyl tRNA synthetase. The three genomes of the recently proposed Thermoplasmata-related 7th order of methanogens contain the complete genetic set for Pyl synthesis and its translational use. Here, we have analyzed the genomic features of the Pyl-coding system in these three genomes with those previously known from Bacteria and Archaea and analyzed the phylogeny of each component. This shows unique peculiarities, notably an amber tRNAPyl with an imperfect anticodon stem and a shortened tRNAPyl synthetase. Phylogenetic analysis indicates that a Pyl-coding system was present in the ancestor of the seventh order of methanogens and appears more closely related to Bacteria than to Methanosarcinaceae, suggesting the involvement of lateral gene transfer in the spreading of pyrrolysine between the two prokaryotic domains. We propose that the Pyl-coding system likely emerged once in Archaea, in a hydrogenotrophic and methanol-H2-dependent methylotrophic methanogen. The close relationship between methanogenesis and the Pyl system provides a possible example of expansion of a still evolving genetic code, shaped by metabolic requirements.
doi:10.1155/2014/374146
PMCID: PMC3941956  PMID: 24669202
6.  The Alternative Route to Heme in the Methanogenic Archaeon Methanosarcina barkeri 
Archaea  2014;2014:327637.
In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called “classical” heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460) together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793) was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar_A1458) was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme.
doi:10.1155/2014/327637
PMCID: PMC3942049  PMID: 24669201
7.  Development of β-Lactamase as a Tool for Monitoring Conditional Gene Expression by a Tetracycline-Riboswitch in Methanosarcina acetivorans 
Archaea  2014;2014:725610.
The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.
doi:10.1155/2014/725610
PMCID: PMC3942078  PMID: 24678266
8.  Early Methanogenic Colonisation in the Faeces of Meishan and Yorkshire Piglets as Determined by Pyrosequencing Analysis 
Archaea  2014;2014:547908.
Gut methanogenic archaea of monogastric animals are considered to be related to energy metabolism and adipose deposition of the host; however, information on their development in young piglets is limited. Thus, to investigate early methanogenic colonisation in the faeces of Meishan and Yorkshire piglets, faecal samples were collected from piglets at 1, 3, 7, and 14 days after birth and used to analyse the methanogenic community with 16S rRNA gene pyrosequencing. Results showed that the diversity of the methanogenic community in the faeces of neonatal piglets decreased from one to 14 days of age, as the total methanogen populations increased. The age of piglets, but not the breed, significantly affected the diversity of the methanogenic community which was dominated by the genus Methanobrevibacter. From the ages of one to 14 days, the abundance of M. smithii-related operational taxonomic units (OTUs) increased significantly, while the abundances of M. thaueri- and M. millerae-related OTUs decreased significantly. The substitution of M. smithii for M. thaueri/M. millerae was faster in Yorkshire piglets than in Meishan piglets. These results suggest that the early establishment of microbiota in neonatal piglets is accompanied by dramatic changes in the methanogenic community, and that the changes vary among pigs of different genotypes.
doi:10.1155/2014/547908
PMCID: PMC3941784  PMID: 24678265
9.  Comparative Analysis of Proteomes and Functionomes Provides Insights into Origins of Cellular Diversification 
Archaea  2013;2013:648746.
Reconstructing the evolutionary history of modern species is a difficult problem complicated by the conceptual and technical limitations of phylogenetic tree building methods. Here, we propose a comparative proteomic and functionomic inferential framework for genome evolution that allows resolving the tripartite division of cells and sketching their history. Evolutionary inferences were derived from the spread of conserved molecular features, such as molecular structures and functions, in the proteomes and functionomes of contemporary organisms. Patterns of use and reuse of these traits yielded significant insights into the origins of cellular diversification. Results uncovered an unprecedented strong evolutionary association between Bacteria and Eukarya while revealing marked evolutionary reductive tendencies in the archaeal genomic repertoires. The effects of nonvertical evolutionary processes (e.g., HGT, convergent evolution) were found to be limited while reductive evolution and molecular innovation appeared to be prevalent during the evolution of cells. Our study revealed a strong vertical trace in the history of proteins and associated molecular functions, which was reliably recovered using the comparative genomics approach. The trace supported the existence of a stem line of descent and the very early appearance of Archaea as a diversified superkingdom, but failed to uncover a hidden canonical pattern in which Bacteria was the first superkingdom to deploy superkingdom-specific structures and functions.
doi:10.1155/2013/648746
PMCID: PMC3892558  PMID: 24492748
10.  The Potent In Vitro Skin Permeation of Archaeosome Made from Lipids Extracted of Sulfolobus acidocaldarius 
Archaea  2013;2013:782012.
Archaeosomes are a new generation of liposomes that exhibit higher stabilities under different conditions, such as high temperatures, alkaline or acidic pH, and presence of bile salts in comparison with liposomes, and can be used in biotechnology including drug, gene, and vaccine delivery. The objective of this study was to prepare archaeosomes using lipid extracted from Sulfolobus acidocaldarius and evaluate their physicochemical properties. The lipids were extracted from S. acidocaldarius and assayed by High Performance Thin-Layer Chromatography (HPTLC). Archaeosomes were prepared using film method and methylene blue was used as drug model. They were characterized for their vesicle size and Differential Scanning Calorimetry (DSC) was used to investigate changes in their thermal behavior. The released amount of methylene blue was determined using a dialysis membrane and rat skin. HPTLC analysis of the extracted lipids showed that glycerol ether may be the major lipid with more than 78 percent probability. Results of particle size determination showed a mean size of 158.33 nm and the results of DSC indicated the possible interaction of methylene blue with lipids during the preparation of archaeosome. The addition of cholesterol significantly improved the encapsulation of methylene blue in the archaeosome so that the encapsulation efficiency was 61.66 ± 2.88%. The result of in vitro skin permeation showed that methylene blue could pass through skin model according to Peppas model and there was about 41.66% release after 6 h, whereas no release was observed through dialysis membrane. According to the results of the study, it is concluded that archaeosome may be successfully used as drug delivery system.
doi:10.1155/2013/782012
PMCID: PMC3888715  PMID: 24453698
11.  Archaeal Production of Polyhydroxyalkanoate (PHA) Co- and Terpolyesters from Biodiesel Industry-Derived By-Products 
Archaea  2013;2013:129268.
The archaeon Haloferax mediterranei was selected for production of PHA co- and terpolyesters using inexpensive crude glycerol phase (CGP) from biodiesel production as carbon source. CGP was assessed by comparison with the application of pure glycerol. Applying pure glycerol, a copolyester with a molar fraction of 3-hydroxybutyrate (3HB) of 0.90 mol/mol and 3-hydroxyvalerate (3HV) of 0.10 mol/mol, was produced at a volumetric productivity of 0.12 g/Lh and an intracellular PHA content of 75.4 wt.-% in the sum of biomass protein plus PHA. Application of CGP resulted in the same polyester composition and volumetric productivity, indicating the feasibility of applying CGP as feedstock. Analysis of molar mass distribution revealed a weight average molar mass Mw of 150 kDa and polydispersity Pi of 2.1 for pure glycerol and 253 kDa and 2.7 for CGP, respectively; melting temperatures ranged between 130 and 140°C in both setups. Supplying γ-butyrolactone as 4-hydroxybutyrate (4HB) precursor resulted in a poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate-co-4-hydroxybutyrate] (PHBHV4HB) terpolyester containing 3HV (0.12 mol/mol) and 4HB (0.05 mol/mol) in the poly[(R)-3-hydroxybutyrate] (PHB) matrix; in addition, this process runs without sterilization of the bioreactor. The terpolyester displayed reduced melting (melting endotherms at 122 and 137°C) and glass transition temperature (2.5°C), increased molar mass (391 kDa), and a polydispersity similar to the copolyesters.
doi:10.1155/2013/129268
PMCID: PMC3880725  PMID: 24453697
12.  A Single-Culture Bioprocess of Methanothermobacter thermautotrophicus to Upgrade Digester Biogas by CO2-to-CH4 Conversion with H2 
Archaea  2013;2013:157529.
We optimized and tested a postbioprocessing step with a single-culture archaeon to upgrade biogas (i.e., increase methane content) from anaerobic digesters via conversion of CO2 into CH4 by feeding H2 gas. We optimized a culture of the thermophilic methanogen Methanothermobacter thermautotrophicus using: (1) a synthetic H2/CO2 mixture; (2) the same mixture with pressurization; (3) a synthetic biogas with different CH4 contents and H2; and (4) an industrial, untreated biogas and H2. A laboratory culture with a robust growth (dry weight of 6.4–7.4 g/L; OD600 of 13.6–15.4), a volumetric methane production rate of 21 L/L culture-day, and a H2 conversion efficiency of 89% was moved to an industrial anaerobic digester facility, where it was restarted and fed untreated biogas with a methane content of ~70% at a rate such that CO2 was in excess of the stoichiometric requirements in relation to H2. Over an 8-day operating period, the dry weight of the culture initially decreased slightly before stabilizing at an elevated level of ~8 g/L to achieve a volumetric methane production rate of 21 L/L culture-day and a H2 conversion efficiency of 62%. While some microbial contamination of the culture was observed via microscopy, it did not affect the methane production rate of the culture.
doi:10.1155/2013/157529
PMCID: PMC3806361  PMID: 24194675
13.  Comparative Analysis of Barophily-Related Amino Acid Content in Protein Domains of Pyrococcus abyssi and Pyrococcus furiosus 
Archaea  2013;2013:680436.
Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code.
doi:10.1155/2013/680436
PMCID: PMC3804272  PMID: 24187517
14.  Microbial Community Analysis of a Methane-Producing Biocathode in a Bioelectrochemical System 
Archaea  2013;2013:481784.
A methane-producing biocathode that converts CO2 into methane was studied electrochemically and microbiologically. The biocathode produced methane at a maximum rate of 5.1 L CH4/m2 projected cathode per day (1.6 A/m2) at −0.7 V versus NHE cathode potential and 3.0 L CH4/m2 projected cathode per day (0.9 A/m2) at −0.6 V versus NHE cathode potential. The microbial community at the biocathode was dominated by three phylotypes of Archaea and six phylotypes of bacteria. The Archaeal phylotypes were most closely related to Methanobacterium palustre and Methanobacterium aarhusense. Besides methanogenic Archaea, bacteria seemed to be associated with methane production, producing hydrogen as an intermediate. Biomass density varied greatly with part of the carbon electrode covered with a dense biofilm, while only clusters of cells were found on other parts. Based on our results, we discuss how inoculum enrichment and changing operational conditions may help to increase biomass density and to select for microorganisms that produce methane.
doi:10.1155/2013/481784
PMCID: PMC3800620  PMID: 24187516
15.  Genetic Confirmation of the Role of Sulfopyruvate Decarboxylase in Coenzyme M Biosynthesis in Methanococcus maripaludis 
Archaea  2013;2013:185250.
Coenzyme M is an essential coenzyme for methanogenesis. The proposed biosynthetic pathway consists of five steps, of which the fourth step is catalyzed by sulfopyruvate decarboxylase (ComDE). Disruption of the gene comE by transposon mutagenesis resulted in a partial coenzyme M auxotroph, which grew poorly in the absence of coenzyme M and retained less than 3% of the wild type level of coenzyme M biosynthesis. Upon coenzyme M addition, normal growth of the mutant was restored. Moreover, complementation of the mutation with the wild type comE gene in trans restored full growth in the absence of coenzyme M. These results confirm that ComE plays an important role in coenzyme M biosynthesis. The inability to yield a complete CoM auxotroph suggests that either the transposon insertion failed to completely inactivate the gene or M. maripaludis possesses a promiscuous activity that partially complemented the mutation.
doi:10.1155/2013/185250
PMCID: PMC3787637  PMID: 24151448
16.  Low-Temperature (10°C) Anaerobic Digestion of Dilute Dairy Wastewater in an EGSB Bioreactor: Microbial Community Structure, Population Dynamics, and Kinetics of Methanogenic Populations 
Archaea  2013;2013:346171.
The feasibility of anaerobic digestion of dairy wastewater at 10°C was investigated in a high height : diameter ratio EGSB reactor. Stable performance was observed at an applied organic loading rate (OLR) of 0.5–2 kg COD m−3 d−1 with chemical oxygen demand (COD) removal efficiencies above 85%. When applied OLR increased to values above 2 kg COD m−3 d−1, biotreatment efficiency deteriorated, with methanogenesis being the rate-limiting step. The bioreactor recovered quickly (3 days) after reduction of the OLR. qPCR results showed a reduction in the abundance of hydrogenotrophic methanogenic Methanomicrobiales and Methanobacteriales throughout the steady state period followed by a sharp increase in their numbers (111-fold) after the load shock. Specific methanogenic activity and maximum substrate utilising rate (Amax) of the biomass at the end of trial indicated increased activity and preference towards hydrogenotrophic methanogenesis, which correlated well with the increased abundance of hydrogenotrophic methanogens. Acetoclastic Methanosaeta spp. remained at stable levels throughout the trial. However, increased apparent half-saturation constant (Km) at the end of the trial indicated a decrease in the specific substrate affinity for acetate of the sludge, suggesting that Methanosaeta spp., which have high substrate affinity, started to be outcompeted in the reactor.
doi:10.1155/2013/346171
PMCID: PMC3780618  PMID: 24089597
17.  Archaeal Assemblages Inhabiting Temperate Mixed Forest Soil Fluctuate in Taxon Composition and Spatial Distribution over Time 
Archaea  2013;2013:870825.
This study explored the persistence and spatial distribution of a diverse Archaeal assemblage inhabiting a temperate mixed forest ecosystem. Persistence under native conditions was measured from 2001 to 2010, 2011, and 2012 by comparison of 16S rRNA gene clone libraries. The Archaeal assemblages at each of these time points were found to be significantly different (AMOVA, P < 0.01), and the nature of this difference was dependent on taxonomic rank. For example, the cosmopolitan genus g_Ca. Nitrososphaera (I.1b) was detected at all time points, but within this taxon the abundance of s_SCA1145, s_SCA1170, and s_Ca. N. gargensis fluctuated over time. In addition, spatial heterogeneity (patchiness) was measured at these time points using 1D TRFLP-SSCP fingerprinting to screen soil samples covering multiple spatial scales. This included soil collected from small volumes of 3 cubic centimeters to larger scales—over a surface area of 50 m2, plots located 1.3 km apart, and a separate locality 23 km away. The spatial distribution of Archaea in these samples changed over time, and while g_Ca. Nitrososphaera (I.1b) was dominant over larger scales, patches were found at smaller scales that were dominated by other taxa. This study measured the degree of change for Archaeal taxon composition and patchiness over time in temperate mixed forest soil.
doi:10.1155/2013/870825
PMCID: PMC3747363  PMID: 23983618
18.  Virus-Host and CRISPR Dynamics in Archaea-Dominated Hypersaline Lake Tyrrell, Victoria, Australia 
Archaea  2013;2013:370871.
The study of natural archaeal assemblages requires community context, namely, a concurrent assessment of the dynamics of archaeal, bacterial, and viral populations. Here, we use filter size-resolved metagenomic analyses to report the dynamics of 101 archaeal and bacterial OTUs and 140 viral populations across 17 samples collected over different timescales from 2007–2010 from Australian hypersaline Lake Tyrrell (LT). All samples were dominated by Archaea (75–95%). Archaeal, bacterial, and viral populations were found to be dynamic on timescales of months to years, and different viral assemblages were present in planktonic, relative to host-associated (active and provirus) size fractions. Analyses of clustered regularly interspaced short palindromic repeat (CRISPR) regions indicate that both rare and abundant viruses were targeted, primarily by lower abundance hosts. Although very few spacers had hits to the NCBI nr database or to the 140 LT viral populations, 21% had hits to unassembled LT viral concentrate reads. This suggests local adaptation to LT-specific viruses and/or undersampling of haloviral assemblages in public databases, along with successful CRISPR-mediated maintenance of viral populations at abundances low enough to preclude genomic assembly. This is the first metagenomic report evaluating widespread archaeal dynamics at the population level on short timescales in a hypersaline system.
doi:10.1155/2013/370871
PMCID: PMC3703381  PMID: 23853523
19.  Deposition of Biogenic Iron Minerals in a Methane Oxidizing Microbial Mat 
Archaea  2013;2013:102972.
The syntrophic community between anaerobic methanotrophic archaea and sulfate reducing bacteria forms thick, black layers within multi-layered microbial mats in chimney-like carbonate concretions of methane seeps located in the Black Sea Crimean shelf. The microbial consortium conducts anaerobic oxidation of methane, which leads to the formation of mainly two biomineral by-products, calcium carbonates and iron sulfides, building up these chimneys. Iron sulfides are generated by the microbial reduction of oxidized sulfur compounds in the microbial mats. Here we show that sulfate reducing bacteria deposit biogenic iron sulfides extra- and intracellularly, the latter in magnetosome-like chains. These chains appear to be stable after cell lysis and tend to attach to cell debris within the microbial mat. The particles may be important nuclei for larger iron sulfide mineral aggregates.
doi:10.1155/2013/102972
PMCID: PMC3697272  PMID: 23843725
20.  The Nitrosopumilus maritimus CdvB, but Not FtsZ, Assembles into Polymers 
Archaea  2013;2013:104147.
Euryarchaeota and Crenarchaeota are two major phyla of archaea which use distinct molecular apparatuses for cell division. Euryarchaea make use of the tubulin-related protein FtsZ, while Crenarchaea, which appear to lack functional FtsZ, employ the Cdv (cell division) components to divide. Ammonia oxidizing archaeon (AOA) Nitrosopumilus maritimus belongs to another archaeal phylum, the Thaumarchaeota, which has both FtsZ and Cdv genes in the genome. Here, we used a heterologous expression system to characterize FtsZ and Cdv proteins from N. maritimus by investigating the ability of these proteins to form polymers. We show that one of the Cdv proteins in N. maritimus, the CdvB (Nmar_0816), is capable of forming stable polymers when expressed in fission yeast. The N. maritimus CdvB is also capable of assembling into filaments in mammalian cells. However, N. maritimus FtsZ does not assemble into polymers in our system. The ability of CdvB, but not FtsZ, to polymerize is consistent with a recent finding showing that several Cdv proteins, but not FtsZ, localize to the mid-cell site in the dividing N. maritimus. Thus, we propose that it is Cdv proteins, rather than FtsZ, that function as the cell division apparatus in N. maritimus.
doi:10.1155/2013/104147
PMCID: PMC3684127  PMID: 23818813
21.  The Effect of Saturated Fatty Acids on Methanogenesis and Cell Viability of Methanobrevibacter ruminantium 
Archaea  2013;2013:106916.
Saturated fatty acids (SFAs) are known to suppress ruminal methanogenesis, but the underlying mechanisms are not well known. In the present study, inhibition of methane formation, cell membrane permeability (potassium efflux), and survival rate (LIVE/DEAD staining) of pure ruminal Methanobrevibacter ruminantium (DSM 1093) cell suspensions were tested for a number of SFAs. Methane production rate was not influenced by low concentrations of lauric (C12; 1 μg/mL), myristic (C14; 1 and 5 μg/mL), or palmitic (C16; 3 and 5 μg/mL) acids, while higher concentrations were inhibitory. C12 and C14 were most inhibitory. Stearic acid (C18), tested at 10–80 μg/mL and ineffective at 37°C, decreased methane production rate by half or more at 50°C and ≥50 μg/mL. Potassium efflux was triggered by SFAs (C12 = C14 > C16 > C18 = control), corroborating data on methane inhibition. Moreover, the exposure to C12 and C14 decreased cell viability to close to zero, while 40% of control cells remained alive after 24 h. Generally, tested SFAs inhibited methanogenesis, increased cell membrane permeability, and decreased survival of M. ruminantium in a dose- and time-dependent way. These results give new insights into how the methane suppressing effect of SFAs could be mediated in methanogens.
doi:10.1155/2013/106916
PMCID: PMC3655487  PMID: 23710130
22.  Archaeal Community Structures in the Solfataric Acidic Hot Springs with Different Temperatures and Elemental Compositions 
Archaea  2013;2013:723871.
Archaeal 16S rRNA gene compositions and environmental factors of four distinct solfataric acidic hot springs in Kirishima, Japan were compared. The four ponds were selected by differences of temperature and total dissolved elemental concentration as follows: (1) Pond-A: 93°C and 1679 mg L−1, (2) Pond-B: 66°C and 2248 mg L−1, (3) Pond-C: 88°C and 198 mg L−1, and (4) Pond-D: 67°C and 340 mg L−1. In total, 431 clones of 16S rRNA gene were classified into 26 phylotypes. In Pond-B, the archaeal diversity was the highest among the four, and the members of the order Sulfolobales were dominant. The Pond-D also showed relatively high diversity, and the most frequent group was uncultured thermoacidic spring clone group. In contrast to Pond-B and Pond-D, much less diverse archaeal clones were detected in Pond-A and Pond-C showing higher temperatures. However, dominant groups in these ponds were also different from each other. The members of the order Sulfolobales shared 89% of total clones in Pond-A, and the uncultured crenarchaeal groups shared 99% of total Pond-C clones. Therefore, species compositions and biodiversity were clearly different among the ponds showing different temperatures and dissolved elemental concentrations.
doi:10.1155/2013/723871
PMCID: PMC3654644  PMID: 23710131
23.  PH1: An Archaeovirus of Haloarcula hispanica Related to SH1 and HHIV-2 
Archaea  2013;2013:456318.
Halovirus PH1 infects Haloarcula hispanica and was isolated from an Australian salt lake. The burst size in single-step growth conditions was 50–100 PFU/cell, but cell density did not decrease until well after the rise (4–6 hr p.i.), indicating that the virus could exit without cell lysis. Virions were round, 51 nm in diameter, displayed a layered capsid structure, and were sensitive to chloroform and lowered salt concentration. The genome is linear dsDNA, 28,064 bp in length, with 337 bp terminal repeats and terminal proteins, and could transfect haloarchaeal species belonging to five different genera. The genome is predicted to carry 49 ORFs, including those for structural proteins, several of which were identified by mass spectroscopy. The close similarity of PH1 to SH1 (74% nucleotide identity) allowed a detailed description and analysis of the differences (divergent regions) between the two genomes, including the detection of repeat-mediated deletions. The relationship of SH1-like and pleolipoviruses to previously described genomic loci of virus and plasmid-related elements (ViPREs) of haloarchaea revealed an extensive level of recombination between the known haloviruses. PH1 is a member of the same virus group as SH1 and HHIV-2, and we propose the name halosphaerovirus to accommodate these viruses.
doi:10.1155/2013/456318
PMCID: PMC3622292  PMID: 23585730
24.  Temporal and Spatial Coexistence of Archaeal and Bacterial amoA Genes and Gene Transcripts in Lake Lucerne 
Archaea  2013;2013:289478.
Despite their crucial role in the nitrogen cycle, freshwater ecosystems are relatively rarely studied for active ammonia oxidizers (AO). This study of Lake Lucerne determined the abundance of both amoA genes and gene transcripts of ammonia-oxidizing archaea (AOA) and bacteria (AOB) over a period of 16 months, shedding more light on the role of both AO in a deep, alpine lake environment. At the surface, at 42 m water depth, and in the water layer immediately above the sediment, AOA generally outnumbered AOB. However, in the surface water during summer stratification, when both AO were low in abundance, AOB were more numerous than AOA. Temporal distribution patterns of AOA and AOB were comparable. Higher abundances of amoA gene transcripts were observed at the onset and end of summer stratification. In summer, archaeal amoA genes and transcripts correlated negatively with temperature and conductivity. Concentrations of ammonium and oxygen did not vary enough to explain the amoA gene and transcript dynamics. The observed herbivorous zooplankton may have caused a hidden flux of mineralized ammonium and a change in abundance of genes and transcripts. At the surface, AO might have been repressed during summer stratification due to nutrient limitation caused by active phytoplankton.
doi:10.1155/2013/289478
PMCID: PMC3603158  PMID: 23533328
25.  Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi 
Archaea  2013;2013:568053.
Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.
doi:10.1155/2013/568053
PMCID: PMC3603647  PMID: 23533329

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