Phylogenetic analysis of Group I marine archaeal rRNA sequences emphasizes the hidden diversity within the primary group Archaea

Archaea form one of the three primary groups of extant life and are commonly associated with the extreme environments which many of their members inhabit. Currently, the Archaea are classified into two kingdoms, Crenarchaeota and Euryarchaeota, based on phylogenetic analysis of ribosomal RNA (rRNA) sequences. Molecular techniques allowing the retrieval and analysis of rRNA sequences from diverse environments are increasing our knowledge of archaeal diversity. This report describes the presence of marine Archaea in north-east Atlantic waters. Quantitative estimates indicated that the marine Archaea constitute 8 per cent of the total prokaryotic rRNA in Irish coastal waters. Phylogenetic analysis of the archaeal rRNA gene sequences revealed sufficient genetic diversity within Archaea to indicate that the current two-kingdom classification of Crenarchaeota and Euryarchaeota is restrictive.     

Population Structure and Phylogenetic Characterization of Marine Benthic Archaea in Deep-Sea Sediments

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.     

中国9种嗜子宫线虫系统发育的初步研究

为了探讨鱼类寄生嗜子宫线虫的系统发育关系,测定了8种嗜子宫线虫的ITS rDNA(核糖体转录内间隔区核 糖核酸)序列和9种嗜子宫线虫的18S rDNA(小亚基核糖体核糖核酸)部分序列,并构建了18S rDNA序列的系统发 育树。在比较和分析ITS rDNA和18S rDNA两种分子标记对嗜子宫科线虫系统发育适用性的基础上,分析了嗜子 宫线虫的系统发育关系。结果表明:中国嗜子宫线虫是单系起源;黄颡鱼似嗜子宫线虫、赣州似嗜子宫线虫和棍头 嗜子宫线虫亲缘关系非常接近,可能是较晚形成的种;似嗜子宫线虫属可能应该被细分为更多的属。     

Characterization of extremely halophilic Archaea isolated from the Ayvalik Saltern,Turkey

Seven extremely halophilic strains were isolated from the Ayvalik Saltern in the north-eastern part of Turkey. Chemical analyses of the brine and salt samples were performed to measure their salt content, hardness and pH. Isolated strains were tested for their antibiotic sensitivities; cell and colony morphologies; hydrolysis of casein, starch, gelatin, Tween 20 and Tween 80; and oxidase and catalase activity. All strains were found to belong to the domain Archaea. Characterization of polar lipids by thin layer chromatography indicated that all isolates contained phytanyl diether derivatives of phosphatidylglycerol (PG), the methyl ester of phosphatidyl glycerophosphate (PGP-Me), and phosphatidylglycerosulphate (PGS). Four isolates had triglycosyl diether (TGD-2) as glycolipid, and the other three contained a sulphated diglycosyl diether instead. All isolates were examined for the presence of plasmids by agarose gel electrophoresis. Four strains were found to harbour plasmids ranging in size from 13.8 to 15.3 kbp. Correlation between the protein profiles in SDS–PAGE and the phenotypic properties of the strains was poor. The data presented here provide the first published account of the microbiota of the Ayvalik saltern, which provides a large part of the salt produced in Turkey.     

Phylogenetic analysis of archaeal PCNA homologues

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27 kDa) and 248 aa (27 kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages. Received: July 10, 2000 / Accepted: September 26, 2000     

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three “classical” morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.     

Molecular Phylogenetic Analysis of Archaea and Bacteria in Wind Cave,South Dakota

The diversity of bacteria and archaea was characterized from sediments collected from Wind Cave located in Wind Cave National Park in the Black Hills of South Dakota. Wind Cave is a limestone dissolution cave with strata that started forming over 300 million years ago, making it one of the oldest in the world. Previous work suggested that the cave was largely a detritus based system ultimately dependent upon allochthonous energy and carbon from photosynthesis of the overlying vegetation, and algae growing near lights along the tour routes. In this work, we used a molecular phylogenetic approach to characterize the microbial structure and infer a corresponding ecosystem function where appropriate. Four bacterial divisions and subdivisions were found in the culture collection, which represented 14 phylotypes, whereas 12 divisions and subdivisions were identified in the clonal analysis comprising 49 phylotypes. The predominant groups were the γ-Proteobacteria and Acidobacteria. Although a few of the clones resembled sequences from other cave and subterranean systems, no cave-specific bacterial community was evident in this work. Archaeal phylotypes (20 Crenarchaeota and 2 Euryarchaeota) were detected, with a large proportion of the Crenarchaeota resembling sequences from a South African gold mine. One archaeal cluster in particular appears to be specific to the subterranean environment. Most of the microbial sequences were not related to known chemolithoautotrophs, therefore we conclude that this particular community is likely detritus based where allochthonous energy and carbon are transported into the cave by infiltrating waters.     

Nanoarchaeal 16S rRNA gene sequences are widely dispersed in hyperthermophilic and mesophilic halophilic environments

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.     

Divergent Histories of rDNA Group I Introns in the Lichen Family Physciaceae

The wide but sporadic distribution of group I introns in protists, plants, and fungi, as well as in eubacteria, likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA, which can support the insertion and/or retention of group I introns. In contrast, we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene. Reviewing Editor: Dr. W. Ford Doolittle     

利用三种分子标记研究缘毛类纤毛虫的系统发育地位

为了探讨缘毛类纤毛虫的系统发育地位 ,利用RAPD方法得到了 9种缘毛类纤毛虫、 1种四膜虫和1种喇叭虫的 3个随机引物的电泳带谱 ;测定了 7种缘毛类纤毛虫rRNA基因中的间隔区 1(ITS1)和小亚基核糖体核糖核酸 (SSrRNA)基因序列 ,并构建了相应的系统树。在比较和分析RAPD、ITS1和SSrRNA基因序列在缘毛类纤毛虫系统发育研究中的适用范围的基础上 ,以SSrRNA基因序列为分子标记研究了缘毛类纤毛虫系统发育地位 ,结果表明 :①缘毛亚纲是单系的 ,作为寡膜纲中一个亚纲的分类地位是合理的 ;②缘毛类纤毛虫可能是寡膜纲中较高等的一个类群。     

Cloning and sequencing of a gene encoding 16S ribosomal RNA from a novel hyperthermophilic archaebacterium NC12

A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring. To characterize this organism, a gene coding for 16S rRNA was cloned and sequenced. The 16S rRNA sequence from NC12 shows the highest similarity with those from Pyrodictium occultum and Desulfurococcus mobilis among the sequences in the database, indicating that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota) in the archaeal domain. However, since the highest identity score was only 91.2%, it is suggested that NC12 may constitute a new genus.     

Molecular characterization of bacterial population in the forest soil of Kashmir,India

The bacterial diversity in the forest soil of Kashmir, India was investigated by 16S rDNA-dependent molecular phylogeny. Small subunit rRNA (16S rDNA) from forest soil metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain bacteria. 30 unique phylotypes were obtained by PCR based RFLP of 16S rRNA genes using endonucleases Hae 111 and Msp 1, which were most suitable to score the genetic diversity. The use of 16S rRNA analysis allowed identification of several bacterial populations in the soil belonging to the following phyla: Firmicutes (33.3%), Bacteroidetes (13.3%), Proteobacterium (6.6%), Planctomycete (3.3%), and Deferribacteraceae (3.3%) in addition to the others that were not classified, beyond Archaea domain, However, 36.6% of the retrieved bacterial sequences could not be grouped with any phylum/lineage. The large amount of unclassified clone sequence could imply that novel groups of bacteria were present in the forest soil.     

Phylogenetic relationships of the Santalales and relatives

Summary Determining relationships among parasitic angiosperms has often been difficult owing to frequent morphological reductions in floral and vegetative features. We report 18S (small-subunit) rRNA sequences for representative genera of three families within the Santalales (Olacaceae, Santalaceae, and Viscaceae) and six outgroup dicot families (Celastraceae, Cornaceae, Nyssaceae, Buxaceae, Apiaceae, and Araliaceae). Using Wagner parsimony analysis, one most parsimonius tree resulted that shows the Santalales to be a holophyletic taxon most closely related toEuronymus (Celastraceae). The santalalean taxa showed approximately 13% more transitional mutations than the group of seven other dicot species. This suggests a higher fixation rate for mutations in these organisms, possibly owing to a relaxation of selection pressures at the molecular level in parasitic vs nonparasitic plants. Outgroup relationships are generally in accord with current taxonomic classifications, such as the grouping of Nyssaceae and Cornaceae together (Cornales) and the grouping of Araliaceae with Apiaceae (Apiales). These data provide the first nucleotide sequences for any parasitic flowering plant and support the contention that rRNA sequence analysis can result in robust phylogenetic comparisons at the family level and above.     

A molecular phylogenetic investigation of Pseudoamphisiella and Parabirojimia (Protozoa, Ciliophora, Spirotrichea), two genera with ambiguous systematic positions

The gene coding for the small subunit ribosomal RNA molecule was sequenced in three marine spirotrichs: Pseudoamphisiella lacazei, P. alveolata, and Parabirojimia similis, the systematic positions of which remain unclear in spite of recent progress. Phylogenetic trees were constructed by means of neighbor-joining (NJ), least-squares (LS), maximum parsimony (MP), and Bayesian inference (BI) methods in order to test previously inferred phylogenetic assignments of Pseudoamphisiella and Parabirojimia based on morphological and morphogenetic data. The main results are that: (1) all trees constructed have similar topologies in which the genus Parabirojimia derives at the base of the class Stichotrichia, which suggests that it might represent a unique taxon at about suborder or even-order level near urostylids; (2) the present study supports the conclusion reported by previous researchers that urostylids could be a paraphyletic assemblage and that Uroleptus and Paruroleptus should be removed from the order Urostylida; (3) Pseudoamphisiellidae/Pseudoamphisiella always clusters with Prodiscocephalus borrori rather than with urostylids, which indicates that they might represent an ancestral form for both discocephaline and urostylid species; (4) the monophyly of the traditional suborder Discocephalina and typical euplotids is consistently rejected. Based on both morphological/morphogenetical and molecular data obtained, a new suborder Parabirojimina n. subord. is suggested, which currently includes one family, Parabirojimidae n. fam., and the single genus Parabirojimia.     

Molecular diversity analysis of rumen methanogenic Archaea from goat in eastern China by DGGE methods using different primer pairs

Aims:  To screen a pair of primers suitable for denaturing gradient gel electrophoretic (DGGE) analysis of ruminal methanogenic Archaea and to detect the archaeal communities in the rumen of goat.
Methods and Results:  Nine primer pairs for 16S rDNA of methanogenic Archaea , including six for directed polymerase chain reaction (PCR) and three for nested PCR were first evaluated by PCR amplification of the total DNA from rumen fluids and bacteria. The DGGE analysis of rumen fluids was then conducted with three primer sets (344fGC/915r, 1106fGC/1378r and 519f/915rGC) of the nine pairs tested. Good separation and quality of patterns were obtained in DGGE analysis with primer pairs 1106fGC/1378r and 519f/915rGC. A total of 40 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic Archaea while primer pair 519f/915rGC had better amplification ranges than the other two primer pairs.
Conclusions:  The procedure of DGGE analysis with primer pair 519f/915rGC was more suitable for investigating methanogenic archaeal community in the rumen. The dominant methanogenic Archaea in the rumen of goat was Methanobrevibacter sp. and an unidentified methanogenic Archaea .
Significance and Impact of the Study:  One pair of primers suitable for DGGE analysis of ruminal methanogenic Archaea was obtained and the molecular diversity of ruminal methanogenic Archaea in goat was investigated by PCR-DGGE.     

Community Structure of Archaea in the Water Column above Gas Hydrates in the Gulf of Mexico

Microorganisms play fundamental roles in the ecosystem of the Gulf of Mexico (GOM), yet their vertical distributions along the depth continuum of water column are not well known. In this study, we presented the 16S rDNA sequences and lipid profiles in the context of water chemistry to characterize the archaeal community structure above a gas hydrate mound (MC 118) in GOM. Our results showed that all archaeal sequences were related to unknown species of Crenarchaeota or Euryarchaeota. Phylogenetically, group II –β Euryarchaeota dominated the surface water and mid-depth (400-m) water (74% and 58% of total archaeal species, respectively) whereas the marine group I-γ Crenarchaeota dominated the bottom (869 m) water (61% of total archaeal species). Estimates of the Shannon index showed the highest diversity of planktonic Archaea at the 400 m depth. Glycerol dialkyl glycerol tetraether (GDGT) lipids were detected from the 400- and 869-m depths only and characterized by relatively high abundances of GDGT-5 (crenarchaeol) and GDGT-0. Our studies suggested a possible zonation of archaeal community in the water column, which did not seem to be affected by the possible venting of hydrocarbons from the hydrate location in GOM.     

Identification of Protein-Tyrosine Phosphatases in Archaea

Protein-tyrosine dephosphorylation is a major mechanism in cellular regulation. A large number of protein-tyrosine phosphatases is known from Eukarya, and more recently bacterial homologues have also been identified. By employing conserved sequence patterns from both eukaryotic and bacterial protein-tyrosine phosphatases, we have identified three homologous sequences in two of the four complete archaeal genomes. Two hypothetical open reading frames in the genome of Methanococcus jannaschii (MJ0215 and MJECL20) and one in the genome of Pyrococcus horikoshii (PH1732) clearly bear all the conserved residues of this family. No homologues were found in the genomes of Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum. This is the first report of protein-tyrosine phosphatase sequences in Archaea. Received: 29 April 1998 / Accepted: 27 November 1998     

Characterization of cofactor-dependent and cofactor-independent phosphoglycerate mutases from Archaea

Phosphoglycerate mutases (PGM) catalyze the reversible conversion of 3-phosphoglycerate and 2-phosphoglycerate as part of glycolysis and gluconeogenesis. Two structural and mechanistically unrelated types of PGMs are known, a cofactor (2,3-bisphosphoglycerate)-dependent (dPGM) and a cofactor-independent enzyme (iPGM). Here, we report the characterization of the first archaeal cofactor-dependent PGM from Thermoplasma acidophilum, which is encoded by ORF TA1347. This ORF was cloned and expressed in Escherichia coli and the recombinant protein was characterized as functional dPGM. The enzyme constitutes a 46 kDa homodimeric protein. Enzyme activity required 2,3-bisphosphoglycerate as cofactor and was inhibited by vanadate, a specific inhibitor of dPGMs in bacteria and eukarya; inhibition could be partially relieved by EDTA. Histidine 23 of the archaeal dPGM of T. acidophilum, which corresponds to active site histidine in dPGMs from bacteria and eukarya, was exchanged for alanine by site directed mutagenesis. The H23A mutant was catalytically inactive supporting the essential role of H23 in catalysis of the archaeal dPGM. Further, an archaeal cofactor-independent PGM encoded by ORF AF1751 from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus was characterized after expression in E. coli. The monomeric 46 kDa protein showed cofactor-independent PGM activity and was stimulated by Mn²⁺ and exhibited high thermostability up to 70°C. A comprehensive phylogenetic analysis of both types of archaeal phosphoglycerate mutases is also presented.     

Single strand conformation polymorphism monitoring of 16S rDNA Archaea during start-up of an anaerobic digester

A laboratory-scale continuously mixed anaerobic digester was inoculated with a mix of anaerobic sludge and fed with glucose. The start-up strategy was progressive and chemical analyses were done to evaluate digester performance from day 1 to day 107. In parallel, Archaeal community dynamics were monitored by SSCP analysis of the V3 region of 16S rDNA genes and further characterized by partial sequencing of 16S rDNA genes. At day 1 the inoculum contained at least five distinct Archaeal peaks close to known methanogenic species. The dominant peak was very close to Methanosaeta concilli, the remaining species being members of the Methanobacteriales and Methanomicrobiales. A rapid shift of the Archaeal population was observed during the experiment. At day 21 Methanobacterium formicicum, which was not detected at day 1, became the dominant methanogenic species in the bioreactor and remained so until the end of the experiment.     

Thymidine Kinases in Archaea

Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea, while none was found in Nanoarchaeum. The identified TK1s have high identity to Gram-positive bacteria TK1s. The TK1s from archaea, Gram-positive bacteria and eukaryotes share the same common ancestor, while the TK1s from Gram-negative bacteria belong to a less-related subgroup. It seems that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell.