The split genes of Nanoarchaeum equitans are an ancestral character

The introns early hypothesis predicts that introns were fundamental in assembling the first genes. In Nanoarchaeum equitans some genes are split into two. If these split genes were the ancestral forms, as suggested by the introns early hypothesis, then the end-beginning of the two parts of the split protein in a multiple alignment with the orthologous proteins from the Eukarya and Arachaea domains should make a clear prediction on where the intron in the homologous eukaryotic gene should be positioned. The analysis has shown that the introns are in this position, which is therefore predictable on the basis of the split proteins of N. equitans. This corroborates the hypothesis that the split genes of N. equitans are the plesiomorphic forms of these genes. If true, this would show that the origin of genes was polyphyletic as the monophyletic origin hypothesis would deny the existence, in a real organism, of these ancestral (split) genes, which imply that they were assembled late on and after the domains of life were established. Furthermore, it would seem that hyperthermophily is also an ancestral trait because it is linked to a split gene in N. equitans.     

Nanoarchaeum equitans and Ignicoccus hospitalis: new insights into a unique, intimate association of two archaea

Nanoarchaeum equitans and Ignicoccus hospitalis represent a unique, intimate association of two archaea. Both form a stable coculture which is mandatory for N. equitans but not for the host I. hospitalis. Here, we investigated interactions and mutual influence between these microorganisms. Fermentation studies revealed that during exponential growth only about 25% of I. hospitalis cells are occupied by N. equitans cells (one to three cells). The latter strongly proliferate in the stationary phase of I. hospitalis, until 80 to 90% of the I. hospitalis cells carry around 10 N. equitans cells. Furthermore, the expulsion of H2S, the major metabolic end product of I. hospitalis, by strong gas stripping yields huge amounts of free N. equitans cells. N. equitans had no influence on the doubling times, final cell concentrations, and growth temperature, pH, or salt concentration ranges or optima of I. hospitalis. However, isolation studies using optical tweezers revealed that infection with N. equitans inhibited the proliferation of individual I. hospitalis cells. This inhibition might be caused by deprivation of the host of cell components like amino acids, as demonstrated by 13C-labeling studies. The strong dependence of N. equitans on I. hospitalis was affirmed by live-dead staining and electron microscopic analyses, which indicated a tight physiological and structural connection between the two microorganisms. No alternative hosts, including other Ignicoccus species, were accepted by N. equitans. In summary, the data show a highly specialized association of N. equitans and I. hospitalis which so far cannot be assigned to a classical symbiosis, commensalism, or parasitism.     

<Emphasis Type="Italic">Ignicoccus hospitalis</Emphasis> and <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis>: ultrastructure,cell–cell interaction,and 3D reconstruction from serial sections of freeze-substituted cells and by electron cryotomography

Ultrastructure and intercellular interaction of Ignicoccus hospitalis and Nanoarchaeum equitans were investigated using two different electron microscopy approaches, by three-dimensional reconstructions from serial sections, and by electron cryotomography. Serial sections were assembled into 3D reconstructions, for visualizing the unusual complexity of I. hospitalis, its huge periplasmic space, the vesiculating cytoplasmic membrane, and the outer membrane. The cytoplasm contains fibres which are reminiscent to a cytoskeleton. Cell division in I. hospitalis is complex, and different to that in Euryarchaeota or Bacteria. An irregular invagination of the cytoplasmic membrane is followed by separation of the two cytoplasms. Simultaneous constriction of cytoplasmic plus outer membrane is not observed. Cells of N. equitans show a classical mode of cell division, by constriction in the mid-plane. Their cytoplasm exhibits two types of fibres, elongated and ring-shaped. Electron micrographs of contact sites between I. hospitalis and N. equitans exhibit two modes of interaction. One is indirect and mediated by thin fibres; in other cells the two cell surfaces are in direct contact. The two membranes of I. hospitalis cells are frequently seen in direct contact, possibly a prerequisite for transporting metabolites or substrates from the cytoplasm of one cell to the other. Rarely, a transport based on cargo vesicles is observed between I. hospitalis and N. equitans.     

The complete set of tRNA species in Nanoarchaeum equitans

The archaeal parasite Nanoarchaeum equitans was found to generate five tRNA species via a unique process requiring the assembly of seperate 5' and 3' tRNA halves [Randau, L., Munch, R., Hohn, M.J., Jahn, D. and Soll, D. (2005) Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5'- and 3'-halves. Nature 433, 537-541]. Biochemical evidence was missing for one of the computationally-predicted, joined tRNAs designated as tRNA(Trp). Our RT-PCR and sequencing results identify this tRNA as tRNA(Lys) (CUU) joined at the alternative position between bases 30 and 31. We show that the intron-containing tRNA(Trp) was misidentified in the initial Nanoarchaeum equitans genome annotation [E. Waters et al. (2003) The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism. Proc. Natl. Acad. Sci. USA 100, 12984-12988]. Along with a previously unidentified joined tRNA(Gln) (UUG), Nanoarchaeum equitans exhibits 44 tRNAs and is enabled to read all 61 sense codons. Features unique to this set of tRNA molecules are discussed.     

The tree of life might be rooted in the branch leading to Nanoarchaeota

It is suggested that the tree of life might be rooted in the domain of the Archaea, in the branch leading to the phylum of Nanoarchaeota. This hypothesis seems to be corroborated by the uniqueness and ancestrality of some traits possessed by Nanoarchaeum equitans, such as split genes separately codifying for the 5' and 3' halves of the tRNA molecule. These half genes are the oldest ancestral form of gene we have ever seen. This, along with the absence of operons from the genome of N. equitans, would seem to indicate that this genome is a molecular fossil of the evolutionary stage which the ancestral genomes had reached when the first lines of divergence were established. Moreover, the late appearance of DNA coinciding with the rooting of the universal phylogenetic tree would make the genome of N. equitans a witness to this fundamental event.     

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.     

新世纪微生物学者的一项重要任务——未培养微生物的分离培养

扼要介绍近年来有关分离培养未培养的微生物 (Unculturablemicroorganisms)、扩大生态环境微生物多样性的认识等方面取得的重要进展。首先 ,是采用非常规的 ,甚至认为有毒性的电子传递物质 ,获得了多种新生理型(physiotypes)的纯种微生物。另外 ,在多种生态域中分离出非同寻常的微生物 ,扩大了这些生境微生物的多样性的视野 ,如普遍存在于大洋的浮游细菌SAR11类群 ,其微小菌体呈半月形 ,在海水中细菌浓度可达 (10⁵～10⁶) mL ,基因组估计为 1.54Mb。更值得注意的是从极端高温环境分离的嗜热纳米古细菌属 (Nanoarchaeota) ,其基因组仅有500kb ,是已知原核生物的最小者。应重视的是在分离这些多样性微生物的过程中所发展的新型培养技术 ,如微滴胶囊化法和扩散小室法都是具有革命性的方法 ,既有通用性 ,又无需昂贵设备 ,对今后这一领域的发展将起重要推动作用     

RNA processing in the minimal organism Nanoarchaeum equitans

ABSTRACT: BACKGROUND: The minimal genome of the tiny, hyperthermophilic archaeon Nanoarchaeum equitans contains several fragmented genes and revealed unusual RNA processing pathways. These include the maturation of tRNA molecules via the trans-splicing of tRNA halves and genomic rearrangements to compensate for the absence of RNase P. RESULTS: Here, the RNA processing events in the N. equitans cell are analyzed using RNA-Seq deep sequencing methodology. All tRNA half precursor and tRNA termini were determined and support the tRNA trans-splicing model. The processing of CRISPR RNAs from two CRISPR clusters was verified. Twenty-seven C/D box small RNAs (sRNAs) and a H/ACA box sRNA were identified. The C/D box sRNAs were found to flank split genes, to form dicistronic tRNA-sRNA precursors and to be encoded within the tRNAMet intron. CONCLUSIONS: The presented data provide an overview of the production and usage of small RNAs in a cell that has to survive with a highly reduced genome. N. equitans lost many essential metabolic pathways but maintains highly active CRISPR/Cas and rRNA modification systems that appear to play an important role in genome fragmentation.     

Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion.     

Gene transfers from nanoarchaeota to an ancestor of diplomonads and parabasalids

Rare evolutionary events, such as lateral gene transfers and gene fusions, may be useful to pinpoint, and correlate the timing of, key branches across the tree of life. For example, the shared possession of a transferred gene indicates a phylogenetic relationship among organismal lineages by virtue of their shared common ancestral recipient. Here, we present phylogenetic analyses of prolyl-tRNA and alanyl-tRNA synthetase genes that indicate lateral gene transfer events to an ancestor of the diplomonads and parabasalids from lineages more closely related to the newly discovered archaeal hyperthermophile Nanoarchaeum equitans (Nanoarchaeota) than to Crenarchaeota or Euryarchaeota. The support for this scenario is strong from all applied phylogenetic methods for the alanyl-tRNA sequences, whereas the phylogenetic analyses of the prolyl-tRNA sequences show some disagreements between methods, indicating that the donor lineage cannot be identified with a high degree of certainty. However, in both trees, the diplomonads and parabasalids branch together within the Archaea, strongly suggesting that these two groups of unicellular eukaryotes, often regarded as the two earliest independent offshoots of the eukaryotic lineage, share a common ancestor to the exclusion of the eukaryotic root. Unfortunately, the phylogenetic analyses of these two aminoacyl-tRNA synthetase genes are inconclusive regarding the position of the diplomonad/parabasalid group within the eukaryotes. Our results also show that the lineage leading to Nanoarchaeota branched off from Euryarchaeota and Crenarchaeota before the divergence of diplomonads and parabasalids, that this unexplored archaeal diversity, currently only represented by the hyperthermophilic organism Nanoarchaeum equitans, may include members living in close proximity to mesophilic eukaryotes, and that the presence of split genes in the Nanoarchaeum genome is a derived feature.     

Evolutionary and functional genomics of the Archaea

In the past two years, archaeal genomics has achieved several breakthroughs. On the evolutionary front the most exciting development was the sequencing and analysis of the genome of Nanoarchaeum equitans, a tiny parasitic organism that has only approximately 540 genes. The genome of Nanoarchaeum shows signs of extreme rearrangement including the virtual absence of conserved operons and the presence of several split genes. Nanoarchaeum is distantly related to other archaea, and it has been proposed to represent a deep archaeal branch that is distinct from Euryarchaeota and Crenarchaeota. This would imply that many features of its gene repertoire and genome organization might be ancestral. However, additional genome analysis has provided a more conservative suggestion - that Nanoarchaeum is a highly derived euryarchaeon. Also there have been substantial developments in functional genomics, including the discovery of the elusive aminoacyl-tRNA synthetase that is involved in both the biosynthesis of cysteine and its incorporation into proteins in methanogens, and the first experimental validation of the predicted archaeal exosome.     

A Comparison Among the Models Proposed to Explain the Origin of the tRNA Molecule: A Synthesis

A comparison is made among all the models proposed to explain the origin of the tRNA molecule. The conclusion reached is that, for the model predicting that the tRNA molecule originated after the assembly of two hairpin-like structures, molecular fossils have been found in the half-genes of the tRNAs of Nanoarchaeum equitans. These might be the witnesses of the transition stage predicted by the model through which the evolution of the tRNA molecule passed, thus providing considerable corroboration for this model.     

Detection of 16S rDNA sequences representing the novel phylum "Nanoarchaeota": indication for a wide distribution in high temperature biotopes

We screened samples from high temperature biotopes for 16S rRNA genes of the novel archaeal phylum "Nanoarchaeota". Positive PCR amplifications were obtained from Yellowstone National Park, Uzon Caldera, and an abyssal vent system. These sequences form a cluster with the sequence of "Nanoarchaeum equitans", indicating a wide distribution of this phylum.     

Separate and combined biochemical activities of the subunits of a naturally split reverse gyrase

Reverse gyrase reanneals denatured DNA and induces positive supercoils in DNA, an activity that is critical for life at very high temperatures. Positive supercoiling occurs by a poorly understood mechanism involving the coordination of a topoisomerase domain and a helicase-like domain. In the parasitic archaeon Nanoarchaeum equitans, these domains occur as separate subunits. We express the subunits, and characterize them both in isolation and as a heterodimer. Each subunit tightly associates and interacts with the other. The topoisomerase subunit enhances the catalytic specificity of the DNA-dependent ATPase activity of the helicase-like subunit, and the helicase-like subunit inhibits the relaxation activity of the topoisomerase subunit while promoting positive supercoiling. DNA binding preference for both single- and double-stranded DNA is partitioned between the subunits. Based on a sensitive topological shift assay, the binding preference of helicase-like subunit for underwound DNA is modulated by its binding with ATP cofactor. These results provide new insight into the mechanism of positive supercoil induction by reverse gyrase.     

Archaeal symbionts and parasites

Several species of Archaea are involved in symbiotic or parasitic associations with representatives of Eukarya, Bacteria and other Archaea. Eukaryal interactions include different members of methanogens, found in the gut of arthropods, in the rumen of cattle, and in the human intestine, while Cenarchaeum symbiosum is a partner of a marine sponge. Examples for bacterial-archaeal associations are the anaerobic methane oxidation consortia and the SM1 Euryarchaeon with its highly unusual 'hami' as extracellular appendages. The so far only known and cultivated association between two Archaea is composed of Nanoarchaeum equitans and its obligate host Ignicoccus hospitalis. All these consortia can often not be assigned to the 'classical' concepts of mutalism, commensialism or parasitism and represent highly specialized interspecies associations.     

Signature genes as a phylogenomic tool

Gene content has been shown to contain a strong phylogenetic signal, yet its usage for phylogenetic questions is hampered by horizontal gene transfer and parallel gene loss and until now required completely sequenced genomes. Here, we introduce an approach that allows the phylogenetic signal in gene content to be applied to any set of sequences, using signature genes for phylogenetic classification. The hundreds of publicly available genomes allow us to identify signature genes at various taxonomic depths, and we show how the presence of signature genes in an unspecified sample can be used to characterize its taxonomic composition. We identify 8,362 signature genes specific for 112 prokaryotic taxa. We show that these signature genes can be used to address phylogenetic questions on the basis of gene content in cases where classic gene content or sequence analyses provide an ambiguous answer, such as for Nanoarchaeum equitans, and even in cases where complete genomes are not available, such as for metagenomics data. Cross-validation experiments leaving out up to 30% of the species show that approximately 92% of the signature genes correctly place the species in a related clade. Analyses of metagenomics data sets with the signature gene approach are in good agreement with the previously reported species distributions based on phylogenetic analysis of marker genes. Summarizing, signature genes can complement traditional sequence-based methods in addressing taxonomic questions.     

Beneficial interactions between insects and gut bacteria

Insects are amongst the most successful of animals, both in terms of diversity and in colonizing all ecological niches. Recent studies have highlighted the benefi ciary roles that bacteria play in the success and establishment of insects. By adopting techniques like 16S rRNA sequencing we are now in a position to understand the diversity of bacteria present in insect guts. It has been shown that some of these bacteria, like Wolbachia and Cardinium are involved in manipulating insect populations and distorting their sex ratio. Attempts have been made to culture these bacteria in insect cell lines, as they are recalcitrant to culture under normal microbiological conditions. The diversity of bacteria associated with insects and the functional role played by them in the insect is discussed below.     

Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.     

Nitrogen fixation and hydrogen metabolism in photosynthetic bacteria.

The photosynthetic bacteria are found in a wide range of specialized aquatic environments. These bacteria represent important members of the microbial community since they are capable of carrying out two of the most important processes on earth, namely, photosynthesis and nitrogen fixation, at the expense of solar energy. Since the discovery that these bacteria could fix atmospheric nitrogen, there has been an intensification of studies relating to both the biochemistry and physiology of this process. The practical importance of this field is emphasized by a consideration of the tremendous energy input required for the production of artificial nitrogenous fertilizer. The present communication aims to briefly review the current state of knowledge relating to certain aspects of nitrogen fixation by the photosynthetic bacteria. The topics that will be discussed include a general survey of the nitrogenase system in the various photosynthetic bacteria, the regulation of both nitrogenase biosynthesis and activity, recent advances in the genetics of the nitrogen fixing system, and the hydrogen cycle in these bacteria. In addition, a brief discussion of some of some of the possible practical applications provided by the photosynthetic bacteria will be presented.