Inhibitory effects of C2 to C10 1-alkynes on ammonia oxidation in two Nitrososphaera species.

A previous study showed that ammonia oxidation by the Thaumarchaeota Nitrosopumilus maritimus (group 1.1a) was resistant to concentrations of the C8 1-alkyne, octyne, which completely inhibits activity by ammonia-oxidizing bacteria. In this study, the inhibitory effects of octyne and other C2 to C10 1-alkynes were evaluated on the nitrite production activity of two pure culture isolates from Thaumarchaeota group 1.1b, Nitrososphaera viennensis strain EN76 and Nitrososphaera gargensis. Both N. viennensis and N. gargensis were insensitive to concentrations of octyne that cause complete and irreversible inactivation of nitrite production by ammonia-oxidizing bacteria. However, octyne concentrations (≥20 µM) that did not inhibit N. maritimus partially inhibited nitrite production in N. viennensis and N. gargensis in a manner that did not show the characteristics of irreversible inactivation. In contrast to previous studies with an ammonia-oxidizing bacterium, Nitrosomonas europaea, octyne inhibition of N. viennensis was: (i) fully and immediately reversible, (ii) not competitive with NH4 (+), and (iii) without effect on the competitive interaction between NH4 (+) and acetylene. Both N. viennensis and N. gargensis demonstrated the same overall trend in regard to 1-alkyne inhibition as previously observed for N. maritimus, being highly sensitive to ≤C5 alkynes and more resistant to longer-chain length alkynes. Reproducible differences were observed among N. maritimus, N. viennensis, and N. gargensis in regard to the extent of their resistance/sensitivity to C6 and C7 1-alkynes, which may indicate differences in the ammonia monooxygenase binding and catalytic site(s) among the Thaumarchaeota.

Archaea predominate among ammonia-oxidizing prokaryotes in soils.

Ammonia oxidation is the first step in nitrification, a key process in the global nitrogen cycle that results in the formation of nitrate through microbial activity. The increase in nitrate availability in soils is important for plant nutrition, but it also has considerable impact on groundwater pollution owing to leaching. Here we show that archaeal ammonia oxidizers are more abundant in soils than their well-known bacterial counterparts. We investigated the abundance of the gene encoding a subunit of the key enzyme ammonia monooxygenase (amoA) in 12 pristine and agricultural soils of three climatic zones. amoA gene copies of Crenarchaeota (Archaea) were up to 3,000-fold more abundant than bacterial amoA genes. High amounts of crenarchaeota-specific lipids, including crenarchaeol, correlated with the abundance of archaeal amoA gene copies. Furthermore, reverse transcription quantitative PCR studies and complementary DNA analysis using novel cloning-independent pyrosequencing technology demonstrated the activity of the archaea in situ and supported the numerical dominance of archaeal over bacterial ammonia oxidizers. Our results indicate that crenarchaeota may be the most abundant ammonia-oxidizing organisms in soil ecosystems on Earth.

Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus.

Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.

Genome sequence of Picrophilus torridus and its implications for life around pH 0.

The euryarchaea Picrophilus torridus and Picrophilus oshimae are able to grow around pH 0 at up to 65 degrees C, thus they represent the most thermoacidophilic organisms known. Several features that may contribute to the thermoacidophilic survival strategy of P. torridus were deduced from analysis of its 1.55-megabase genome. P. torridus has the smallest genome among nonparasitic aerobic microorganisms growing on organic substrates and simultaneously the highest coding density among thermoacidophiles. An exceptionally high ratio of secondary over ATP-consuming primary transport systems demonstrates that the high proton concentration in the surrounding medium is extensively used for transport processes. Certain genes that may be particularly supportive for the extreme lifestyle of P. torridus appear to have been internalized into the genome of the Picrophilus lineage by horizontal gene transfer from crenarchaea and bacteria. Finally, it is noteworthy that the thermoacidophiles from phylogenetically distant branches of the Archaea apparently share an unexpectedly large pool of genes.

High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements.

We have isolated uracil-auxotrophic mutants of the hyperthermophilic archaeon Sulfolobus solfataricus in order to explore the genomic stability and mutational frequencies of this organism and to identify complementable recipients for a selectable genetic transformation system. Positive selection of spontaneous mutants resistant to 5-fluoroorotate yielded uracil auxotrophs with frequencies of between 10(-4) and 10(-5) per sensitive, viable cell. Four different, nonhomologous insertion sequences (ISs) were identified at different positions within the chromosomal pyrEF locus of these mutants. They ranged in size from 1,058 to 1,439 bp and possessed properties typical of known transposable elements, i.e., terminal inverted repeats, flanking duplicated target sequences, and putative transposase genes encoding motifs that are indicative of the IS4-IS5 IS element families. Between 12 and 25 copies of each IS element were found in chromosomal DNAs by Southern analyses. While characteristic fingerprint patterns created by IS element-specific probes were observed with genomic DNA of different S. solfataricus strains, no homologous sequences were identified in DNA of other well-characterized strains of the order Sulfolobales.

Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors.

Directed open reading frame (ORF) disruption and a serial selection technique in Escherichia coli and the extremely thermophilic archaeon Sulfolobus solfataricus allowed the identification of otherwise cryptic crucial and noncrucial viral open reading frames in the genome of the archaeal virus SSV1. It showed that the 15. 5-kbp viral genome can incorporate a 2.96-kbp insertion without loss of viral function and package this DNA properly into infectious virus particles. The selection technique, based on the preferential binding of ethidium bromide to relaxed DNA and the resulting inhibition of endonuclease cleavage to generate a pool of mostly singly cut molecules, should be generally applicable. A fully functional viral shuttle vector for S. solfataricus and E. coli was made. This vector spreads efficiently through infected cultures of S. solfataricus, its replication is induced by UV irradiation, it forms infectious virus particles, and it is stable at high copy number in both S. solfataricus and E. coli. The classification of otherwise unidentifiable ORFs in SSV1 facilitates genetic analysis of this virus, and the shuttle vector should be useful for the development of genetic systems for Crenarchaeota.

Diversity of radA genes from cultured and uncultured archaea: comparative analysis of putative RadA proteins and their use as a phylogenetic marker.

Archaea-specific radA primers were used with PCR to amplify fragments of radA genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15 radA genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species of Crenarcheota. The two most deeply branching archaeal radA genes found had some amino acid deletion and insertion patterns characteristic of bacterial recA genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.

Genomic analysis reveals chromosomal variation in natural populations of the uncultured psychrophilic archaeon Cenarchaeum symbiosum.

Molecular phylogenetic surveys have recently revealed an ecologically widespread crenarchaeal group that inhabits cold and temperate terrestrial and marine environments. To date these organisms have resisted isolation in pure culture, and so their phenotypic and genotypic characteristics remain largely unknown. To characterize these archaea, and to extend methodological approaches for characterizing uncultivated microorganisms, we initiated genomic analyses of the nonthermophilic crenarchaeote Cenarchaeum symbiosum found living in association with a marine sponge, Axinella mexicana. Complex DNA libraries derived from the host-symbiont population yielded several large clones containing the ribosomal operon from C. symbiosum. Unexpectedly, cloning and sequence analysis revealed the presence of two closely related variants that were consistently found in the majority of host individuals analyzed. Homologous regions from the two variants were sequenced and compared in detail. The variants exhibit >99.2% sequence identity in both small- and large-subunit rRNA genes and they contain homologous protein-encoding genes in identical order and orientation over a 28-kbp overlapping region. Our study not only indicates the potential for characterizing uncultivated prokaryotes by genome sequencing but also identifies the primary complication inherent in the approach: the widespread genomic microheterogeneity in naturally occurring prokaryotic populations.

Dibiphytanyl ether lipids in nonthermophilic crenarchaeotes.

The kingdom Crenarchaeota is now known to include archaea which inhabit a wide variety of low-temperature environments. We report here lipid analyses of nonthermophilic crenarchaeotes, which revealed the presence of cyclic and acyclic dibiphytanylglycerol tetraether lipids. Nonthermophilic crenarchaeotes appear to be a major biological source of tetraether lipids in marine planktonic environments.

Dibiphytanyl ether lipids in nonthermophilic crenarchaeotes.

[This corrects the article on p. 1136 in vol. 64.].

Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum.

Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments. Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution. Here we report on the first protein to be characterized from one of these organisms. The DNA polymerase gene of C. symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig. Its deduced amino acid sequence is highly similar to those of the archaeal family B (alpha-type) DNA polymerases. It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively). The conserved motifs of B (alpha-)-type DNA polymerases and 3'-5' exonuclease were identified in the 845-amino-acid sequence. The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags. It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate. Single-strand- and double-strand-dependent 3'-5' exonuclease activity was detected, as was a marginal 5'-3' exonuclease activity. The enzyme was rapidly inactivated at temperatures higher than 40 degrees C, with a half-life of 10 min at 46 degrees C. It was found to be less thermostable than polymerase I of E. coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes. Although phylogenetic studies suggest a thermophilic ancestry for C. symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution.

Recovery of crenarchaeotal ribosomal DNA sequences from freshwater-lake sediments.

We report several novel environmental sequences of archaea from the kingdom Crenarchaeota, recovered from anaerobic freshwater-lake sediments in Michigan. A nested PCR approach with Archaea- and Crenar-chaeota-specific primers was used to amplify partial Small-subunit ribosomal DNAs. Phylogenetic analysis of seven sequences shows that these DNAs represent a monophyletic lineage diverging prior to all recently identified crenarchaeotal phylotypes isolated from temperate environments. Including our lineage, all uncultured crenarchaeotal sequences recovered from moderate or cold environments form a distinct, monophyletic group separate from the "genuine" thermophilic crenarchaeota. Our finding extends the emerging picture that crenarchaeota, thought until recently to be solely extreme thermophiles, have radiated into an unexpectedly large variety of ecologically important, temperate environments.

Viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic Archaea.

We review and update the work on genetic elements, e.g., viruses and plasmids (exluding IS elements and transposons) in the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in the kingdom Euryarchaeota of the archael domain, including unpublished data from our laboratory. The viruses of Crenarchaeota represent four novel virus families. The Fuselloviridae represented by SSVI of S. shibatae and relatives in other Sulfolobus strains have the form of a tailed spindle. The envelope is highly hydrophobic. The DNA is double-stranded and circular. Members of this group have also been found in Methanococcus and Haloarcula. The Lipothrivciridae (e.g., T TV1 to 3) have the form of flexible filaments. They have a core containing linear double-stranded DNA and DNA-binding proteins which is wrapped into a lipid membrane. The "Bacilloviridae" (e.g., TTV4 and SIRV) are stiff rods lacking this membrane, but also featuring linear double-stranded DNA and DNA-binding proteins. Both virus types carry on both ends structures involved in the attachment to receptors. Both types are represented in Thermoproteus and Sulfolobus. The droplet-formed novel Sulfolobus virus SNDV represents the "Guttaviridae" containing circular double-stranded DNA. Though head and tail viruses distantly resembling T phages or lambdoid phages were seen electronmicroscopically in solfataric water samples, no such virus has so far been isolated. SSV1 is temperate, TTV1 causes lysis after induction, the other viruses found so far exist in carrier states. The hosts of all but TTV1 survive virus production. We discuss the implications of the nature of these viruses for understanding virus evolution. The plasmids found so far range in size from 4.5 kb to about 40 kb. Most of them occur in high copy number, probably due to the way of their detection. Most are cryptic, pNOB8 is conjugative, the widespread pDL10 alleviates in an unknown way autotrophic growth of its host Desulfurolobus by sulfur reduction. The plasmid pTIK4 appears to encode a killer function. pNOB8 has been used as a vector for the transfer of the lac S (beta-galactosidase) gene into a mutant of S. solfataricus.

Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector.

We describe a transformation system for extremely thermophilic archaea of the genus Sulfolobus in the kingdom Crenarchaeota. We have constructed in vitro a recombinant derivative of the recently described conjugative plasmid pNOB8, containing a beta-galactosidase gene downstream of a strong promotor. Transformation of a beta-galactosidase negative mutant of Sulfolobus solfataricus with this construct resulted in its spreading through the culture containing the primary transformants and in efficient restoration of beta-galactosidase activity.

Complete nucleotide sequence of the Sulfolobus islandicus multicopy plasmid pRN1.

The complete sequence of the 5350-bp plasmid pRN1 from the crenarchaeote Sulfolobus islandicus has been determined. This plasmid is the first to be sequenced from this group of thermoacidophilic archaebacteria (Archaea) and its high copy number and wide host range make it a good candidate for a cloning vector. pRN1 contains several open reading frames, including one that spans over half the plasmid and has significant similarity to the helicase domain of viral primase proteins. Directly upstream of this putative primase is a homologue of Cop, a family of small proteins from promiscuous eubacterial plasmids which control copy number by repressing the expression of the replication initiation protein. In eubacterial plasmids cop is found upstream of the replication initiator protein. The location of a cop homologue upstream of a primase-like gene in pRN1 suggests that it controls DNA replication in a manner similar to these eubacterial plasmids, but does so using a mixture of components from plasmids and viruses.

Picrophilus gen. nov., fam. nov.: a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around pH 0.

Two species belonging to a novel genus of archaea, designated Picrophilus oshimae and Picrophilus torridus, have been isolated from two different solfataric locations in northern Japan. One habitat harboring both organisms was a dry, extremely acidic soil (pH < 0.5) that was heated by solfataric gases to about 55 degrees C. In the laboratory both species grew heterotrophically on yeast extract and poorly on tryptone under aerobic conditions at temperatures between 45 and 65 degrees C; they grew optimally at 60 degrees C. The pH optimum was 0.7, but growth occurred even around pH 0. Under optimal conditions, the generation time was about 6 h, yielding densities of up to 10(10) cells per ml. The cells were surrounded by a highly filigreed regular tetragonal S-layer, and the core lipids of the membrane were mainly bis-phytanyltetraethers. The 16S rRNA sequences of the two species were about 3% different. The complete 16S rRNA sequence of P. oshimae was 9.3% different from that of the closest relative, Thermoplasma acidophilum. The morphology and physiological properties of the two species characterize Picrophilus as a novel genus that is a member of a novel family within the order Thermoplasmales.

A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating.

A plasmid of 45 kb, designated pNOB8, was found in high copy number in a new heterotrophic Sulfolobus isolate, NOB8H2, from Japan. Dissemination of the plasmid occurred in six cultures of nine different Sulfolobus strains when small amounts of the donor were added. These mixed cultures exhibited a high average copy number of the plasmid, between 20 and 40 per chromosome, and showed a marked growth retardation. Horizontal transfer of pNOB8 was proved by isolating transcipients from mating mixtures via single colonies. In these isolates, the copy number of the plasmid appeared to be subject to a control mechanism. Cell-free filtrates of donor cultures did not transmit the plasmid, and plating of the donor on lawns of recipients did not result in plaque formation, suggesting that the transfer was not mediated by a virus. Rapid formation of cell-to-cell contacts between differently stained donor and recipient partners was demonstrated after the two strains were mixed. Electron microscopic analysis of mating mixtures revealed many cell aggregates made up of 2 to 30 cells and intercellular cytoplasmic bridges connecting two or more cells. Cells that had been transformed with purified plasmid DNA as well as transcipients isolated from mating mixtures were shown to serve as donors for further transmission of pNOB8. The plasmid undergoes extensive genetic variations, since deletions and insertions were frequently observed in plasmid preparations from the donor strain and from mating mixtures.