The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota

Sulfolobus acidocaldarius is an aerobic thermoacidophilic crenarchaeon which grows optimally at 80 degrees C and pH 2 in terrestrial solfataric springs. Here, we describe the genome sequence of strain DSM639, which has been used for many seminal studies on archaeal and crenarchaeal biology. The circular genome carries 2,225,959 bp (37% G+C) with 2,292 predicted protein-encoding genes. Many of the smaller genes were identified for the first time on the basis of comparison of three Sulfolobus genome sequences. Of the protein-coding genes, 305 are exclusive to S. acidocaldarius and 866 are specific to the Sulfolobus genus. Moreover, 82 genes for untranslated RNAs were identified and annotated. Owing to the probable absence of active autonomous and nonautonomous mobile elements, the genome stability and organization of S. acidocaldarius differ radically from those of Sulfolobus solfataricus and Sulfolobus tokodaii. The S. acidocaldarius genome contains an integrated, and probably encaptured, pARN-type conjugative plasmid which may facilitate intercellular chromosomal gene exchange in S. acidocaldarius. Moreover, it contains genes for a characteristic restriction modification system, a UV damage excision repair system, thermopsin, and an aromatic ring dioxygenase, all of which are absent from genomes of other Sulfolobus species. However, it lacks genes for some of their sugar transporters, consistent with it growing on a more limited range of carbon sources. These results, together with the many newly identified protein-coding genes for Sulfolobus, are incorporated into a public Sulfolobus database which can be accessed at http://dac.molbio.ku.dk/dbs/Sulfolobus.     

Characterization of the RNase P RNA of Sulfolobus acidocaldarius.

RNase P is the ribonucleoprotein enzyme that cleaves precursor sequences from the 5' ends of pre-tRNAs. In Bacteria, the RNA subunit is the catalytic moiety. Eucaryal and archaeal RNase P activities copurify with RNAs, which have not been shown to be catalytic. We report here the analysis of the RNase P RNA from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The holoenzyme was highly purified, and extracted RNA was used to identify the RNase P RNA gene. The nucleotide sequence of the gene was determined, and a secondary structure is proposed. The RNA was not observed to be catalytic by itself, but it nevertheless is similar in sequence and structure to bacterial RNase P RNA. The marked similarity of the RNase P RNA from S. acidocaldarius and that from Haloferax volcanii, the other known archael RNase P RNA, supports the coherence of Archaea as a phylogenetic domain.     

An unusual 5S rRNA, from Sulfolobus acidocaldarius, and its implications for a general 5S rRNA structure.

The nucleotide sequence of the 5S ribosomal RNA of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was determined. The high degree of evident secondary structure in the molecule has implications for the common higher order structure of other 5S rRNAs, both bacterial and eukaryotic.     

Comparative analysis of ribonuclease P RNA structure in Archaea.

Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.     

Sequences of the 5S rRNAs of Azotobacter vinelandii, Pseudomonas aeruginosa and Pseudomonas fluorescens with some notes on 5S RNA secondary structure

Recently published alignments of available 5 S rRNA sequences have shown that a rigid base pairing pattern, pointing to the existence of a universal five-helix secondary structure for all 5 S RNAs, can be superimposed on such alignments. For a few species, the alignment and the base pairing pattern show distortions with respect to the large majority of sequences. Their 5 S RNAs may form exceptional secondary structures, or there may just be errors in the published sequences. We have examined such a case, Pseudomonas fluorescens, and found the sequence to be in error. The corrected sequence, as well as those of the related species Azotobacter vinelandii and Pseudomonas aeruginosa, fit perfectly in the 5 S RNA sequence alignment and in the five-helix secondary structure model. There exists comparative evidence for the frequent presence of non-standard base pairs at several points of the 5 S RNA secondary structure.     

Probing the structure and function of an archaeal C/D-box methylation guide sRNA

The genome of the hyperthermophilic archaeon Sulfolobus solfataricus contains dozens of small C/D-box sRNAs that use a complementary guide sequence to target 2'-O-ribose methylation to specific locations within ribosomal and transfer RNAs. The sRNAs are approximately 50-60 nucleotides in length and contain two RNA structural kink-turn (K-turn) motifs that are required for assembly with ribosomal protein L7Ae, Nop5, and fibrillarin to form an active ribonucleoprotein (RNP) particle. The complex catalyzes guide-directed methylation to target RNAs. Earlier work in our laboratory has characterized the assembly pathway and methylation reaction using the model sR1 sRNA from Sulfolobus acidocaldarius. This sRNA contains only one antisense region situated adjacent to the D-box, and methylation is directed to position U52 in 16S rRNA. Here we have investigated through RNA mutagenesis, the relationship between the sR1 structure and methylation-guide function. We show that although full activity of the guide requires intact C/D and C'/D' K-turn motifs, each structure plays a distinct role in the methylation reaction. The C/D motif is directly implicated in the methylation function, whereas the C'/D' element appears to play an indirect structural role by facilitating the correct folding of the RNA. Our results suggest that L7Ae facilitates the folding of the K-turn motifs (chaperone function) and, in addition, is required for methylation activity in the presence of Nop5 and Fib.     

Structural and functional exchangeability of 5 S RNA species from the eubacterium E.coli and the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures. It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins. Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures.     

The expanding world of small RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus

Archaeal L7Ae is a multifunctional protein that binds to a distinctive K-turn motif in RNA and is found as a component in the large subunit of the ribosome, and in ribose methylation and pseudouridylation guide RNP particles. A collection of L7Ae-associated small RNAs were isolated from Sulfolobus solfataricus cell extracts and used to construct a cDNA library; 45 distinct cDNA sequences were characterized and divided into six groups. Group 1 contained six RNAs that exhibited the features characteristic of the canonical C/D box archaeal sRNAs, two RNAs that were atypical C/D box sRNAs and one RNA representative of archaeal H/ACA sRNA family. Group 2 contained 13 sense strand RNA sequences that were encoded either within, or overlapping annotated open reading frames (ORFs). Group 3 contained three sequences form intergenic regions. Group 4 contained antisense sequences from within or overlapping sense strand ORFs or antisense sequences to C/D box sRNAs. More than two-thirds of these sequences possessed K-turn motifs. Group 5 contained two sequences corresponding to internal regions of 7S RNA. Group 6 consisted of 11 sequences that were fragments from the 5' or 3' ends of 16S and 23S ribosomal RNA and from seven different tRNAs. Our data suggest that S. solfataricus contains a plethora of small RNAs. Most of these are bound directly by the L7Ae protein; the others may well be part of larger, transiently stable RNP complexes that contain the L7Ae protein as core component.     

Novel trimeric adenylate kinase from an extremely thermoacidophilic archaeon,Sulfolobus solfataricus: molecular cloning,nucleotide sequencing,expression in Escherichia coli,and characterization of the recombinant enzyme

A gene coding for adenylate kinase was cloned from an extremely thermoacidophilic archaeon Sulfolobus solfataricus. The open reading frame of the sequenced gene consisted of 585 nucleotides coding for a polypeptide of 195 amino acid residues with a calculated molecular weight of 21,325. Although the S. solfataricus adenylate kinase, which belonged to the small variants of the adenylate kinase family, had low sequence identities with bacterial and eukaryotic enzymes, a functionally important glycine-rich region and also two invariant arginine residues were conserved in the sequence of the S. solfataricus enzyme. The recombinant enzyme, overexpressed in Escherichia coli and purified to homogeneity, had high affinity for AMP and high thermal stability, comparable to the extremely thermostable enzyme from a similar archaeon, S. acidocaldarius. Furthermore, gel filtration and sedimentation analyses showed that the S. solfataricus adenylate kinase was a homotrimer in solution, which is a novel subunit structure for nucleoside monophosphate kinases.     

The nucleotide sequences of the 5 S rRNAs of seven molds and a yeast and their use in studying ascomycete phylogeny.

The sequences of the 5 S rRNAs isolated from 8 ascomycete species belonging to the genera Aspergillus, Penicillium, Acremonium and Candida are reported. Two of the examined strains each yielded a mixture of 3 slightly different 5 S RNAs, which were individually sequenced after fractionation. A previously published sequence for Aspergillus nidulans 5 S RNA was found to contain errors. Reconstruction of an evolutionary tree based on 5 S RNA sequences showed that the 16 presently examined ascomycetes form three clusters. The same threefold partition can be observed in the secondary structure pattern, each cluster showing a slightly different variant of the general 5-helix model for 5 S rRNA (De Wachter, Chen and Vandenberghe (1982) Biochimie 64, 311-329), and different sets of secondary structure equilibrium forms in helices C and E of the aforementioned model.     

Multiple origins of replication in archaea

Until recently, the only archaeon for which a bona fide origin of replication was reported was Pyrococcus abyssi, where a single origin was identified. Although several in silico analyses have suggested that some archaeal species might contain more than one origin, this has only been demonstrated recently. Two studies have shown that multiple origins of replication function in two archaeal species. One study identified two origins of replication in the archaeon Sulfolobus solfataricus, whereas a second study used a different technique to show that both S. solfataricus and Sulfolobus acidocaldarius have three functional origins. These are the first reports of archaea having multiple origins. This finding has implications for research on the mechanisms of DNA replication and evolution.     

Nucleotide sequence, secondary structure and evolution of the 5S ribosomal RNA from five bacterial species

The nucleotide sequences of the 5S ribosomal RNAs of the bacteria Agrobacterium tumefaciens, Alcaligenes faecalis, Pseudomonas cepacia, Aquaspirillum serpens and Acinetobacter calcoaceticus have been determined. The sequences fit in a generally accepted model for 5S RNA secondary structure. However, a closer comparative examination of these and other bacterial 5S RNA primary structures reveals the potential of additional base pairing and of multiple equilibria between a set of slightly different alternative secondary structures in one area of the molecule. The phylogenetic position of the examined bacteria is derived from a 5S RNA sequence alignment by a clustering method and compared with the position derived on the basis of 16S ribosomal RNA oligonucleotide catalogs.     

Highly conserved 5S ribosomal RNA sequences in four rust fungi and atypical 5S rRNA secondary structure in Microstroma juglandis.

The 5S ribosomal RNA nucleotide sequences of five basidiomycetous fungi, Coleosporium tussilaginis , Gymnosporangium clavariaeforme , Puccinia poarum , Endophyllum sempervivi and Microstroma juglandis were determined. Despite high differentiation in their host spectra the four rust species are highly conserved with respect to their 5S rRna sequences, which fit with the basidiomycete cluster 5 described by Walker and Doolittle (1). The sequences obtained from the first three rust fungi were proven to be identical while the sequence from Endophyllum sempervivi showed two base substitutions compared with the other rust fungi. The Microstroma juglandis 5S rRNA sequence differs from all other basidiomycete 5S rRNA sequences published so far in respect to its secondary structure which shows an atypical 'CCA' loop in helix D, but it reveals typical basidiomycetous signature nucleotides. Therefore Microstroma juglandis represents a cluster of its own within the Basidiomycetes. A dendrogram was constructed based on Kimura's "Neutral Theory of Molecular Evolution".     

Isolation, characterization and microsequence analysis of a small basic methylated DNA-binding protein from the Archaebacterium, Sulfolobus solfataricus

DNA-binding proteins have been extracted from the thermoacidophilic archaebacterium Sulfolobus solfataricus strain P1, grown at 86 degrees C and pH 4.5. These proteins, which may have a histone-like function, were isolated and purified under standard, non-denaturing conditions, and can be grouped into three molecular mass classes of 7, 8 and 10 kDa. We have purified to homogenity the main 7 kDa protein and determined its DNA-binding affinity by filter binding assays and electron microscopy. The Stokes radius of gyration indicates that the protein occurs as a monomer. The complete amino-acid sequence of this protein contains 14 lysine residues out of 63 amino acids and the calculated Mr is 7149. Five of the lysine residues are partially monomethylated to varying extents and the methylated residues are located exclusively in the N-terminal (positions 4 and 6) and the C-terminal (positions 60, 62 and 63) regions only. The protein is strongly homologous to the 7 kDa proteins of Sulfolobus acidocaldarius with the highest homology to protein 7d. Accordingly, the name of this protein from S. solfataricus was assigned as DNA-binding protein Sso7d.     

Nucleotide sequence and structure of cytoplasmic 5S RNA and 5.8S RNA of Chlamydomonas reinhardii.

Three small RNAs of the cytoplasmic 8OS ribosomes of the green unicellular alga Chlamydomonas reinhardii have been sequenced. They include two species of ribosomal 5S RNA, a major and a minor one of 122 and 121 nucleotides respectively, which differ from each other by 17 bases, and also the ribosomal 5.8S RNA of 156 nucleotides. Novel structural features can be recognized in the 5S RNAs of C. reinhardii by a comparison with published 5S RNA sequences. In addition the secondary structure of these small RNA molecules has been examined using a newly developed method based on differential nuclease susceptibility.