A small basic ribosomal protein from the extreme thermophilic archaebacterium Sulfolobus solfataricus that has no equivalent in Escherichia coli.

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene has been confirmed by partial amino acid sequencing. The protein shows no sequence similarity to any of the ribosomal proteins from eubacteria (Escherichia coli) or to those that have been reported from eukaryotes.     

Unidentified Open Reading Frames in the Genome of Methanococcus jannaschii Are Similar in Sequence to an Archaebacterial Gene for tRNA Nucleotidyltransferase

The protein sequence of ATP/CTP:tRNA nucleotidyltransferase (cca) from Sulfolobus shibatae was used to search open reading frames in the genome of Methanococcus jannaschii. Translations of two unidentified open reading frames showed significant sequence similarity to portions of the Sulfolobus cca protein. When the two open reading frames were joined together, the expanded open reading frame was similar in sequence to the entire Sulfolobus cca protein and displayed features of the active site signature sequence proposed for members of class I enzymes within the superfamily of nucleotidyltransferases (Yue et al., 1996, RNA 2, 895–908). A possible UUG start codon was identified based on significant sequence similarity of the resulting amino-terminal region to that of Sulfolobus, and on a six-base complementarity between an adjacent upstream sequence and Methanococcus 16S rRNA. Received: 10 February 1997     

腾冲热海一株嗜酸热硫化叶菌的分离与鉴定

从云南腾冲热海酸性温泉中分离纯化出一株极端嗜酸热菌株K4-1,并对其进行形态观察、生长特征、碳源和能源利用及16S rRNA基因分析.该菌株细胞形态为不规则球形,有单生鞭毛,严格好氧,兼性自养,能利用元素硫作为能源,也能利用酵母膏、精氨酸或核糖作为碳源和能源.其最适生长温度为75℃,最适pH为3.5.通过16S rRNA基因序列相似性对比对该菌株进行鉴定,结果表明该菌株与硫化叶茵属标准菌株的相似性介于86.6%～94.3%之间,与分离自腾冲热海的腾冲硫化叶菌Sulfolobus tengchongensis RT8-4菌株序列相似性最高,达到98.9%,可将菌株K4-1鉴定为硫化叶菌属菌株.菌株K4-1的16S rRNA基因序列号为EU729124.     

The structure of the gene for ribosomal protein L5 in the archaebacterium Sulfolobus acidocaldarius.

The gene for the ribosomal protein L5 from the archaebacterium Sulfolobus acidocaldarius has been isolated and sequenced. The gene codes for a basic protein of molecular weight 29 165 Da. This protein shows substantial similarity to the equivalent protein from other archaebacteria as well as from yeast, and considerably less similarity to the equivalent eubacterial protein. These results support the concept of the archaebacteria as a monophyletic kingdom more closely related to eukaryotes than to eubacteria.     

Identification of the archaeal NMN adenylyltransferase gene

Increasing evidence on the importance of fluctuations in NAD+ levels in the living cell is accumulating. Therefore a deeper knowledge on the regulation of coenzyme synthesis and recycling is required. In this context the study of NMN adenylyltransferase (EC 2.7.7. 1), a key enzyme in the NAD+ biosynthetic pathway, assumes a remarkable relevance. We have previously purified to homogeneity and characterized the protein from the thermophilic archaeon Sulfolobus solfataricus. The determination of partial sequence of the S. solfataricus enzyme, together with the recent availability of the genome sequence of the archaeon Methanococcus jannaschii allowed us, based on sequence similarity, to identify the M. jannaschii NMN adenylyltransferase gene. As far as we know from literature, this is the first report on the NMN adenylyltransferase gene.     

Cloning and sequencing of the gene coding for aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

The gene coding for aspartate aminotransferase (EC 2.6.1.1) has been cloned from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus strain MT4. Partial sequence data obtained directly from the purified protein and from the two cyanogen-bromide-generated peptides confirm the primary structure of aspartate aminotransferase inferred from the nucleotide sequence of its gene. A comparison of the enzyme with other aminotransferases revealed an interesting similarity with tyrosine aminotransferase from rat liver (EC 2.6.1.5) and allowed some tentative assignments of the residues implied in the catalysis. The aspartate aminotransferase gene-flanking regions were compared to those of other archaebacterial genes already described in the literature with the aim of identifying potential regulatory sites.     

Cellulose degradation by Sulfolobus solfataricus requires a cell-anchored endo-β-1-4-glucanase

A sequence encoding a putative extracellular endoglucanase (sso1354) was identified in the complete genome sequence of Sulfolobus solfataricus. The encoded protein shares signature motifs with members of glycoside hydrolases family 12. After an unsuccessful first attempt at cloning the full-length coding sequences in Escherichia coli, an active but unstable recombinant enzyme lacking a 27-residue N-terminal sequence was generated. This 27-amino-acid sequence shows significant similarity with corresponding regions in the sugar binding proteins AraS, GlcS, and TreS of S. solfataricus that are responsible for anchoring them to the plasma membrane. A strategy based on an effective vector/host genetic system for Sulfolobus and on expression control by the promoter of the S. solfataricus gene which encodes the glucose binding protein allowed production of the enzyme in sufficient quantities for study. In fact, the enzyme expressed in S. solfataricus was stable and highly thermoresistant and showed optimal activity at low pH and high temperature. The protein was detected mainly in the plasma membrane fraction, confirming the structural similarity to the sugar binding proteins. The results of the protein expression in the two different hosts showed that the SSO1354 enzyme is endowed with an endo-β-1-4-glucanase activity and specifically hydrolyzes cellulose. Moreover, it also shows significant but distinguishable specificity toward several other sugar polymers, such as lichenan, xylan, debranched arabinan, pachyman, and curdlan.     

Structural conservation of the isolated zinc site in archaeal zinc-containing ferredoxins as revealed by x-ray absorption spectroscopic analysis and its evolutionary implications.

The zfx gene encoding a zinc-containing ferredoxin from Thermoplasma acidophilum strain HO-62 was cloned and sequenced. It is located upstream of two genes encoding an archaeal homolog of nascent polypeptide-associated complex alpha subunit and a tRNA nucleotidyltransferase. This gene organization is not conserved in several euryarchaeoteal genomes. The multiple sequence alignments of the zfx gene product suggest significant sequence similarity of the ferredoxin core fold to that of a low potential 8Fe-containing dicluster ferredoxin without a zinc center. The tightly bound zinc site of zinc-containing ferredoxins from two phylogenetically distantly related Archaea, T. acidophilum HO-62 and Sulfolobus sp. strain 7, was further investigated by x-ray absorption spectroscopy. The zinc K-edge x-ray absorption spectra of both archaeal ferredoxins are strikingly similar, demonstrating that the same zinc site is found in T. acidophilum ferredoxin as in Sulfolobus sp. ferredoxin, which suggests the structural conservation of isolated zinc binding sites among archaeal zinc-containing ferredoxins. The sequence and spectroscopic data provide the common structural features of the archaeal zinc-containing ferredoxin family.     

An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family

An open reading frame upstream of the Methanococcus vannielii L12 gene has been detected. The beginning of this open reading frame agrees with the N-terminal region of a protein (MvaL10) which has been isolated from the 50 S ribosomal subunit of M. vannielii and sequenced. The length of this gene is 1008 nucleotides, coding for 336 amino acids. Excellent sequence similarities were found to the L10-like ribosomal proteins from Halobacterium halobium and man. The N-terminal part of the MvaL10 protein shows significant sequence similarities to the E. coli L10 protein. MvaL10 is more than twice as long as E. coli L10 but is of length similar to those of the homologous halobacterial and human proteins. Interestingly, the C-terminal region of MvaL10 shows exceptionally high similarity to the C-terminal sequence of the MvaL12 protein. This is not the case for the E. coli proteins but was also observed for the human, Halobacterium and Sulfolobus proteins.     

Identification of a novel alpha-galactosidase from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable alpha-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for alpha-linked galactosides, which are optimally hydrolyzed at pH 5 and 90 degrees C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable alpha-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.     

A small basic ribosomal protein in Sulfolobus solfataricus equivalent to L46 in yeast: Structure of the protein and its gene

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene (L46e) has been confirmed by partial amino acid sequencing. The protein shows substantial sequence homology to the eukaryotic ribosomal proteins L39 in rat and L46 in yeast. There is no sequence homology to any of the eubacterial ribosomal proteins suggesting that this protein is absent in the eubacterial ribosome.     

Phylogenetic analysis of carbamoylphosphate synthetase genes: complex evolutionary history includes an internal duplication within a gene which can root the tree of life

Carbamoylphosphate synthetase (CPS) catalyzes the first committed step in pyrimidine biosynthesis, arginine biosynthesis, or the urea cycle. Organisms may contain either one generalized or two specific CPS enzymes, and these enzymes may be heterodimeric (encoded by linked or unlinked genes), monomeric, or part of a multifunctional protein. In order to help elucidate the evolution of CPS, we have performed a comprehensive phylogenetic analysis using the 21 available complete CPS sequences, including a sequence from Sulfolobus solfataricus P2 which we report in this paper. This is the first report of a complete CPS gene sequence from an archaeon, and sequence analysis suggests that it encodes an enzyme similar to heterodimeric CPSII. We confirm that internal similarity within the synthetase domain of CPS is the result of an ancient gene duplication that preceded the divergence of the Bacteria, Archaea, and Eukarya, and use this internal duplication in phylogenetic tree construction to root the tree of life. Our analysis indicates with high confidence that this archaeal sequence is more closely related to those of Eukarya than to those of Bacteria. In addition to this ancient duplication which created the synthetase domain, our phylogenetic analysis reveals a complex history of further gene duplications, fusions, and other events which have played an integral part in the evolution of CPS.      

Molecular analysis of pDL10 from Acidianus ambivalens reveals a family of related plasmids from extremely thermophilic and acidophilic archaea.

The 7598-bp plasmid pDL10 from the extremely thermophilic, acidophilic, and chemolithoautotrophic Archaeon Acidianus ambivalens was sequenced. It contains 10 open reading frames (ORFs) organized in five putative operons. The deduced amino acid sequence of the largest ORF (909 aa) showed similarity to bacterial Rep proteins known from phages and plasmids with rolling-circle (RC) replication. From the comparison of the amino acid sequences, a novel family of RC Rep proteins was defined. The pDL10 Rep protein shared 45-80% identical residues with homologous protein genes encoded by the Sulfolobus islandicus plasmids pRN1 and pRN2. Two DNA regions capable of forming extended stem-loop structures were also conserved in the three plasmids (48-69% sequence identity). In addition, a putative plasmid regulatory protein gene (plrA) was found, which was conserved among the three plasmids and the conjugative Sulfolobus plasmid pNOB8. A homolog of this gene was also found in the chromosome of S. solfataricus. Single-stranded DNA of both pDL10 strands was detected with a mung bean nuclease protection assay using PCR detection of protected fragments, giving additional evidence for an RC mechanism of replication.     

Malonyl-coenzyme A reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus spp

Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO2 fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO2 into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H+ --> malonate-semialdehyde + CoA + NADP+. In its native state it is associated with small RNA. Its activity was stimulated by Mg2+ and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (Km = 25 microM) and malonyl-CoA (Km = 40 microM). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.     

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.     

Cloning and nucleotide sequence of a gene encoding a glycogen debranching enzyme in the trehalose operon from Arthrobacter sp. Q36

A gene located just upstream of the treYZ operon was isolated from Arthrobacter sp. strain Q36. The gene, designated treX, encoded an 823-amino acid protein. The amino acid sequence of the protein had 50% identity with the TreX protein (isoamylase) from Sulfolobus acidocaldarius ATCC 33909 which has a treZXY operon on the genome. We suggest that Arthrobacter treX is an isoamylase gene, and that it is a component of a treXYZ operon.     

SSoNΔ and SSoNΔlong: two thermostable esterases from the same ORF in the archaeon Sulfolobus solfataricus?

Previously, we reported from the Sulfolobus solfataricus open reading frame (ORF) SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL) family and apparently having a deletion at the N-terminus, which we named SSoNDelta. Searching the recently reported Sulfolobus acidocaldarius genome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105) sharing high sequence similarity (82%) with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, named SSoNDeltalong, was 15-fold more active with the substrate p-nitrophenyl hexanoate than SSoNDelta. Furthermore, SSoNDeltalong and SSoNDelta displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship between S. solfataricus and S. acidocaldarius suggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression of SSoNDeltalong under specific metabolic conditions could be hypothesized.     

The exopolyphosphatase gene from sulfolobus solfataricus: characterization of the first gene found to be involved in polyphosphate metabolism in archaea

Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Although the presence of polyP in members of the domain Archaea has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain. We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (ppx) from thermophilic Sulfolobus solfataricus. The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa). The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60 degrees C. The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs. In contrast, informatic analysis revealed that the deduced amino acid sequence of S. solfataricus PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs. To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the ARCHAEA: