The genome of Salinibacter ruber: convergence and gene exchange among hyperhalophilic bacteria and archaea.

Saturated thalassic brines are among the most physically demanding habitats on Earth: few microbes survive in them. Salinibacter ruber is among these organisms and has been found repeatedly in significant numbers in climax saltern crystallizer communities. The phenotype of this bacterium is remarkably similar to that of the hyperhalophilic Archaea (Haloarchaea). The genome sequence suggests that this resemblance has arisen through convergence at the physiological level (different genes producing similar overall phenotype) and the molecular level (independent mutations yielding similar sequences or structures). Several genes and gene clusters also derive by lateral transfer from (or may have been laterally transferred to) haloarchaea. S. ruber encodes four rhodopsins. One resembles bacterial proteorhodopsins and three are of the haloarchaeal type, previously uncharacterized in a bacterial genome. The impact of these modular adaptive elements on the cell biology and ecology of S. ruber is substantial, affecting salt adaptation, bioenergetics, and photobiology.

Do orthologous gene phylogenies really support tree-thinking?

BACKGROUND: Since Darwin's Origin of Species, reconstructing the Tree of Life has been a goal of evolutionists, and tree-thinking has become a major concept of evolutionary biology. Practically, building the Tree of Life has proven to be tedious. Too few morphological characters are useful for conducting conclusive phylogenetic analyses at the highest taxonomic level. Consequently, molecular sequences (genes, proteins, and genomes) likely constitute the only useful characters for constructing a phylogeny of all life. For this reason, tree-makers expect a lot from gene comparisons. The simultaneous study of the largest number of molecular markers possible is sometimes considered to be one of the best solutions in reconstructing the genealogy of organisms. This conclusion is a direct consequence of tree-thinking: if gene inheritance conforms to a tree-like model of evolution, sampling more of these molecules will provide enough phylogenetic signal to build the Tree of Life. The selection of congruent markers is thus a fundamental step in simultaneous analysis of many genes. RESULTS: Heat map analyses were used to investigate the congruence of orthologues in four datasets (archaeal, bacterial, eukaryotic and alpha-proteobacterial). We conclude that we simply cannot determine if a large portion of the genes have a common history. In addition, none of these datasets can be considered free of lateral gene transfer. CONCLUSION: Our phylogenetic analyses do not support tree-thinking. These results have important conceptual and practical implications. We argue that representations other than a tree should be investigated in this case because a non-critical concatenation of markers could be highly misleading.

The complete genome of the crenarchaeon Sulfolobus solfataricus P2.

The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.

Identification and molecular characterization of the first alpha -xylosidase from an archaeon.

We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31. xylS belongs to a cluster of four genes in the S. solfataricus genome, including a beta-glycosidase, an hypothetical membrane protein homologous to the major facilitator superfamily of transporters, and an open reading frame of unknown function. The alpha-xylosidase was overexpressed in Escherichia coli showing optimal activity at 90 degrees C and a half-life at this temperature of 38 h. The purified enzyme follows a retaining mechanism of substrate hydrolysis, showing high hydrolytic activity on the disaccharide isoprimeverose and catalyzing the release of xylose from xyloglucan oligosaccharides. Synergy is observed in the concerted in vitro hydrolysis of xyloglucan oligosaccharides by the alpha-xylosidase and the beta-glycosidase from S. solfataricus. The analysis of the total S. solfataricus RNA revealed that all the genes of the cluster are actively transcribed and that xylS and orf3 genes are cotranscribed.

Gene content and organization of a 281-kbp contig from the genome of the extremely thermophilic archaeon, Sulfolobus solfataricus P2.

The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed. Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis, pyrimidine biosynthesis, and arginine biosynthesis, an ATPase operon, numerous genes for enzymes of lipopolysaccharide biosynthesis, and six insertion sequences. The content and organization of this contig are compared with sequences from crenarchaeotes, euryarchaeotes, bacteria, and eukaryotes.

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.

Two DNA polymerase sliding clamps from the thermophilic archaeon Sulfolobus solfataricus.

Herein, we report the identification and characterization of two DNA polymerase processivity factors from the thermoacidophilic archaeon Sulfolobus solfataricus. They, referred to as 039p (244 amino acid residues, 27 kDa) and 048p (249 amino acid residues, 27 kDa), present significant primary structure similarity to eukaryotic proliferating cell nuclear antigen (PCNA). We demonstrate that both 039p and 048p form oligomers in solution and are able to substantially activate the synthetic activity of the single-subunit family B DNA polymerase from S. solfataricus (Sso DNA pol B1) on poly(dA)-oligo(dT) as a primer-template. This stimulatory effect is the result of enhanced DNA polymerase processivity, as indicated by the analysis of the elongation products on polyacrylamide gels. Activation of Sso DNA pol B1 synthetic activity was also observed on linear primed single-stranded M13 mp18 DNA as a template. By immunoblot analysis using specific rabbit antisera, 039p and 048p were both detected in the logarithmic and stationary phases of S. solfataricus growth curve. This is the first report of the identification and biochemical characterization of two distinct DNA polymerase processivity factors from the same organism. The significance of these findings for the understanding of the DNA replication process in Archaea is discussed.

Glucose transport in the extremely thermoacidophilic Sulfolobus solfataricus involves a high-affinity membrane-integrated binding protein.

The archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars. Cells rapidly accumulate glucose. This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with very high affinity (Kd of 0. 43 microM) and retains high glucose affinity at very low pH values (as low as pH 0.6). The binding protein was extracted with detergent and purified to homogeneity as a 65-kDa glycoprotein. The gene coding for the binding protein was identified in the S. solfataricus P2 genome by means of the amino-terminal amino acid sequence of the purified protein. Sequence analysis suggests that the protein is anchored to the membrane via an amino-terminal transmembrane segment. Neighboring genes encode two membrane proteins and an ATP-binding subunit that are transcribed in the reverse direction, whereas a homologous gene cluster in Pyrococcus horikoshii OT3 was found to be organized in an operon. These data indicate that S. solfataricus utilizes a binding-protein-dependent ATP-binding cassette transporter for the uptake of glucose.

An Lrp-like protein of the hyperthermophilic archaeon Sulfolobus solfataricus which binds to its own promoter.

Regulation of gene expression in the domain Archaea, and specifically hyperthermophiles, has been poorly investigated so far. Biochemical experiments and genome sequencing have shown that, despite the prokaryotic cell and genome organization, basal transcriptional elements of members of the domain Archaea (i.e., TATA box-like sequences, RNA polymerase, and transcription factors TBP, TFIIB, and TFIIS) are of the eukaryotic type. However, open reading frames potentially coding for bacterium-type transcription regulation factors have been recognized in different archaeal strains. This finding raises the question of how bacterial and eukaryotic elements interact in regulating gene expression in Archaea. We have identified a gene coding for a bacterium-type transcription factor in the hyperthermophilic archaeon Sulfolobus solfataricus. The protein, named Lrs14, contains a potential helix-turn-helix motif and is related to the Lrp-AsnC family of regulators of gene expression in the class Bacteria. We show that Lrs14, expressed in Escherichia coli, is a highly thermostable DNA-binding protein. Bandshift and DNase I footprint analyses show that Lrs14 specifically binds to multiple sequences in its own promoter and that the region of binding overlaps the TATA box, suggesting that, like the E. coli Lrp, Lrs14 is autoregulated. We also show that the lrs14 transcript is accumulated in the late growth stages of S. solfataricus.

Completing the sequence of the Sulfolobus solfataricus P2 genome.

The Sulfolobus solfataricus P2 genome collaborators are poised to sequence the entire 3-Mbp genome of this crenarchaeote archaeon. About 80% of the genome has been sequenced to date, with the rest of the sequence being assembled fast. In this publication we introduce the genomic sequencing and automated analysis strategy and present intial data derived from the sequence analysis. After an overview of the general sequence features, metabolic pathway studies are explained, using sugar metabolism as an example. The paper closes with an overview of repetitive elements in S. solfataricus.

Sulfolobus genome: from genomics to biology.

Major progress in sequencing the genome of Sulfolobus solfataricus has been closely concerted with the characterization and sequencing of many extrachromosomal genetic elements, including viruses, cryptic plasmids and conjugative plasmids, as well as mobile archaeal introns and transposons. The latter have provided a basis for developing the first generation of vectors that are now being used to study the genetics of Sulfolobus and other Archaea.

Characterization of two heat shock genes from Haloferax volcanii: a model system for transcription regulation in the Archaea.

The expression of two heat-responsive cct (chaperonin-containing Tcp-1) genes from the archaeon Haloferax volcanii was investigated at the transcription level. The cct1 and cct2 genes, which encode proteins of 560 and 557 amino acids, respectively, were identified on cosmid clones of an H. volcanii genomic library and subsequently sequenced. The deduced amino acid sequences of these genes exhibited a high degree of similarity to other archaeal and eucaryal cct family members. Expression of the cct genes was characterized in detail for the purpose of developing a model for studying transcription regulation in the domain Archaea. Northern (RNA) analysis demonstrated that the cct mRNAs were maximally induced after heat shock from 37 to 55 degrees C and showed significant heat inducibility after 30 min at 60 degrees C. Transcription of cct mRNAs was also stimulated in response to dilute salt concentrations. Transcriptional analysis of cct promoter regions coupled to a yeast tRNA reporter gene demonstrated that 5' flanking sequences up to position -233 (cct1) and position -170 (cct2) were sufficient for promoting heat-induced transcription. Transcript analysis indicated that both basal transcription and stress-induced transcription of the H. volcanii cct genes were directed by a conserved archaeal consensus TATA motif (5'-TTTATA-3') centered at -25 relative to the mapped initiation site. Comparison of the cct promoter regions also revealed a striking degree of sequence conservation immediately 5' and 3' of the TATA element.

Evolutionary analysis of the hisCGABdFDEHI gene cluster from the archaeon Sulfolobus solfataricus P2.

While sequencing the genome of the archaeon Sulfolobus solfataricus P2, we found an 8,313-bp sequence containing a cluster of nine histidine biosynthesis genes in an order different from that of any known his operon. Results of phylogenetic analysis of the coding regions in the putative operon give conflicting evolutionary histories for individual his genes.

An archaebacterial homolog of pelota, a meiotic cell division protein in eukaryotes.

An open reading frame (pelA) specifying a homolog of pelota and DOM34, proteins required for meiotic cell division in Drosophila melanogaster and Saccharomyces cerevisiae, respectively, has been cloned, sequenced and identified from the archaebacterium Sulfolobus solfataricus. The S. solfataricus PelA protein is about 20% identical with pelota, DOM34 and the hypothetical protein R74.6 of Caenorhabditis elegans. The presence of a pelota homolog in archaebacteria implies that the meiotic functions of the eukaryotic protein were co-opted from, or added to, other functions existing before the emergence of eukaryotes. The nuclear localization signal and negatively charged carboxy-terminus characteristic of eukaryotic pelota-like proteins are absent from the S. solfataricus homolog, and hence may be indicative of the acquired eukaryotic function(s).

Organizational characteristics and information content of an archaeal genome: 156 kb of sequence from Sulfolobus solfataricus P2.

We have initiated a project to sequence the 3 Mbp genome of the thermoacidophilic archaebacterium Sulfolobus solfataricus P2. Cosmids were selected from a provisional set of minimally overlapping clones, subcloned in pUC18, and sequenced using a hybrid (random plus directed) strategy to give two blocks of contiguous unique sequence, respectively, 100,389 and 56,105 bp. These two contigs contain a total of 163 open reading frames (ORFs) in 26-29 putative operons; 56 ORFs could be identified with reasonable certainty. Clusters of ORFs potentially encode proteins of glycogen biosynthesis, oxidative decarboxylation of pyruvate, ATP-dependent transport across membranes, isoprenoid biosynthesis, protein synthesis, and ribosomes. Putative promoters occur upstream of most ORFs. Thirty per cent of the predicted strong and medium-strength promoters can initiate transcription at the start codon or within 10 nucleotides upstream, indicating a process of initial mRNA-ribosome contact unlike that of most eubacterial genes. A novel termination motif is proposed to account for 15 additional terminations. The two contigs differ in densities of ORFs, insertion elements and repeated sequences; together they contain two copies of the previously reported insertion sequence ISC 1217, five additional IS elements representing four novel types, four classes of long non-IS repeated sequences, and numerous short, perfect repeats.

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.

Comparative genomic analysis of the Haloferax volcanii DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement.

Anonymous probes from the genome of Halobacterium salinarium GRB and 12 gene probes were hybridized to the cosmid clones representing the chromosome and plasmids of Halobacterium salinarium GRB and Haloferax volcanii DS2. The order of and pairwise distances between 35 loci uniquely cross-hybridizing to both chromosomes were analyzed in a search for conservation. No conservation between the genomes could be detected at the 15-kbp resolution used in this study. We found distinct sets of low-copy-number repeated sequences in the chromosome and plasmids of Halobacterium salinarium GRB, indicating some degree of partitioning between these replicons. We propose alternative courses for the evolution of the haloarchaeal genome: (i) that the majority of genomic differences that exist between genera came about at the inception of this group or (ii) that the differences have accumulated over the lifetime of the lineage. The strengths and limitations of investigating these models through comparative genomic studies are discussed.

The Sulfolobus solfataricus P2 genome project.

Over 800 kbp of the 3-Mbp genome of Sulfolobus solfataricus have been sequenced to date. Our approach is to sequence subclones of mapped cosmids, followed by sequencing directly on cosmid templates with custom primers. Using a prototype automated system for genome-scale analysis, known as MAGPIE, along with other tools, we have discovered one open reading frame of at least 100 amino acids per kbp of sequence, and have been able to associate 50% of these with known genes through database searches. An examination of completely sequenced cosmids suggests a clustering of genes by function in the S. solfataricus genome.

Supercoiling and map stability in the bacterial chromosome.

A major goal of comparative genomics is an understanding of the forces which control gene order. This assumes that gene order is important, a supposition backed by the existence of genomic colinearity between many related species. In the bacterial chromosome, a polarity in the order of genes has been suggested, influenced by distance and orientation relative to the origin of DNA replication. We propose a model of the bacterial chromosome in which gene order is maintained by the adaptation of gene expression to local superhelical context. This force acts not directly at the genomic level but rather at the local gene level. A full understanding of gene-order conservation must therefore come from the bottom up.

Genomic stability in the archaeae Haloferax volcanii and Haloferax mediterranei.

Through hybridization of available probes, we have added nine genes to the macrorestriction map of the Haloferax mediterranei chromosome and five genes to the contig map of Haloferax volcanii. Additionally, we hybridized 17 of the mapped cosmid clones from H. volcanii to the H. mediterranei genome. The resulting 35-point chromosomal comparison revealed only two inversions and a few translocations. Forces known to promote rearrangement, common in the haloarchaea, have been ineffective in changing global gene order throughout the nearly 10(7) years of these species' divergent evolution.