Peter Palm

The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity

BackgroundThe square halophilic archaeon Haloquadratum walsbyi dominates NaCl-saturated and MgCl2 enriched aquatic ecosystems, which imposes a serious desiccation stress, caused by the extremely low water activity. The genome sequence was analyzed and physiological and physical experiments were carried out in order to reveal how H. walsbyi has specialized into its narrow and hostile ecological niche and found ways to cope with the desiccation stress.ResultsA rich repertoire of proteins involved in phosphate metabolism, phototrophic growth and extracellular protective polymers, including the largest archaeal protein (9159 amino acids), a homolog to eukaryotic mucins, are amongst the most outstanding features. A relatively low GC content (47.9%), 15–20% less than in other halophilic archaea, and one of the lowest coding densities (76.5%) known for prokaryotes might be an indication for the specialization in its unique environmentConclusionAlthough no direct genetic indication was found that can explain how this peculiar organism retains its square shape, the genome revealed several unique adaptive traits that allow this organism to thrive in its specific and extreme niche.

Thermoproteales: A novel type of extremely thermoacidophilic anaerobic archaebacteria isolated from Icelandic solfataras

Summary Two types of anaerobic extremely thermoacidophilic bacteria have been isolated from more than 50% of the samples collected in solfataric fields throughout Iceland. They both possess highly stable cell envelopes with a characteristic subunit structure, are usually rods of variable length (about 1 to more than 50 μ ) without septa, but can give rise to spheric bodies which exist either attached to the rods or in free state. Under certain conditions, truly branched filaments are frequently observed. The similarities in morphology and growth characteristics indicate that the two types are related to each other. In view of its variable appearance the larger organism (diameter about 0.4 μ m) was called Thermoproteus tenax . Thermoproteus tenax forms H 2 S and CO 2 from elemental sulfur and organic substrates, like glucose, ethanol, malate or formamide. The absence of murein, the presence of polyisoprenoid ether lipids, the complete resistance against the antibiotics rifampicin, streptomycin, vancomycin and chloramphenicol and the component pattern and rifampicin insensitivity of the DNA-dependent RNA polymerase are evidence that Thermoproteus tenax belongs to the archaebacteria. It represents a novel order of the thermoacidophilic branch of this urkingdom.

Complete nucleotide sequence of the virus SSV1 of the archaebacterium Sulfolobus shibatae.

The DNA sequence of the Sulfolobus shibatae virus SSV1 is the first complete sequence of an archaebacterial virus genome. The viral DNA is a closed double-stranded DNA circle of 15465 bp. The features of the sequence, the positions of all 11 transcripts, the three characterized proteins, and the open reading frames are described.

Methanothermus fervidus, sp. nov., a novel extremely thermophilic methanogen isolated from an Icelandic hot spring

Summary A rod-shaped extremely thermophilic methanogen is described, growing between 65 and 97 °C with an optimal temperature around 83 °C and a doubling time of 170 min. The GC-content of its DNA is 33 mol %. The isolated cell wall sacculus contains pseudomurein. The complex cell envelope exhibits two layers, each about 12 nm thick; the inner represents the pseudomurein sacculus and the outer a protein envelope. An enriched fraction of RNA polymerase does not react with antiserum against RNA polymerase from Metbanobacterium thermoautotrophicum, indicating that the isolate belongs to a new family, the Methanothermaceae, within the order Methanobacteriales. The new organism is named Methanothermus fervidus.

Isolate B12, which harbours a virus-like element, represents a new species of the archaebacterial genus Sulfolobus, Sulfolobus shibatae, sp. nov.

The Sulfolobus isolate B12 and its endogenous virus-like element SSV1 have provided a fruitful system for detailed analysis of certain aspects of archaebacterial molecular biology, especially those concerning gene expression. In the course of clarifying this isolates taxonomic position, we determined DNA base composition, ability to grow autotrophically, nucleotide sequence of 16S ribosomal RNA, and level of total genomic homology to other Sulfolobus strains. Although the results generally demonstrate a similarity to S. solfataricus, DNA-DNA hybridisation and 16S rRNA sequence data indicate that isolate B12 in fact represents a distinct species.

Evolution in the laboratory: The genome of Halobacterium salinarum strain R1 compared to that of strain NRC-1

We report the sequence of the Halobacterium salinarum strain R1 chromosome and its four megaplasmids. Our set of protein-coding genes is supported by extensive proteomic and sequence homology data. The structures of the plasmids, which show three large-scale duplications (adding up to 100 kb), were unequivocally confirmed by cosmid analysis. The chromosome of strain R1 is completely colinear and virtually identical to that of strain NRC-1. Correlation of the plasmid sequences revealed 210 kb of sequence that occurs only in strain R1. The remaining 350 kb shows virtual sequence identity in the two strains. Nevertheless, the number and overall structure of the plasmids are largely incompatible. Also, 20% of the protein sequences differ despite the near identity at the DNA sequence level. Finally, we report genome-wide mobility data for insertion sequences from which we conclude that strains R1 and NRC-1 originate from the same natural isolate. This exemplifies evolution in the laboratory.

Desulfurococcaceae, the Second Family of the Extremely Thermophilic, Anaerobic, Sulfur-Respiring Thermoproteales*

Summary Two novel species of anaerobic thermophilic archaebacteria isolated from acidic hot springs of Iceland, Desulfurococcus mucosus and Desulfurococcus mobilis , representing a second family, termed Desulfurococcaceae , of the order Thermoproteales are described. They utilize yeast extract or casein or its tryptic digest, but not casamino acids, as carbon sources, by sulfur respiration with the production of H 2 S and CO 2 , or by fermentation. The pH optimum of growth is pH 5.5 to 6, the temperature optimum 85 °C. The archaebacterial nature of the Desulfurococcaceae is evident from their insensitivity towards vancomycin, streptomycin and chloramphenicol, the lack of a murein cell wall, the presence of phytanol and polyisoprenoid dialcohols in the lipids, and the composition and the properties of the DNA dependent RNA polymerase. They are closely related to the recently described anaerobic thermoacidophilic sulfur-respiring Thermoproteus tenax representing the first family, Thermoproteaceae of the Thermoproteales . Of the other divisions of archaebacteria, Sulfolobus is the nearest relative. Desulfurococcus mucosus has a slimy polymer attached to its envelope. Desulfurococcus mobilis possesses flagellae in monopolar polytrichous arrangement.

Haloquadratum walsbyi: Limited Diversity in a Global Pond

Background Haloquadratum walsbyi commonly dominates the microbial flora of hypersaline waters. Its cells are extremely fragile squares requiring >14%(w/v) salt for growth, properties that should limit its dispersal and promote geographical isolation and divergence. To assess this, the genome sequences of two isolates recovered from sites at near maximum distance on Earth, were compared. Principal Findings Both chromosomes are 3.1 MB in size, and 84% of each sequence was highly similar to the other (98.6% identity), comprising the core sequence. ORFs of this shared sequence were completely synteneic (conserved in genomic orientation and order), without inversion or rearrangement. Strain-specific insertions/deletions could be precisely mapped, often allowing the genetic events to be inferred. Many inferred deletions were associated with short direct repeats (4–20 bp). Deletion-coupled insertions are frequent, producing different sequences at identical positions. In cases where the inserted and deleted sequences are homologous, this leads to variant genes in a common synteneic background (as already described by others). Cas/CRISPR systems are present in C23T but have been lost in HBSQ001 except for a few spacer remnants. Numerous types of mobile genetic elements occur in both strains, most of which appear to be active, and with some specifically targetting others. Strain C23T carries two ∼6 kb plasmids that show similarity to halovirus His1 and to sequences nearby halovirus/plasmid gene clusters commonly found in haloarchaea. Conclusions Deletion-coupled insertions show that Hqr. walsbyi evolves by uptake and precise integration of foreign DNA, probably originating from close relatives. Change is also driven by mobile genetic elements but these do not by themselves explain the atypically low gene coding density found in this species. The remarkable genome conservation despite the presence of active systems for genome rearrangement implies both an efficient global dispersal system, and a high selective fitness for this species.

SSV1-encoded site-specific recombination system in Sulfolobus shibatae.

SummaryWe present evidence for the existence of a conservative site-specific recombination system in Archaea by demonstrating integrative recombination of Sulfolobus shibatae virus SSV1 DNA with the host chromosome, catalysed by the SSVI-encoded integrase in vitro. The putative int gene of SSV1 was expressed in Escherichia coli yielding a protein of about 39 kDa. This protein alone efficiently recombined linear DNA substrates containing chromosomal (attA) and viral (attP) attachment sites; recombination with either negatively or positively supercoiled SSV1 DNA was less efficient. Intermolecular attA × attA and attP × attP recombination was also promoted by the SSV integrase. The invariant 44 by “common attachment core” present in all att sites contained sufficient information to allow recombination, whilst the flanking sequences effected the efficiency. These features clearly distinguish the SSV1 — encoded site — specific recombination system from others and make it suitable for the study of regulatory mechanisms of SSV1 genome — host chromosome interaction and investigations of the evolution of the recombination machinery.

Gene expression in archaebacteria: physical mapping of constitutive and UV-inducible transcripts from the Sulfolobus virus-like particle SSV1.

SummaryThe transcription of the genome of the UV-inducible Sulfolobus virus-like particle SSV1 was studied. Eight different transcripts could be distinguished by Northern analysis that were present in uninduced cells and that coordinately increased in amount after UV induction of SSV1. Using single-stranded DNA probes from different parts of the genome, the approximate map positions of these RNAs and the directions of transcription were determined. In two cases, terminator read-through resulted in the formation of more than one RNA species from a single 5′ end and therefore the eight different RNAs corresponded to only five different transcriptional starts. Two RNAs sharing a common 5′ end encode SSV1 structural proteins. The 5′ end of these transcripts was determined by S1 nuclease analysis. About 20 nucleotides upstream of the transcriptional start of these RNAs, there is an AT-rich region resembling putative promoter sequences which have been found at a similar distance 5′ to the genes encoding stable RNAs in Thermoproteus. In addition to the eight constitutive transcripts, a UV-inducible RNA of 0.3 kb was mapped on the SSV1 genome. In contrast to all other RNAs, it was not detectable in uninduced cells and it is expressed shortly before the amplification and packaging of the SSV1 genome commences.