Genetic elements in the extremely thermophilic archaeon Sulfolobus

This minireview summarizes what is known about genetic elements in the archaeal crenarchaeotal genus Sulfolobus, including recent work on viruses, cryptic plasmids, a novel type of virus satellite plasmids or satellite viruses, and conjugative plasmids (CPs), mostly from our laboratory. It does not discuss IS elements and transposons. Received: January 22, 1998 / Accepted: February 16, 1998     

New insights into the diversity and evolution of the archaeal mobilome from three complete genomes of Saccharolobus shibatae

Saccharolobus (formerly Sulfolobus) shibatae B12, isolated from a hot spring in Beppu, Japan in 1982, is one of the first hyperthermophilic and acidophilic archaeal species to be discovered. It serves as a natural host to the extensively studied spindle-shaped virus SSV1, a prototype of the Fuselloviridae family. Two additional Sa. shibatae strains, BEU9 and S38A, sensitive to viruses of the families Lipothrixviridae and Portogloboviridae, respectively, have been isolated more recently. However, none of the strains has been fully sequenced, limiting their utility for studies on archaeal biology and virus–host interactions. Here, we present the complete genome sequences of all three Sa. shibatae strains and explore the rich diversity of their integrated mobile genetic elements (MGE), including transposable insertion sequences, integrative and conjugative elements, plasmids, and viruses, some of which were also detected in the extrachromosomal form. Analysis of related MGEs in other Sulfolobales species and patterns of CRISPR spacer targeting revealed a complex network of MGE distributions, involving horizontal spread and relatively frequent host switching by MGEs over large phylogenetic distances, involving species of the genera Saccharolobus, Sulfurisphaera and Acidianus. Furthermore, we characterize a remarkable case of a virus-to-plasmid transition, whereby a fusellovirus has lost the genes encoding for the capsid proteins, while retaining the replication module, effectively becoming a plasmid.     

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 (excluding IS elements and transposons) in the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in the kingdom Euryachaeota of the archael domain, including unpublished data from our laboratory. The viruses of Crenarchaeota represent four novel virus families. The Fuselloviridae represented by SSV1 of S. shibatae and relatives in other Sulfolobus strains have the form of a failed 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 Lipothrixviridae (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 type 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 (β-galactosidase) gene into a mutant of S. solfataricus.     

Genomic analysis of <Emphasis Type="Italic">Acidianus hospitalis</Emphasis> W1 a host for studying crenarchaeal virus and plasmid life cycles

The Acidianus hospitalis W1 genome consists of a minimally sized chromosome of about 2.13 Mb and a conjugative plasmid pAH1 and it is a host for the model filamentous lipothrixvirus AFV1. The chromosome carries three putative replication origins in conserved genomic regions and two large regions where non-essential genes are clustered. Within these variable regions, a few orphan orfB and other elements of the IS200/607/605 family are concentrated with a novel class of MITE-like repeat elements. There are also 26 highly diverse vapBC antitoxin–toxin gene pairs proposed to facilitate maintenance of local chromosomal regions and to minimise the impact of environmental stress. Complex and partially defective CRISPR/Cas/Cmr immune systems are present and interspersed with five vapBC gene pairs. Remnants of integrated viral genomes and plasmids are located at five intron-less tRNA genes and several non-coding RNA genes are predicted that are conserved in other Sulfolobus genomes. The putative metabolic pathways for sulphur metabolism show some significant differences from those proposed for other Acidianus and Sulfolobus species. The small and relatively stable genome of A. hospitalis W1 renders it a promising candidate for developing the first Acidianus genetic systems.     

Surface resistance to SSVs and SIRVs in pilin deletions of Sulfolobus islandicus

Characterizing the molecular interactions of viruses in natural microbial populations offers insights into virus–host dynamics in complex ecosystems. We identify the resistance of Sulfolobus islandicus to Sulfolobus spindle-shaped virus (SSV9) conferred by chromosomal deletions of pilin genes, pilA1 and pilA2 that are individually able to complement resistance. Mutants with deletions of both pilA1 and pilA2 or the prepilin peptidase, PibD, show the reduction in the number of pilins observed in TEM and reduced surface adherence but still adsorb SSV9. The proteinaceous outer S-layer proteins, SlaA and SlaB, are not required for adsorption nor infection demonstrating that the S-layer is not the primary receptor for SSV9 surface binding. Strains lacking both pilins are resistant to a broad panel of SSVs as well as a panel of unrelated S. islandicus rod-shaped viruses (SIRVs). Unlike SSV9, we show that pilA1 or pilA2 is required for SIRV8 adsorption. In sequenced Sulfolobus strains from around the globe, one copy of each pilA1 and pilA2 is maintained and show codon-level diversification, demonstrating their importance in nature. By characterizing the molecular interactions at the initiation of infection between S. islandicus and two different types of viruses we hope to increase the understanding of virus–host interactions in the archaeal domain.     

<Emphasis Type="Italic">Bacillus megaterium</Emphasis>—from simple soil bacterium to industrial protein production host

Bacillus megaterium has been industrially employed for more than 50 years, as it possesses some very useful and unusual enzymes and a high capacity for the production of exoenzymes. It is also a desirable cloning host for the production of intact proteins, as it does not possess external alkaline proteases and can stably maintain a variety of plasmid vectors. Genetic tools for this species include transducing phages and several hundred mutants covering the processes of biosynthesis, catabolism, division, sporulation, germination, antibiotic resistance, and recombination. The seven plasmids of B. megaterium strain QM B1551 contain several unusual metabolic genes that may be useful in bioremediation. Recently, several recombinant shuttle vectors carrying different strong inducible promoters and various combinations of affinity tags for simple protein purification have been constructed. Leader sequences-mediated export of affinity-tagged proteins into the growth medium was made possible. These plasmids are commercially available. For a broader application of B. megaterium in industry, sporulation and protease-deficient as well as UV-sensitive mutants were constructed. The genome sequence of two different strains, plasmidless DSM319 and QM B1551 carrying seven natural plasmids, is now available. These sequences allow for a systems biotechnology optimization of the production host B. megaterium. Altogether, a “toolbox” of hundreds of genetically characterized strains, genetic methods, vectors, hosts, and genomic sequences make B. megaterium an ideal organism for industrial, environmental, and experimental applications.     

Characterization of Two Compatible Small Plasmids from Sphingobium yanoikuyae

We isolated and characterized two small cryptic indigenous plasmids, pYAN-1 (4,896 bp) and pYAN-2 (4,687 bp), from Sphingobium yanoikuyae, and developed a versatile system that permitted genetic manipulation of the genus Sphingomonas. Nucleotide sequencing of both plasmids revealed that they contained mobA, mobs, and repA genes, which are predicted to encode proteins associated with mobilization and replication, in common. Transformation with each plasmid harboring the antibiotic resistance gene by electroporation was fully successful, using Novosphingobium capsulatum as a host.     

Living Side by Side with a Virus: Characterization of Two Novel Plasmids from Thermococcus prieurii,a Host for the Spindle-Shaped Virus TPV1

Microbial cells often serve as an evolutionary battlefield for different types of mobile genetic elements, such as viruses and plasmids. Here, we describe the isolation and characterization of two new archaeal plasmids which share the host with the spindle-shaped Thermococcus prieurii virus 1 (TPV1). The two plasmids, pTP1 and pTP2, were isolated from the hyperthermophilic archaeon Thermococcus prieurii (phylum Euryarchaeota), a resident of a deep-sea hydrothermal vent located at the East Pacific Rise at 2,700-m depth (7°25′24 S, 107°47′66 W). pTP1 (3.1 kb) and pTP2 (2.0 kb) are among the smallest known plasmids of hyperthermophilic archaea, and both are predicted to replicate via the rolling-circle mechanism. The two plasmids and the virus TPV1 do not have a single gene in common and stably propagate in infected cells without any apparent antagonistic effect on each other. The compatibility of the three genetic elements and the high copy number of pTP1 and pTP2 plasmids (50 copies/cell) might be useful for developing new genetic tools for studying hyperthermophilic euryarchaea and their viruses.     

Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the β-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus–E. coli plasmid shuttle vectors were constructed, which genetically complemented ΔpyrEFΔlacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.     

Characterization of the Sulfolobus host–SSV2 virus interaction

The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNA^Gly (CCC) gene. In this paper we report the findings of a comparative study of SSV2 physiology in the natural host, Sulfolobus islandicus REY15/4, versus the foreign host, Sulfolobus solfataricus, and provide evidence of differently regulated SSV2 life cycles in the two hosts. In fact, whereas a significant induction of SSV2 replication takes place during the growth of the natural host REY15/4, the cellular content of SSV2 DNA remains fairly low throughout the incubation of the foreign host. The accumulation of episomal DNA in the former case cannot be traced to decreased packaging activity because of a simultaneous increase in the virus titre in the medium. In addition, the interaction between SSV2 and its natural host is characterized by the concurrence of host growth inhibition and the induction of viral DNA replication. When this virus–host interaction was investigated using S. islandicus REY15A, a strain which is closely related to the natural host, it was found that the SSV2 replication process was induced in the same way as in the natural host REY15/4.     

Plasmid-borne resistance to arsenate, arsenite, cadmium, and chloramphenicol in a Rhodococcus species

Summary A primarily genetic approach was employed to obtain plasmids in Rhodococcus erythropolis ATCC 12674 which carried genes conferring increased resistance to sodium arsenate and arsenite, cadmium chloride, and chloramphenicol. The plasmids were large, migrating more slowly than chromosomal DNA in agarose gels, and were made up of resistance determinants from the host organism together with part of the genome of nocardiophage Q4. Purified plasmid was used to transform a suitable recipient to increased resistance to sodium arsenate, sodium arsenite, and cadmium chloride.     

Recent advances in genetic analyses of hyperthermophilic Archaea and Bacteria

Hyperthermophilic Archaea and Bacteria are an extraordinarily important class of organisms for which genetic tools remain to be developed. Unique technological obstacles to this goal are posed by the thermophilic and, in some cases, strictly anaerobic nature of these organisms. However, recent advances in the cultivation of hyperthermophiles, in the discovery of genetic elements for vector development, and in the construction of genetic markers point toward the achievement of this goal in the near future. Transformation protocols have already been reported for Sulfolobus and Pyrococcus, and plasmid-mediated conjugation was recently found in Sulfolobus. Plasmids are available for Sulfolobus, Pyrococcus, and the bacterial hyperthermophile Thermotoga, and these provide the bases for vector construction in these hosts. A Desulfurococcus mobile intron may provide a novel means to introduce genes into a variety of archaeal hosts. With full genome sequences of several hyperthermophiles available soon, genetic tools will allow full exploitation of this information to study these organisms in depth and to utilize their unique properties in biotechnological applications. Received: 27 January 1997 / Accepted: 24 April 1997     

Intra- and Interbacterial Genetic Exchange of Lyme Disease Spirochete <Emphasis Type="Italic">erp</Emphasis> Genes Generates Sequence Identity Amidst Diversity

All isolates of the spirochete Borrelia burgdorferi contain multiple, different plasmids of the cp32 family, each of which contains a locus encoding Erp surface proteins. Many of these proteins are known to bind host complement regulatory factor H, enabling the bacteria to avoid killing by the alternative complement pathway during vertebrate infection. In the present study, we characterized the erp loci and cp32 plasmids of strains N40, Sh-2-82, and 297 and compared them to the previously determined cp32 sequences of type strain B31. Bacteria of strain N40 contain 6 different cp32s, those of Sh-2-82 contain 10, and 297 bacteria contain 9 cp32s. Significant conservation between all strains was noted for the cp32 loci responsible for plasmid maintenance, indicating close relationships that appear to correspond with incompatibility groups. In contrast, considerable diversity was found between erp gene sequences, both within individual bacteria and between different strains. However, examples of identities among erp loci were found, with strains Sh-2-82, 297, and B31 each containing three identical loci that likely arose through intrabacterial genetic rearrangements. These studies also found the first evidence of large-scale genetic exchanges between Lyme disease spirochetes in nature, including the apparent transfer of an entire cp32 plasmid between two different bacteria.     

Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti

The Class II transposable element, piggyBac, was used to transform the yellow fever mosquito, Aedes aegypti. In two transformed lines only 15–30 of progeny inherited the transgene, with these individuals displaying mosaic expression of the EGFP marker gene. Southern analyses, gene amplification of genomic DNA, and plasmid rescue experiments provided evidence that these lines contained a high copy number of piggyBac transformation constructs and that much of this DNA consisted of both donor and helper plasmids. A detailed analysis of one line showed that the majority of piggyBac sequences were unit-length donor or helper plasmids arranged in a large tandem array that could be lost en masse in a single generation. Despite the presence of a transposase source and many intact donor elements, no conservative (cut and paste) transposition of piggyBac was observed in these lines. These results reveal one possible outcome of uncontrolled and/or unexpected recombination in this mosquito, and support the conclusion that further investigation is necessary before transposable elements such as piggyBac can be used as genetic drive mechanisms to move pathogen-resistance genes into mosquito populations.     

Construction of a bifunctional genetically labelled plasmid for Bacillus thuringiensis subsp. israelensis

A small cryptic plasmid of Bacillus thuringiensis subsp. israelensis was labelled in vitro with two genetic markers. One of the recombinant plasmids was mapped and transformed in Escherichia coli, Bacillus subtilis and Bacillus thuringiensis. This and similar shuttle plasmids could be very useful as vectors for the investigation of the toxin genes in their own host.Abbreviations BTI Bacillus thuringiensis subsp. israelensis - MDal megadaltons     

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we speculate that Mavericks represent an evolutionary missing link between seemingly disparate invasive DNA elements that include bacteriophages, adenoviruses and eukaryotic linear plasmids.     

Excision of the <Emphasis Type="Italic">nifD</Emphasis> element in the heterocystous cyanobacteria

Heterocyst differentiation in cyanobacteria is accompanied by developmentally regulated DNA rearrangements that occur within the nifD, fdxN, and hupL genes. These genetic elements are excised from the genome by site-specific recombination during the latter stages of differentiation. The nifD element is excised by the recombinase, XisA, located within the element. Our objective was to examine the XisA-mediated excision of the nifD element. To accomplish this, we observed the ability of XisA to excise substrate plasmids that contained the flanking regions of the nifD element in an E. coli host. Using PCR directed mutagenesis, nucleotides in the nifD element flanking regions in substrate plasmids were altered and the effect on recombination was determined. Results indicate that only certain nucleotides within and surrounding the direct repeats are involved in excision. In some nucleotide positions, the presence of a purine versus a pyrimidine greatly affected recombination. Our results also indicated that the site of excision and branch migration occurs in a 6 bp region within the direct repeats. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of the DING protein from the archaeon Sulfolobus solfataricus with human phosphate-binding protein and Pseudomonas fluorescence DING counterparts

DING proteins represent a new group of 40 kDa-related members, ubiquitous in living organisms. The family also include the DING protein from Sulfolobus solfataricus, functionally related to poly(ADP-ribose) polymerases. Here, the archaeal protein has been compared with the human Phosphate-Binding Protein and the Pseudomonas fluorescence DING enzyme, by enzyme assays and immune cross-reactivity. Surprisingly, as the Sulfolobus enzyme, the Human and Pseudomonas proteins display poly(ADP-ribose) polymerase activity, whereas a phosphatase activity was only present in Sulfolobus and human protein, despite the conserved phosphate-binding site residues in Pseudomonas DING. All proteins were positive to anti-DING antibodies and gave a comparable pattern of anti-poly(ADP-ribose) polymerase immunoreactivity with two bands, at around 40 kDa and roughly at the double of this molecular mass. The latter signal was present in all Sulfolobus enzyme preparations and proved not due to either a contaminant or a precursor protein, but likely being a dimeric form of the 40 kDa polypeptide. The common immunological and partly enzymatic behavior linking human, Pseudomonas and Sulfolobus DING proteins, makes the archaeal protein an important model system to investigate DING protein function and evolution within the cell.