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Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein.

ABSTRACT: There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of -6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85 degrees C.

SUBMITTER: Lipps G

PROVIDER: S-EPMC29613 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein.

Lipps G G Stegert M M Krauss G G

Nucleic acids research 20010201 4

There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichi ...[more]

PMID: 11160922

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Project description:We investigated the interaction between Sulfolobus spindle-shaped virus (SSV9) and its native archaeal host Sulfolobus islandicus. We show that upon exposure to SSV9, S. islandicus strain RJW002 has a significant growth delay where the majority of cells are dormant (viable but not growing) for 24 to 48 hours postinfection (hpi) compared to the growth of controls without virus. We demonstrate that in this system, dormancy (i) is induced by both active and inactive virus particles at a low multiplicity of infection (MOI), (ii) is reversible in strains with active CRISPR-Cas immunity that prevents the establishment of productive infections, and (iii) results in dramatic and rapid host death if virus persists in the culture even at low levels. Our results add a new dimension to evolutionary models of virus-host interactions, showing that the mere presence of a virus induces host cell stasis and death independent of infection. This novel, highly sensitive, and risky bet-hedging antiviral response must be integrated into models of virus-host interactions in this system so that the true ecological impact of viruses can be predicted and understood. Viruses of microbes play key roles in microbial ecology; however, our understanding of viral impact on host physiology is based on a few model bacteria that represent a small fraction of the life history strategies employed by hosts or viruses across the three domains that encompass the microbial world. We have demonstrated that rare and even inactive viruses induce dormancy in the model archaeon S. islandicus. Similar virus-induced dormancy strategies in other microbial systems may help to explain several confounding observations in other systems, including the surprising abundance of dormant cell types found in many microbial environments, the difficulty of culturing microorganisms in the laboratory, and the paradoxical virus-to-host abundances that do not match model predictions. A more accurate grasp of virus-host interactions will expand our understanding of the impact of viruses in microbial ecology.

Dataset Information

Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein.

Publications

Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein.

Similar Datasets

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