Dataset Information

The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein.

ABSTRACT: The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetrical binding sites separated by approximately 60 bp were found upstream of the orf80 gene. Both binding sites contain two TTAA motifs as well as other conserved bases. Fluorescence measurements show that short duplex DNAs derived from a single binding site sequence are bound with submicromolar affinity and moderate cooperativity by ORF80. On DNA fragments carrying both binding sites, a rather large protein-DNA complex is formed in a highly cooperative manner. ORF80 contains an N-terminal leucine zipper motif and a highly basic domain at its C-terminus. Compared to all known basic leucine zipper proteins the order of the domains is reversed in ORF80. ORF80 may therefore constitute a new subclass of basic leucine zipper DNA-binding proteins.

SUBMITTER: Lipps G

PROVIDER: S-EPMC97583 | biostudies-literature | 2001 Dec

REPOSITORIES: biostudies-literature

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The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein.

Lipps G G Ibanez P P Stroessenreuther T T Hekimian K K Krauss G G

Nucleic acids research 20011201 24

The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetri ...[more]

PMID: 11812827

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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.

Project description:EndoMS is a recently identified mismatch specific endonuclease in Thermococcales of Archaea and Mycobacteria of Bacteria. The homologs of EndoMS are conserved in Archaea and Actinobacteria, where classic MutS-MutL-mediated DNA mismatch repair pathway is absent or non-functional. Here, we report a study on the in vitro mismatch cleavage activity and in vivo function of an EndoMS homolog (SisEndoMS) from Sulfolobus islandicus REY15A, the model archaeon belonging to Crenarchaeota. SisEndoMS is highly active on duplex DNA containing G/T, G/G, and T/T mismatches. Interestingly, the cleavage activity of SisEndoMS is stimulated by the heterotrimeric PCNAs, and when Mn2+ was used as the co-factor instead of Mg2+, SisEndoMS was also active on DNA substrates containing C/T or A/G mismatches, suggesting that the endonuclease activity can be regulated by ion co-factors and accessory proteins. We compared the spontaneous mutation rate of the wild type strain REY15A and ∆endoMS by counter selection against 5-fluoroorotic acid (5-FOA). The endoMS knockout mutant had much higher spontaneous mutation rate (5.06 × 10-3) than that of the wild type (4.6 × 10-6). A mutation accumulation analysis also showed that the deletion mutant had a higher mutation occurrence than the wild type, with transition mutation being the dominant, suggesting that SisEndoMS is responsible for mutation avoidance in this hyperthermophilic archaeon. Overexpression of the wild type SisEndoMS in S. islandicus resulted in retarded growth and abnormal cell morphology, similar to strains overexpressing Hje and Hjc, the Holliday junction endonucleases. Transcriptomic analysis revealed that SisEndoMS overexpression led to upregulation of distinct gene including the CRISPR-Cas IIIB system, methyltransferases, and glycosyltransferases, which are mainly localized to specific regions in the chromosome. Collectively, our results support that EndoMS proteins represent a noncanonical DNA repair pathway in Archaea. The mechanism of the mismatch repair pathway in Sulfolobus which have a single chromosome is discussed.

Project description:UnlabelledSulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicusImportanceCurrently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains.

Dataset Information

The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein.

Publications

The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein.

Similar Datasets

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