Expanding and understanding the genetic toolbox of the hyperthermophilic genus <i>Sulfolobus</i>

Wagner, Michaela; Berkner, Silvia; Ajon, Małgorzata; Driessen, Arnold J. M.; Lipps, Georg; Albers, Sonja‐Verena

doi:10.1042/bst0370097

Cited by 76 publications

(64 citation statements)

References 18 publications

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“…To inactivate the Saci_1271 and Saci_1272 genes named sulA and sulB, genomic DNA was isolated with a QuickPick SML genomic DNA (gDNA) kit (Bio-Nobile, Turku, Finland) from the S. acidocaldarius ⌬pyrEF strain MR31 (19). A fragment of 923 bp containing a portion of the upstream region and of sulB was amplified using the forward primer ForSulB, which contained a SacII restriction site, and the reverse primer RevSulB, which contained a PstI restriction site (all primer sequences are shown in Table S1 in (25). S. acidocaldarius strain MW001 (M. Wagner and S. V. Albers, unpublished results) was transformed with the resulting plasmid, and the sulAB disruption mutant was isolated as described previously (8).…”

Section: Methodsmentioning

confidence: 99%

The Sulfolobicin Genes of Sulfolobus acidocaldariusEncode Novel Antimicrobial Proteins

et al. 2011

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Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.

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Section: Methodsmentioning

confidence: 99%

The Sulfolobicin Genes of Sulfolobus acidocaldariusEncode Novel Antimicrobial Proteins

et al. 2011

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“…Evidence of both reciprocal and non-reciprocal pathways of HR in Sulfolobus species has been reported: transformation of S. acidocaldarius by short oligonucleotides (Grogan & Stengel, 2008;Mao & Grogan, 2012), implies a non-reciprocal (geneconversion) pathway on mechanistic grounds, whereas integration of circular DNAs targeted by inserts of host sequence (Wagner et al, 2009) implies reciprocal strand exchange (crossing over) via the well-studied DSBR pathway (Allers & Lichten, 2001;Aylon & Kupiec, 2004;Haber et al, 2004). In attempts to detect integration via crossing over in S. acidocaldarius, we constructed an E. coli plasmid bearing a heterologous selectable marker (pyrE of S. solfataricus) separated from a non-essential region of the S. acidocaldarius chromosome providing a site for integration.…”

Section: Circularity and Orientation Of Donor Sequencesmentioning

confidence: 99%

Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications

2013

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Although homologous recombination (HR) is known to influence the structure, stability, and evolution of microbial genomes, few of its functional properties have been measured in cells of hyperthermophilic archaea. The present study manipulated various properties of the parental DNAs in high-resolution assays of Sulfolobus acidocaldarius transformation, and measured the impact on the efficiency and pattern of marker transfer to the recipient chromosome. The relative orientation of homologous sequences, the type and position of chromosomal mutation being replaced, and the length of DNA flanking the marked region all affected the efficiency, linkage, tract continuity, and other parameters of marker transfer. Effects predicted specifically by the classical reciprocal-exchange model of HR were not observed. One analysis observed only 90 % linkage between markers defined by adjacent bases; in another series of experiments, sequence divergence up to 4 % had no detectable impact on overall efficiency of HR or on the co-transfer of a distal non-selected marker. The effects of introducing DNA via conjugation, rather than transformation, were more difficult to assess, but appeared to increase co-transfer (i.e. linkage) of relatively distant non-selected markers. The results indicate that HR events between gene-sized duplex DNAs and the S. acidocaldarius chromosome typically involve neither crossing over nor interference from a mismatch-activated anti-recombination system. Instead, the donor DNA may anneal to a transient chromosomal gap, as in the mechanism proposed for oligonucleotidemediated transformation of Sulfolobus and other micro-organisms.

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“…To date, genetic systems of various levels of sophistication have been developed for representatives of all major groups of archaea, including halophiles, methanogens, thermoacidophiles, and hyperthermophiles (2,6,30,40,43,46). A variety of transformation methods are being used, including electroporation, heat shock with or without CaCl 2 treatment, phage-mediated transduction, spheroplast transformation, liposomes, and, very recently, even conjugation with Escherichia coli (2, 12).…”

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confidence: 99%

Natural Competence in the Hyperthermophilic ArchaeonPyrococcus furiosusFacilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases

Lipscomb

Stirrett

Schut

et al. 2011

Appl Environ Microbiol

158

251

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In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single-and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.It would be difficult to overestimate the contribution of genetic manipulation to the study of any biological system, and it is an essential tool for the metabolic engineering of biosynthetic and substrate utilization pathways. This is particularly true for the archaea since, in spite of their environmental and industrial importance, coupled with their unique molecular features, much remains to be learned about their biology (2). The marine hyperthermophilic anaerobe Pyrococcus furiosus is of special interest not only for its ability to grow optimally at 100°C and the implications of this trait for its biology but also for industrial applications of its enzymes, as well as its capacity to produce hydrogen efficiently (4, 13, 44). The ability to apply genetic analyses of P. furiosus to underpin existing biochemical and molecular studies will contribute greatly to the establishment of P. furiosus as a model organism, particularly for biological hydrogen production.The development of genetic systems in the archaea, in general, presents many unique challenges given the extreme growth requirements of many of these organisms. To date, genetic systems of various levels of sophistication have been developed for representatives of all major groups of archaea, including halophiles, methanogens, thermoacidophiles, and hyperthermophiles (2,6,30,40,43,46). A variety of transformation methods are being used, including electroporation, heat shock with or without CaCl 2 treatment, phage-mediated transduction, spheroplast transformation, liposomes, and, very recently, even conjugation with Escherichia coli (2, 12). Transformation via natural competence has been reported in three archaeal species, in comparison to over 60 bacterial species that are known to exhibit this trait (16,36). Two of them are the methanogens Methanococcus voltae PS (7, 27) and Methanobacterium thermoautotrophicum Marburg (47); however, transformation frequencies were low, and there have been no follow-up studies regarding natural competence. The other is the hyperthermophile Thermococcus kodakarensis, which has an optimal growth temperature of 85°C. Its natural competence has enabled the development of genetic tools fo...

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Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus

Cited by 76 publications

References 18 publications

The Sulfolobicin Genes of Sulfolobus acidocaldariusEncode Novel Antimicrobial Proteins

The Sulfolobicin Genes of Sulfolobus acidocaldariusEncode Novel Antimicrobial Proteins

Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications

Natural Competence in the Hyperthermophilic ArchaeonPyrococcus furiosusFacilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases

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