Transformation of the archaebacterium Halobacterium volcanii with genomic DNA

Abstract

We describe optimization of a transformation system for the halophilic archaebacterium Halobacterium volcanii. Transformation of spheroplasts in the presence of polyethylene glycol permits the uptake and expression of high-molecular-weight linear fragments of genomic DNA as well as plasmid or bacteriophage DNA. Transformations can be performed with either fresh or frozen cell preparations. Auxotrophic mutants were transformed to prototrophy with genomic DNA from wild-type cells with efficiencies of 5 x 104/,ug of DNA and frequencies of 8 x 10-5 per regenerated spheroplast. The overall efficiency of transformation with genomic DNA implies that genetic recombination is an efficient process in H. volcanii.The recent development of broadly useful genetic tools for studying the halobacteria promises to yield new insights into the molecular biology of the archaebacterial kingdom. A mating system (13) and techniques for protoplast fusion (16) have been described for Halobacterium volcanii, while bacteriophage DNA transfection (3, 5) and plasmid transformation (3, 9) have been developed for both H. volcanii and Halobacterium halobium. Most recently, selectable shuttle vectors which can be maintained in either H. volcanii or Escherichia coli have been constructed (W. L. Lam and W. F. Doolittle, Proc. Natl. Acad. Sci. USA, in press).Long-standing interest in bacteriorhodopsin, the membrane-bound light-driven proton pump of H. halobium, suggests that these new genetic techniques will find considerable application with the purple membrane-producing halophiles. As a model organism for general genetic studies of halophilic archaebacteria, however, H. volcanii perhaps shows even greater promise. The ability of H. volcanii to grow on simple, defined media has enabled Mevarech and co-workers to generate a variety of auxotrophic mutants (13,17). In addition, Charlebois and co-workers have made substantial progress in preparing a physical map of the H. volcanii genome by ordering a library of overlapping cosmid clones (2).With eubacteria, transformation with linear fragments of chromosomal DNA has long been a basic technique for genetic mapping and an invaluable tool for strain construction. Here we describe transformation of H. volcanii auxotrophs to prototrophy with wild-type chromosomal DNA and establish conditions under which transformation, even of previously frozen cells, occurs at high efficiency. MATERIALS AND METHODSStrains, phage, media, and growth conditions. WFD11, the strain of H. volcanii (synonymous with Haloferax volcanii) used here as wild type, is a derivative of strain DS2 which has been cured of the plasmid pHV2 (3). Auxotrophic H. volcanii strains (17) were from M. Mevarech. H. halobium R1 (20) and its phage XH1 (19) were from W. Zillig.The H. volcanii minimal medium of Kauri and Kushner

Natrinema versiforme sp. nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China.

“…Sequencing of 16S rDNA. Total DNAs were extracted by the method of Cline et al (1989). The 16S rRNA genes were amplified by PCR with the following forward and reverse primers : 5h-TCCGGTTGATCCTGCCG (positions 8-24 according to Escherichia coli numbering) and 5h-GGAGG-TGATCCAGCCG (positions 1540-1525).…”

Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea

“…Hf. volcanii WFD11, lacking the endogenous plasmid pHV2 (Cline et al, 1989), was grown in rich medium containing (l − ") : 175 g NaCl, 37 g MgSO % ;7 H # O, 3n7 g KCl, 5 g Bacto tryptone, 3 g Bacto yeast extract, 25 ml 1 M Tris\HCl pH 7n2, 5 ml 10 % CaCl # ;H # O and 100 µl 100 µM MnCl # . Transformants were selected on agar plates containing 0n2 µg novobiocin ml − " and\or 6 µg mevinolin ml − ".…”

Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

“…Both fragments were fused by PCR, generating an in-frame deletion fragment that was ligated into pTA131 (4) followed by transformation in E. coli XL1-Blue MRF=. After plasmid preparation from Escherichia coli and sequencing, each plasmid was transformed in H. volcanii H26 (17,18). Growth in uracil-free medium was used to select for clones that had integrated the vector into their genome via homologous recombination at the indicated locus (pop-in).…”