We present a practical description of polyethylene glycol mediated spheroplast transformation of Halobacterium halobium and Halobacterium volcanii. This method has been applied to phage DNA transfection, plasmid DNA transformation, and transformation with linear fragments of high molecular weight genomic DNA. Efficient spheroplast regeneration allows uncomplicated recovery of transformed progeny. Transformations can be performed equally well using fresh or frozen cell preparations. These methods should find application in molecular cloning, genetic fine mapping, and strain construction.

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

We determined the complete nucleotide sequence of the 6354-base-pair plasmid pHV2 of the archaebacterium Halobacterium vokcanii. This plasmid is present in approximately six copies per chromosome. We have generated a strain, H. volcanii WFD11, cured of pHV2 by treatment of liquid cultures with ethidium bromide. We describe PEGmediated transformation of H. volcanii WFD11 with intact pHV2 and with a form of pHV2 marked by a 93-base-pair deletion generated in vitro.Since the recognition that the archaebacteria are a form of life distinct from both eubacteria and eukaryotes (1, 2), a considerable amount of research has focused on elucidating the biological and biochemical characteristics of this ancient lineage (for reviews see refs. 3 and 4). Although there has been some progress in understanding the molecular genetics ofarchaebacteria, this work has been hampered until recently by the inability to introduce exogenous DNA into cells. We have demonstrated transfection of halobacteria (5) with naked DNA from OH, a natural bacteriophage of Halobacterium halobium (6). In addition, Bertani and Baresi (7) have observed low-frequency transformation of Methanococcus voltae histidine and purine auxotrophs to prototrophy, using total wild-type DNA. However, there remained a need to obtain transformation with a plasmid as a first step toward the construction of vectors to shuttle between an archaebacterium and Escherichia coli.The halobacteria have, for purposes of research in molecular biology, an advantage over other groups of archaebacteria in that they can be maintained and manipulated in the laboratory easily, by methods similar to those for E. coli. Mevarech and Werczberger (8) (5). E. coli, strain JM101, was grown and maintained according to standard procedures (13).Preparation of DNA. Total DNA was isolated from halobacteria by quickly lysing resuspended cell pellets in 50 mM Tris'HCI, pH 8/50 mM EDTA/0.2% N-lauroylsarcosine, followed by digestion with proteinase K at 0.1 mg/ml for several hours with gentle shaking at 37°C, before phenol extraction and ethanol precipitation. The 6.4-kilobase-pair (kbp) plasmid pHV2 (14) was prepared from H. volcanii DS2 using the following methods: (i) CsCl/ethidium bromide buoyant density ultracentrifugation (14), (ii) alkaline extraction (method B without column purification of ref. 15), or (iii) rate zonal centrifugation on a sucrose density gradient (16). Phage 4OH was prepared as described (6). Plasmids and M13 RF forms from E. coli JM101 were prepared by alkaline extraction. The single-stranded M13 DNA was isolated and purified for sequencing as described by Messing (13).Cloning and Sequencing. The 6.4-kbp plasmid pHV2 of H. volcanii DS2 cloned at its unique Pst I site into a pUC plasmid vector was kindly provided by C. J. Daniels (Ohio State University). Ordered series of deletions for each strand of three restriction fragments subcloned into various pUC or M13mp series vectors (17) were prepared using BAL-31 (18) or exonuclease III followed by nuclease S1 (19). Deleted...

We developed an efficient polyethylene glycol-mediated spheroplast transfection method for the extremely halophilic archaebacterium Halobacterium halobium. The 59-kilobase-pair linear phage IH DNA molecule routinely produced between 5 X 106 and 2 x 107 transfectants per ,ug of DNA. Between 0.5 and 1% of spheroplasts were transfected per ,Ig of OH DNA. Under our conditions, survival and regeneration of H. halobium spheroplasts were also quite efficient, suggesting that this method will be useful for introducing other DNAs into these bacteria.

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