Characterization of pHV2 from Halobacterium volcanii and its use in demonstrating transformation of an archaebacterium.

Abstract

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...

Mutations in DNA gyrase result in novobiocin resistance in halophilic archaebacteria

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Enhanced production of histamine dehydrogenase by <i>Natrinema gari</i> BCC 24369 in a non-sterile condition

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Transduction in the archaebacterium Methanobacterium thermoautotrophicum Marburg

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