JOURNAL OF BACTERIOLOGY, Feb. 1990, p. 756-761 Vol. 172, No. 2 0021-9193/90/020756-06$02.00/0 Copyright © 1990, American Society for Microbiology A Plasmid Vector with a Selectable Marker for Halophilic Archaebacteria MELISSA L. HOLMES* AND MICHAEL L. DYALL-SMITH Department of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia Received 25 May 1989/Accepted 10 November 1989 A mutant resistant to the gyrase inhibitor novobiocin was selected from a halophilic archaebacterium belonging to the genus Haloferax. Chromosomal DNA from this mutant was able to transform wild-type ceils to novobiocin resistance, and these transformants formed visible colonies in 3 to 4 days on selective plates. The resistance gene was isolated on a 6.7-kilobase DNA KpnI fragment, which was inserted into a cryptic multicopy plasmid (pHK2) derived from the same host strain. The recombinant plasmid transformed wild-type cells at a high efficiency (>106/pg), was stably maintained, and could readily be reisolated from transformants. It could also transform Halobacterium volcanii and appears to be a useful system for genetic analysis in halophilic archaebacteria. Archaebacteria are phylogenetically distinct from eubac- cle was the lack of selectable traits, such as antibiotic teria and eucaryotes and can be broadly divided into three resistance. groups: methanogens, sulfur-dependent thermoacidophiles, The aim of this study was to find a selectable marker for and extreme halophiles (for a review, see reference 22). use in constructing plasmid vectors suitable for isolating and Numerous halobacteria have been isolated from hypersaline studying genes in halobacteria. We report the successful environments around the world, but it is only recently that construction of such a vector by using a gene conferring their taxonomy has been analyzed in a rigorous and compre- resistance to novobiocin and a cryptic plasmid from Halo- hensive fashion. The studies of Tindall et al. (20) and ferax isolate Aa 2.2. Torreblanca et al. (21) identified six genera: Halobacterium, Halococcus, Haloarcula, Haloferax, Natronobacterium, MATERIALS AND METHODS and Natronococcus. Bacterial strains. The following halobacterial strains were Genetic analysis of halobacteria has been complicated in used in this study: Haloferax phenon K isolate Aa 2.2 from strains such as Halobacterium halobium by the high rate of M. Torreblanca, University of Alicante, Alicante, Spain; spontaneous mutation due to insertion sequences and be- Halobacterium (now Haloferax) volcanii NCMB2012 and cause H. halobium strains have complex nutrient require- Halobacterium halobium NCMB777 from the National Col- ments (15). However, there are halobacteria with low muta- lection of Marine Bacteria, Aberdeen, Scotland; and Halo- tion rates that grow in a mineral salts medium containing a bacterium halobium DSM617 and strain RI from R. Schin- single carbon source (16), and these are obvious choices for zel, Wurzburg, Federal Republic of Germany. studying gene structure and function if only suitable gene Escherichia coli XL1-Blue, used in all E. coli transforma- transfer systems, such as transformation or transduction, tion experiments, was obtained from Stratagene, La Jolla, were available. Fortunately, an efficient DNA transforma- California. tion method was recently described by Cline and Doolittle Media and growth conditions. A 25% (wt/vol) solution of (4), using both H. halobium and Halobacterium volcanii (3), artificial salt water (SW) was prepared by using a slightly which opens the way to methods of contemporary genetic modified formulation of that described by Rodriguez-Valera analysis. Plasmid transformation was possible, but with no et al. and contained liter: 197 g of NaCl, 17 g of selectable marker, the detection of transformants was ex- (16) per MgCl2 .6H20, 26 g of MgSO4 - 7H20, 0.1 g of CaCI2, 5 g of tremely laborious (3). To date, the transformation of other KCI, and 5 g of NH4Cl. All Haloferax media were prepared archaebacteria is either extremely inefficient, such as in by using this stock solution. Growth medium (GM) con- methanogens (1), or not currently possible, as is the case tained SW (18%, wt/vol), yeast extract (0.5%, wt/vol; Oxoid with the sulphur-dependent thermoacidophiles; so, of the Ltd., London, England) and 25 mM Tris hydrochloride three main branches of archaebacteria, the halobacteria buffer (pH 7.5). Solid media were prepared by adding show the most promise for genetic research. Bacto-Agar (15 g/liter; Difco Laboratories, Detroit, Mich.). Members of the genus Haloferax are aerobic chemoorgan- Cultures were incubated at 37°C in an orbital shaker (liquid otrophs, only moderately halophilic, requiring 2.0 to 2.5 M media) or in plastic containers (solid media). NaCl for optimum growth, and are capable of growth on Haloferax transformation medium (TM) contained SW single carbon sources. We chose to use an isolate (Aa 2.2) (18%, wt/vol), sucrose (15%, wt/vol), MnCl2 (1.7 ,uM), Tris belonging to phenon K (21). It grew rapidly in liquid and on hydrochloride (25 mM, pH 7.5), yeast extract (0.3%, wt/vol) solid media, had a low frequency of spontaneous mutation, and tryptone (Oxoid; 0.5%, wt/vol). Novobiocin was in- and contained a small multicopy plasmid. These were con- cluded in GM and TM at 0.1 ,ug/ml when required. sidered to be useful properties for constructing a model All solutions and growth media for H. halobium and H. genetic system in the archaebacteria, but a remaining obsta- volcanii were made up as previously described (3, 4). For the H. halobium strains, novobiocin was included in selective media at 0.2 ,ug/ml. * Corresponding author. E. coli XL1-Blue was grown at 37°C in YT medium (9) 756 VOL. 172, 1990 PLASMID VECTOR WITH SELECTABLE MARKER FOR HALOBACTERIA 757 containing tetracycline (15 ,ug/ml) or on YT-TAXI plates, chasing sequencing reactions with terminal deoxynucleoti- which contained YT medium supplemented with tetracycline dyl transferase (Boehringer Mannheim) (5). (15 ,ug/ml), ampicillin (50 ,ug/ml), 5-bromo-4-chloro-3-in- Plasmid map determination. The 10.5-kilobase (kb) cryptic dolyl-p-D-galactopyranoside (50 ,ug/ml), isopropyl-13-D-thio- plasmid ofHaloferax strain Aa 2.2, pHK2, was isolated from galactopyranoside (0.1 mM), and Bacto-Agar (15 g/liter). an agarose gel by electrophoresis onto a dialysis membrane Transformation of bacteria. Haloferax transformations (9), linearized with HindlIl, and cloned into the E. coli were performed by using the polyethylene glycol (PEG) plasmid pUC18 (23) by using T4 DNA ligase (Bresatec, Ltd., method described by Charlebois et al. (3). Freshly inocu- Adelaide, South Australia). A physical map was determined lated cultures were shaken at 37°C until the cells had reached by restricting 0.5 to 1 ,ug of DNA with various restriction a density (A550) of 1 to 1.5 (corresponding to a viable count endonucleases (both single and double digests) and resolving at 1 x 109 to 2 x 109 CFU/ml) and were then centrifuged the fragments on agarose or polyacrylamide gels. Sizes of (4,000 x g, 10 min) and suspended in a 1/10 volume of fragments were then estimated by using HindIlI-cut lambda buffered spheroplasting solution. The spheroplasting solu- DNA or 1-kilobase-pair-ladder DNA standards (Boehringer tion had reduced NaCl (1 M) but was otherwise the same as Mannheim). described previously (3). After PEG treatment, spheroplasts As some of the Sall and AvaI fragments were rather small were diluted in a solution containing SW (18%, wt/vol), and difficult to place, the nuclease digestion method (9) was sucrose (15%, wt/vol), and Tris hydrochloride (20 mM, pH also used. HindIII-cut pUC18-pHK2 (10 ,ug) was incubated 7.5) and allowed to recover for 5 to 12 h at 37°C, and then with Bal3l nuclease (New England BioLabs, Inc., Beverly, 100-1.l samples were spread onto TM plates containing Mass.; 0.75 U) prior to restriction with Sall or AvaI. novobiocin (0.1 ,ugIml) and incubated for 3 to 5 days at 37°C Samples were removed at various time intervals and ana- in plastic containers. lyzed on 10% polyacrylamide gels. The progressive disap- For transforming H. volcanii and H. halobium strains, we pearance of DNA bands then helped to position the restric- followed exactly the procedures described by Cline and tion fragments within pHK2. Doolittle (4) and Charlebois et al. (3). Determination of plasmid copy number. The copy number Isolation of plasmid DNA. For large-scale plasmid isolation of pHK2 (number of plasmids per cell) was estimated by from Haloferax strain Aa 2.2, DNA was isolated from carefully extracting total DNA from mid-log-phase cultures late-log-phase cultures by the following procedure. Cells of Haloferax cells grown in GM, running serial dilutions of were harvested by centrifugation at 2,800 x g for 15 min and the DNA preparation on an agarose gel (0.5%, wt/vol), and were washed once with 1 M NaCl. The cells were then comparing intensities of DNA bands stained with ethidium suspended in a buffer containing 1 M NaCl and 0.1 M EDTA bromide to those of DNA standards of known concentra- and lysed by the addition of 0.007 M sodium deoxycholate. tions. The number of viable cells used in the DNA prepara- This solution was left on ice for 30 min before spinning down tion was determined by plating dilutions onto GM plates, cellular debris and chromosomal DNA at 28,000 x g for 30 incubating for 2 to 3 days at 37°C, and counting colonies. min. The cleared lysate was transferred to a clean tube, and Selection of novobiocin-resistant Haloferax mutants. The DNA was precipitated by the addition of 10% (wt/vol) PEG MIC of novobiocin was first determined and found to be 6000 and incubation on ice for 60 min.