Department of Microbiology and Immunology, University of Melbourne, Melbourne, VIC 3010, Australia1
Departamento de Genetica Medica y Biologia Molecular, Centro Clinico San Carlos, Alicante, Spain2
Author for correspondence: D. Wendoloski. Tel: +61 3 8344 5693. Fax: +61 3 8347 1540. e-mail: d.wendoloski{at}pgrad.unimelb.edu.au
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ABSTRACT |
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Keywords: HMG-CoA reductase, selectable marker gene, mevinolin, halobacteria, Archaea
Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; wt, wild type
The GenBank accession number for the sequence reported in this paper is AF123438.
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INTRODUCTION |
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Mevinolin (Lovastatin, Merck) and its relatives, fluvastatin, pravastatin, and simvastatin, competitively inhibit HMG-CoA reductase, an enzyme found in Eucarya, Archaea and some Bacteria, and used to synthesize mevalonic acid from acetyl-CoA (see Cabrera et al., 1986 ; Lam & Doolittle, 1989
, and references therein). In humans, these drugs help lower cholesterol, but in Archaea they can completely halt growth as they block production of isoprenoid lipids (Cabrera et al., 1986
), the major lipid in the cell membrane. Resistance in Haloferax volcanii arises from overproduction of the enzyme, and an up-promoter mutation in hmgA has been described and the gene used to construct the first halobacterial shuttle vectors (e.g. pWL102; Lam & Doolittle, 1989
, 1992
).
The mevinolin-resistance determinant, hmg, as well as the other two resistance determinants, were originally isolated from the chromosome of resistant mutants of Haloferax spp., and since this host is recombination proficient, homologous recombination events are possible when vectors containing these genes are introduced into Hfx. volcanii (e.g. Lam & Doolittle, 1989 ; Dyall-Smith & Doolittle, 1994
). This can severely compromise genetic strategies that rely on selection for drug resistance. Hfx. volcanii is a preferred host for the genetic study of halobacteria, and while a recombination-deficient (radA) mutant of this host has been isolated, it is slow growing and is unable to maintain the replication of certain plasmids, e.g. pWL102 (Woods & Dyall-Smith, 1997
). In order to lower the recombination rate between the Haloferax chromosome and introduced vector plasmids, we sought a selectable marker that showed less sequence similarity to the Hfx. volcanii genome. We report here the isolation, cloning, sequence and use in Hfx. volcanii of a mevinolin-resistance marker from Haloarcula hispanica.
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METHODS |
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Plasmids and DNA isolation and analysis.
All the plasmids referred to (pMDS95, 99, 100 and 108) are based on pOK12 (Vieira & Messing, 1991 ). Plasmids were isolated from E. coli strains using the alkaline lysis method as described by Ausubel et al. (1989)
, and from Hfx. volcanii using the alkaline lysis method as described by Holmes & Dyall-Smith (1990)
. Restriction endonucleases (AMRAD Pharmacia Biotech or New England Biolabs) were used according to the manufacturers instructions. Vent DNA polymerase (New England Biolabs) was used for polymerase chain reactions, and PCR products were cloned into plasmid pGEM T-Easy (Promega). DNA sequencing was performed using the Dye-deoxy terminator cycle sequencing kit from Applied Biosystems, with custom oligonucleotide primers. Reactions were analysed on an ABI 373A automated sequencer (Perkin Elmer).
Isolation of a simvastatin-resistant mutant and cloning the Har. hispanica resistance determinant.
A simvastatin-resistant mutant of Har. hispanica was produced by sequential passage in the presence of increasing concentrations of simvastatin. The final culture grew readily in the presence of 20 µg simvastatin ml-1, whereas the MIC of the wt strain was between 0·5 and 1 µg ml-1. Colonies were isolated on solid media and the DNA of one mutant showed no difference in MluI digestion profile to the wt strain, indicating that resistance was not likely to be due to gene amplification. DNA from this mutant was then used to clone the hmgA gene. Chromosomal DNA was cut with a number of restriction enzymes and Southern blots prepared. These were hybridized at moderate stringency to a radiolabelled DNA probe prepared from the Hfx. volcanii hmgA gene (carried on plasmid pWL102; see Lam & Doolittle, 1989 , 1992
). BglII digestion produced a single band of about 4·3 kb, i.e. large enough to contain the HMG-CoA reductase gene (approx. 1·3 kb; data not shown). BglII-digested DNA of 45 kb was cut out from a preparative agarose gel, ligated to BglII-cut plasmid pOK12 (Vieira & Messing, 1991
), introduced into E. coli DH5
, and transformant colonies probed using the Hfx. volcanii hmgA. A strongly hybridizing colony was identified which contained a plasmid with an insert of 4·3 kb. The location of the hmgA gene was narrowed further by Southern blot hybridization to a 1·6 kb NotIPstI fragment and the gene was completely sequenced (GenBank accession no. AF123438).
Curing Hfx. volcanii of the smallest plasmid, pHV2.
The commonly used host Hfx. volcanii strain WFD11 was cured of the smallest endogenous plasmid, pHV2, by ethidium bromide treatment (Lam & Doolittle, 1989 ). Recently, it has been found that one of the large plasmids in this strain, pHV3, is unstable and lost at a significant rate, most likely due to the use of the potential mutagen ethidium bromide in its construction. Cells without pHV3 grow more slowly, tend to filament, and lyse when spheroplasted during PEG-mediated transformation procedures (R. Charlebois, personal communication). A new derivative of the wt strain was produced which lacked pHV2 but was not treated with mutagenic agents. Firstly, plasmid pWL102 (a plasmid containing a pHV2 replicon and hmgA) was introduced into wt Hfx. volcanii NCIMB 2012 cells and simvastatin-resistant colonies selected on solid media. Several transformant colonies were subcultured into liquid medium and grown up under selection. These were passaged a further two times, after which dilutions of each culture were plated for single colonies on solid medium (with drug added). Plasmid minipreps from colonies derived from each culture were analysed by agarose gel electrophoresis and a strain that lacked pHV2, but contained pWL102, was selected. This was then grown in liquid medium without drug selection. After three passages this strain was plated for single colonies on solid media and 50 of these were patched onto plates with or without drug present. Sensitive isolates were tested for the presence of pHV2 by agarose gel electrophoresis of plasmid extracts and one was selected which showed no small plasmids (i.e. pHV2 or pWL102) present, although the larger pHV1 plasmid was retained. This was labelled strain DS70. In contrast to strain WFD11, in which about 10% of colonies are small and slow growing, the new strain did not show any significant frequency of slow-growing colonies on solid media, and was indistinguishable in growth and transformation characteristics from the parent.
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RESULTS |
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The Hfx. volcanii hmgA gene, like many other halobacterial genes, produces a leaderless mRNA, i.e. with no 5' leader sequence upstream of the start codon. Transcription begins at the A of the start codon (Lam & Doolittle, 1992 ), and about 25 nt upstream in the gene is a small AT-rich sequence, typical of strong archaeal promoters (Danner & Soppa, 1996
; Palmer & Daniels, 1995
; Reiter et al., 1990
; reviewed by Soppa, 1999
). Upstream of the start codon of the Har. hispanica hmgA there was a similar AT-rich sequence at a very similar distance to the promoter of Hfx. volcanii hmgA (see Fig. 1
). Consistent with this being the promoter, the sequence of the drug-resistant Har. hispanica gene displayed a single base substitution (compared to the wt gene) in this AT-rich region, forming a sequence closer to the consensus for strong archaeal promoters. This change is similar in position and type to the up-promoter mutation observed in the hmgA determinant of Hfx. volcanii (Fig. 1
; Lam & Doolittle, 1992
). To avoid confusion with the previous literature, the marker based on the Har. hispanica hmgA gene will be referred to here as the Mevr locus, even though the drug used for selection was simvastatin. Mevinolin and simvastatin are very similar chemically, and functionally equivalent, but mevinolin is not sold commercially in Australia.
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Recombination with Hfx. volcanii
To determine whether the Har. hispanica simvastatin-resistance gene recombined at a high frequency with the genome of Hfx. volcanii (most likely at the hmgA locus), plasmid pMDS95, containing this gene between the PstI site and the NotI site of pOK12, but unable to replicate in halobacteria because it lacks a halophilic origin of replication, was introduced into Hfx. volcanii cells and resistant colonies selected on plates with 4 µg simvastatin ml-1. The results of a typical experiment are shown in Table 2. Resistant colonies were observed in very low numbers, i.e. 03 colonies per 100 µl of plated transformation mixture (
102 transformants per µg DNA), and this did not change if the plasmid was linearized beforehand (by digestion with NotI). These values were comparable to those with control cells that had either no DNA added or a plasmid containing only the novobiocin-resistance determinant (pMDS20; Holmes et al., 1990). Cell competency was checked using replicating plasmids with a mevinolin-resistance marker (i.e. pWL102, pMDS99), and these produced high counts (>104 transformant colonies µg-1).
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DISCUSSION |
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The Har. hispanica gene is similar to other archaeal hmgA genes and retains the critical catalytic residues in the predicted protein, i.e. amino acids 66 (Glu), 101 (Glu) and 193 (Asp) (see Wang et al., 1990 ). The cloned wt gene conferred increased resistance upon Hfx. volcanii cells, showing that simply increasing the gene copy is sufficient to produce a resistant phenotype. The copy number of the pHV2 replicon has previously been estimated to be about six per cell (Charlebois et al., 1991
). The underlying mutation in the isolated resistance gene appears to be the same as that observed for the mevinolin-resistance gene of Hfx. volcanii (Lam & Doolittle, 1992
), i.e. a single up-promoter mutation causing overproduction of HMG-CoA reductase. Further evidence that the mutation occurred within the promoter of the gene was obtained by introducing an additional change nearby, forming a sequence motif even closer to the consensus for strong halobacterial promoters (Danner & Soppa, 1996
; Palmer & Daniels, 1995
; Soppa, 1999
). The additional up-promoter mutation increased the level of drug resistance about twofold.
The WFD11 strain of Hfx. volcanii was derived from the wt strain by ethidium bromide treatment, to cure it of the smallest cryptic plasmid pHV2 (Charlebois et al., 1987 ). Unfortunately, this process appears to have introduced mutations into the genome, and resulted in the observation (R. Charlebois, personal communication) that a significant proportion of the cells in a population of the WFD11 strain are relatively slow growing, and lack the 442 kb plasmid pHV3. Such strains are less suitable for genetic work. The pHV3 plasmid has now been fully sequenced and contains many recognizable ORFs (R. Charlebois & J. Shaw, personal communication) and one tRNA gene. We avoided the use of mutagens, which may affect the maintenance of pHV3, and eliminated pHV2 from H. volcanii NCIMB 2012 using plasmid incompatibility (with pWL102) followed by screening for spontaneous plasmid loss. Strain DS70 does not show the small-colony (slow-growing) pHV3- variant seen in strain WFD11, and performs equally as well in transformations, plasmid isolations, etc.
The halobacterial replicon of pMDS99 was obtained from pWL102, and this derives from a segment of the Hfx. volcanii cryptic plasmid pHV2 (Charlebois et al., 1987 ; Lam & Doolittle, 1989
). The latter plasmid is extremely stable in its natural host but the cloned replicon is less so. A recent study of pWL102 stability in Hfx. volcanii showed a rate of loss of 49% over 28 generations, or 1·75% per generation (Ortenberg et al., 1999
). Our results show a similar loss of 1·3% per generation, but both this and the previous values are far higher than the rates originally reported by Lam & Doolittle (1989)
in Hfx. volcanii, and by Cline & Doolittle (1992)
in Haloarcula spp. (i.e. <5% loss over >30 generations, or <0·16 % per generation). While this instability can be a problem when cells are not kept under selection, it has the advantage of allowing easy recovery of cured hosts.
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ACKNOWLEDGEMENTS |
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Received 11 October 2000;
revised 20 December 2000;
accepted 2 January 2001.