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Applied and Environmental Microbiology, September 2000, p. 3856-3867, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of the Minimal Replicon of a Cryptic Deinococcus radiodurans SARK Plasmid and Development of Versatile Escherichia coli-D. radiodurans Shuttle Vectors

Rob Meima1 and Mary E. Lidstrom1,2,*

Departments of Chemical Engineering1 and Microbiology,2 University of Washington, Seattle, Washington 98195-1750

Received 10 April 2000/Accepted 8 June 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Ever since its discovery in 1956 (3), Deinococcus radiodurans and other members of the Deinococcaceae have become the paradigm of natural resistance to high-level ionizing and UV radiation. D. radiodurans cells are able to survive levels of gamma radiation 1,000 times higher than the lethal dose for humans and were shown to survive and accurately repair massive DNA damage. At 1.5 megarads, up to 130 double-strand breaks occur per cell, which are repaired without mutagenesis or loss of viability (13). In contrast, the presence of a mere two double-strand breaks is lethal to Escherichia coli (21). As a consequence, this pink-pigmented, non-spore-forming bacterium has received considerable attention from the scientific community, from both a fundamental and an applied point of view. Because of their radioresistance, Deinococcus species hold great potential for bioremediation of complex waste mixtures containing organic solvents, heavy metals, and radioisotopes. Recently, Lange et al. (23) have demonstrated toluene dioxygenase (TDO) activity in engineered strains of D. radiodurans R1. TDO is a broad-spectrum dioxygenase capable of degrading trichloroethylene (42), a common organopollutant at many Department of Energy waste sites (32). In these strains, TDO activity was not inhibited by high levels of radiation, demonstrating the potential of D. radiodurans as a tool for remediation of mixed wastes.

Recent advances in molecular biology and genomics have greatly facilitated the study of D. radiodurans. The complete genome sequence has been available since 1998, and the annotated version has recently been published, revealing a genome consisting of two chromosomes, a megaplasmid, and a 45.7-kb plasmid (44). However, the lack of small, versatile plasmid cloning and expression systems has hampered the exploitation of D. radiodurans to its full potential. An E. coli-D. radiodurans shuttle vector, pI3, and derivatives for promoter cloning were constructed and characterized previously by Masters and Minton (26). However, due to their large size and the lack of knowledge concerning the sequence and the minimal replication functions, these plasmids have not been convenient for genetic engineering of D. radiodurans. The availability of a broader repertoire of more well-defined and convenient genetic systems would significantly facilitate the genetic amenability of D. radiodurans, for both fundamental and applied research. The present paper describes the characterization of the minimal replicon of pI3, which is a derivative of pUE10, a cryptic plasmid from D. radiodurans SARK (25). We report (i) sequencing and functional characterization of this deinococcal replicon and (ii) construction of second-generation vectors for use in D. radiodurans and E. coli. We show that the resulting general-purpose vector can be shuttled efficiently between these organisms and that the lacZ reporter gene, fused to endogenous D. radiodurans promoters, is successfully expressed from this plasmid in D. radiodurans.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1.

                              
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  		TABLE 1.   Bacterial strains and plasmids

Chemicals and enzymes. All chemicals used were of analytical grade and, unless indicated otherwise, were obtained from Baker Chemical Co. (Phillipsburg, N.J.) or Fisher Scientific (Fair Lawn, N.J.). 5-Bromo-4-chloro-3-indolyl-beta -D-galactopyranoside (X-Gal) and o-nitrophenyl-beta -D-galactopyranoside were from ISC Bioexpress (Kaysville, Utah) and Sigma Chemical Co. (St. Louis, Mo.), respectively. Enzymes for molecular biology were purchased from Boehringer Mannheim Corp. (Indianapolis, Ind.) and New England BioLabs (Beverly, Mass.) and were used as described by the supplier. Taq DNA polymerase was obtained from Gibco BRL (Grand Island, N.Y.).

Media and growth conditions. LB broth for growth of E. coli consisted of (per liter) 10 g of tryptone (Difco Laboratories, Detroit, Mich.), 5 g of yeast extract (Difco), and 10 g of NaCl (pH 7.4). TGY broth for D. radiodurans contained (per liter) 5 g of tryptone, 3 g of yeast extract, and 1 g of glucose (28). Solid media were prepared by addition of 1.5% agar (Difco) to either LB or TGY broth. Where necessary, media were supplemented with the appropriate antibiotics, all of which were obtained from Sigma. Ampicillin was used at 50 µg/ml for E. coli. Chloramphenicol was added to a final concentration of 3 µg/ml for D. radiodurans. Kanamycin was routinely used at 50 µg/ml for E. coli and at 8 or 4 µg/ml for D. radiodurans grown on solid and liquid media, respectively. Transformations of E. coli were performed either using commercially available cells (JM109 and TOP10 [Promega, Madison, Wis., and Invitrogen, Carlsbad, Calif., respectively]) or by the CaCl2 method (35). D. radiodurans cells were transformed essentially as described previously (40), with the exception that cells from exponentially growing cultures were collected by centrifugation (12,000 × g, 1 min) and concentrated 10-fold in TGY supplemented with 30 mM CaCl2.

DNA manipulations. Miniscale plasmid DNA preparations of E. coli were obtained as described by Sambrook et al. (35) and were resuspended in a total volume of 25 µl. Plasmid DNA from D. radiodurans was isolated using a variant of the alkaline lysis method. Cells were collected by centrifugation (16,000 × g, 2 min) and resuspended in 100 µl of solution I (25 mM Tris-HCl, 10 mM EDTA, 50 mM glucose [pH 8.0]) supplemented with lysozyme (10 mg/ml; Boehringer GmbH, Mannheim, Germany) and proteinase K (5 µg/ml; Boehringer GmbH). The suspension was incubated at 50°C for 30 min, followed by 5 min at 0°C and 1 min at 100°C. Subsequently, lysis was achieved by the addition of 200 µl of solution II (1% [wt/vol] sodium dodecyl sulfate [SDS], 0.2 N NaOH). After precipitation of chromosomal DNA and proteins (150 µl of solution III [60 ml of potassium acetate, 11.5 ml of glacial acetic acid, 28.5 of ml H2O]), the aqueous phase was extracted twice with an equal volume of phenol-chloroform-isoamylalcohol (24:24:1) (Boehringer Mannheim Corp.), and DNA was precipitated with 2.5 vol of 96% (vol/vol) ethanol. Finally, the pelleted DNA was resuspended in 25 µl of T10E1 (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) containing 2 µg of RNase A (Boehringer GmbH) per ml. This procedure produced sufficient amounts of DNA for several restriction analyses from 1 ml of cultures grown overnight at 30°C. PCR products were purified using a Qiaquick PCR purification kit (Qiagen Inc., Valencia, Calif.). Primers for PCR amplification and sequencing purposes were 18-mers and were obtained from Gibco BRL (Frederick, Md.). Nonradioactive nucleotide sequencing was performed by the University of Washington's Department of Biochemistry DNA Sequencing Facility, using an ABI Prism 377 sequencer (PE Biosystems). Southern hybridization analyses using 32P-labeled probes were performed at 60°C (both hybridization and wash steps) as described by Sambrook et al. (35); [alpha 32P]dCTP (6000 Ci/mmol) was from NEN Life Science Products (Boston, Mass.).

pI3 sequencing strategy. The sequence of the pUE10 moiety was determined by using a primer-walking strategy starting from four positions on the pI3 genome. The primers used for the initial sequencing reactions were based on the sequence of a 0.5-kb PstI-NsiI fragment present on pTCP1 (Table 1), on pKK232-8 (GenBank accession no. U13859, position 5060), and on available pI1 sequences (24) GenBank accession no. M94966).

Sequence comparisons and predictions. Computational analyses of DNA and deduced amino acid sequences were performed using the following World Wide Web-based programs. Similarity searches were carried out using the BLAST algorithms described by Altschul et al. (2) at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST/). Multiple alignments were performed using CLUSTAL W, available at the European Bioinformatics Institute website (http://www.ebi.ac.uk/clustalw/). The presence of possible signal peptidase I cleavage sites was analyzed using the parameters described by Nielsen et al. (29) at the Center for Biological Sequence Analysis website (http://www.cbs.dtu.dk/services/SignalP/). Analyses of primary protein structure were performed using the ExPASy ProtParam tool, available at the Swiss Institute of Bioinformatics website (http://www.expasy.ch/cgi-bin/protparam). Preliminary sequence data for D. radiodurans were obtained from The Institute for Genomic Research website (http://www.tigr.org).

Plasmid constructions. Derivatives of pI3 were constructed as follows (Fig. 1). First, a 3.1-kb fragment containing the putative ardU gene was removed by CelII digestion and self-ligation, resulting in pI5. Plasmid pI6 was constructed by NsiI digestion of pI3, followed by T4 DNA polymerase treatment to remove the protruding 3' ends and self-ligation. In a parallel experiment, a 6.3-kb KpnI fragment was removed by partial KpnI cleavage and subsequent ligation of the mixture, yielding pI8. Next, the resU gene was removed by HindIII-SplI digestion, followed by T4 DNA polymerase treatment in the presence of 0.25 mM deoxynucleoside triphosphates to fill in the overhanging ends and ligation. The resulting plasmid, pI10, was subsequently digested with AocI and religated, producing pI12. Finally, the minimal replicon of pI12 was fused to pMTL23, creating pRAD1.


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  		FIG. 1.   Schematic representation of the 11.9-kb pUE10 EcoRI-HindIII fragment present on pI3 and of deletion derivatives. Restriction sites that were mapped previously are indicated (26); those used for the construction of the deletion derivatives are shown in boldface. Abbreviations for restriction enzymes: E, EcoRI; Sm, SmaI; A, AocI; K, KpnI; Cl, ClaI; S, SplI; St, StuI; C, CelII; P, PstI; N, NsiI; and H, HindIII. See text for details on the construction of the derivatives. The methylated ClaI site is marked with an asterisk (Cl*); note that the position of this site, as determined in our sequencing effort, differs substantially from that on the physical map published previously (26). The genotypes of the resulting plasmids and their ability to replicate in D. radiodurans R1, as measured by the occurrence of Cmr transformants, are indicated on the left. repU*, truncated form of the repU gene as a result of the out-of-frame mutation introduced in pI6 by removal of the NsiI-generated protruding ends followed by self-ligation (N*). wt, wild type.

Overexpression of the plasmid-encoded ResU and RepU proteins in E. coli was achieved by fusing PCR fragments containing the genes to an IPTG (isopropyl-beta -D-thiogalactopyranoside)-inducible promoter. These fragments were obtained using the following primers, bearing 5' tags containing restriction sites for BamHI and BglII (underlined): resU5' (GCGAGATCT-ATGTCTGCACAGAATCA), resU3' (CGCGGATCC-TTTTGAAACCGACGCAG), repU5' A (GCCAGATCT-TCTTGAGACACAATCCA), repU3' A (CGCGGATCC-CTTCTCGGCCTTTCTGT), repU5' Ad (GCCAGATCT-TCGTCAGACACAATCCA), and repU5'Bd (GCGAGATCT-TTCACGGTCCGACTCCT); the last two contain mutations (boldface) that eliminate the predicted translational start sites of repU. After PCR amplification, the fragments were first cloned in pCR2.1-TOPO (Invitrogen) and subsequently transferred to the QIAexpress type IV series of His6 tag-based overexpression vectors (Qiagen), thus fusing the hexahistide-encoding tag to the 5' termini of resU and repU, respectively.

Expression from a plasmid-borne reporter gene was studied using the following plasmids. pRADZ1 was constructed by insertion of a 3.2-kb PCR fragment carrying the pMUTIN2mcs-derived beta -galactosidase (lacZ) gene of E. coli fused to an optimized ribosome-binding site (RBS) flanked by suitable restriction sites (41; R. Meima and M. E. Lidstrom, unpublished data) in the BglII-XbaI sites of pRAD1. Subsequently, the promoter region of the putative groESL operon of D. radiodurans R1 was inserted into the BglII site of pRADZ1 to generate pRADZ3 and pRADZ30 (opposite orientation), respectively.

Protein analysis. SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (22), using a Hoefer Mighty Small vertical electrophoresis system (Pharmacia Biotech, San Francisco, Calif.). Protein was visualized by staining with Coomassie brilliant blue (Sigma). Overexpression and purification, under nondenaturing conditions, of the His-tagged RepU and ResU proteins using Ni-nitrilotriacetic acid (Ni-NTA) column chromatography was performed exactly as indicated by the supplier (Qiagen); samples were stored at 0°C for prolonged periods of time without loss of activity in subsequent gel mobility assays. Gel retardation studies using purified N-terminally His-tagged RepU protein were performed as follows. Approximately 300 ng of protein was mixed with 32P-labeled pRAD1AocI-derived MaeI or PCR fragments in a binding buffer that consisted of 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 10 mM EDTA, and 20% (vol/vol) glycerol. Bovine serum albumin and poly(dI-dC) were used as noncompetitive protein and DNA at 100 and 50 ng/µl, respectively. After 30 min of incubation at room temperature, the samples were electrophoresed on a 4% (wt/vol) polyacrylamide gel in 1× TAE (40 mM Tris-acetic acid [pH 8.0], 2 mM EDTA).

Expression assays. Expression of the lacZ reporter gene in E. coli and D. radiodurans colonies was detected using X-Gal (40 µg/ml). Quantitative analyses of lacZ expression were performed as described by Miller (27). Cell extracts of D. radiodurans were obtained by passing concentrated cell suspensions through a French pressure cell at 1,000 lb/in2 using a J5-598A laboratory pressure cell press (Aminco, Silver Spring, Md.). Alternatively, samples for beta -galactosidase assays were prepared by toluene permeabilization of cells. To correct for the absorption caused by the cell suspension added to the reaction mixtures, the A420 value obtained with R1(pRAD1) cells (lacZ mutant) was subtracted from those obtained with the other constructs.

Nucleotide sequence accession number. The complete sequence of the pUE10 moiety of pI3 can be accessed through GenBank (accession no. AF206717).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Determination of the pI3 sequence. Since previous attempts to define the minimal replicon of pI3 by shotgun approaches were unsuccessful (26), we determined the nucleotide sequence to allow a more directed approach. Based on the physical map published by Masters and Minton (26) we were able to subclone a 0.5-kb NsiI-PstI fragment on pTC23 (Table 1; Fig. 1) and to sequence this insert on both strands. The sequence of the remaining portion of the 11.9-kb EcoRI-HindIII fragment was established using a primer-walking approach. This single-stranded sequence was verified in part by several additional runs using a set of primers directed against the minimal replicon (see below), altogether generating approximately 2.8 kb of double-stranded sequence. No mistakes were found, suggesting that the overall sequence of the entire insert has an error rate of less than 1 mistake per 2,800 bp.

The total size of the pUE10-derived HindIII-EcoRI insert in pI3 was determined to be 11,910 bp, part of which is shown in Fig. 2A. The overall G+C content is 64.5% which is comparable to the genomic percent G+C of D. radiodurans R1. Nevertheless, a region of strikingly high percent A+T was found downstream of the putative replication protein-encoding gene (see below). Annotation of the sequence revealed the presence of 12 putative open reading frames (ORFs), all of which are transcribed in the same direction (Fig. 1). These were analyzed using the BLAST algorithms (2) (Table 2), and five ORFs showed significant similarity at the amino acid level to other database entries. The second ORF encodes a protein that is highly similar to a class of hypothetical proteins, representatives of which are found in both the archaeal (e.g., Methanococcus jannaschii protein MTH993; 55% overall similarity) and eubacterial (e.g., YxiE of Bacillus subtilis and DR2132 and DR2363 of D. radiodurans R1; >= 42% overall similarity) domains. Since the function of these proteins is unknown, the relevance of the gene, which was designated orfB, for pUE10 as yet remains unclear. The third ORF is very similar to periplasmic serine protease genes and was hence designated htrU. In fact, the highest similarity (71% overall) was found with the translated product of a chromosomal htrA-like gene of D. radiodurans R1 (DR1756). In accordance with its expected localization, the predicted protein contains a possible signal peptidase cleavage site (18TDPdown-arrow TE22). The product of ORF8 showed considerable similarity to an antirestriction protein, ArdA, of Yersinia pestis plasmid pMT-1 (24; P. Hu, J. Elliott, P. McCready, E. Skowronski, J. Garnes, A. Kobayashi, A. V. Carrano, R. Brubaker, and E. Garcia, GenBank accession no. AF053947 [http://www.ncbi.nlm.nih.gov/Entrez/index.html]) and to those of pKM101 (5) and Col1bP-9 (1, 4). In addition, the predicted protein is highly acidic (calculated pI of 4.25), which is also strikingly similar to the case for other known plasmid-encoded antirestriction proteins (for comparison, ArdA and ArdB of pKM101 have pIs of 4.05 and 4.84, respectively). Therefore, this gene was designated ardU. The product of ORF11 showed similarity to replication proteins of two Thermus plasmids (15, 43). Although the overall identity was slightly higher with the RepA protein of the ATCC 27737 plasmid, the strongest conservation was found with an internal region of the pTSp45s RepT protein (Fig. 2B). This gene was designated repU. Two possible DnaA-binding sites (14) were found within the coding region of repU (Fig. 2A and C) (see below). No typical direct repeats (iterons) (16, 20) were found in the vicinity of repU, although one imperfect direct repeat (17 bp in length, 13 identical) was present, with one half of the repeat encompassing the DnaA box at position 8716 (Fig. 2A). Finally, the product of ORF12 showed similarity to the XerD family of site-specific recombinases-resolvases, and this ORF was designated resU. The remaining seven putative ORFs shared little or no similarities at the amino acid level with other proteins and/or did not contain recognizable RBS sequences, and they were designated orfA, orfC, orfD, orfE, orfF, orfG, and orfH (Table 2). Of these, orfA is nevertheless of interest, since a gene encoding a protein with 46% overall similarity was found adjacent to the chromosomal origin of replication of Mycobacterium smegmatis (GenBank accession no. X92503) (34). Its role in plasmid replication, however, remains unknown. The predicted product of orfF showed similarity with antifreeze glycoproteins found in arctic fish species such as Boreogadus saida (arctic cod) and Dissostichus mawsoni (nototheniid fish) (11). Although the overall similarity appears to be significant (Table 2), the high alanine content (±40 to 50%) and the Ala-Ala-Thr repeats typical of these proteins were not observed with the predicted product of orfH (17.1% Ala), suggesting that this similarity may not be significant.




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  		FIG. 2.   Nucleotide sequence of the minimal replicon and resolvase gene of D. radiodurans SARK plasmid pUE10. (A) Nucleotide and amino acid sequence of the repU gene and its predicted product. The alternative start codon (TTG) at position 7966 for repU and the first six amino acids are indicated in boldface italics. The putative DnaA-binding sites boxes are boxed; the solid arrows indicate the imperfect repeats (see text for details). Nucleotide numbers correspond to GenBank accession no. AF206717. (B) Alignment of the replication proteins of pUE10 (pI3) and Thermus plasmid pTsp45s. Stretches of >= 3 identical residues or conserved replacements are boxed. (C) Comparison of the two putative DnaA-binding sites present on pUE10, with the DnaA-binding sites of the chromosomal origins of replication of E. coli (Ec) and B. subtilis (Bs), and those of Thermus sp. strain YS45 plasmid pTsp45s.

                              
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  		TABLE 2.   Putative ORFs present on the 11.9-kb pUE10 insert of pI3 and their similarities to database entries

Establishment of the minimal origin of replication. With the availability of the entire sequence, specific combinations of genes could be tested to determine the minimal region required for autonomous replication of pUE10. The results of these analyses are shown in Table 3 and Fig. 1. pI3 transformed D. radiodurans R1 at a frequency of approximately 2.45 × 105 transformants per µg of DNA, which is similar to the frequency described previously for pI3 (26). When part of the ardU gene was removed from pI3 by deletion of a 3.1-kb CelII fragment, transformation of D. radiodurans R1 with the resulting plasmid, pI5, produced Cmr colonies, albeit at a 50- to 100-fold-lower frequency than observed with pI3. Next, the putative repU gene of pI3 was inactivated by an out-of-frame mutation, yielding pI6. This construct produced only a very small number of Cmr colonies (<0.2% of those produced by pI3). Using a second Delta repU derivative, pI9, lacking most of the original fragment present on pI3, no transformants were obtained in D. radiodurans R1. In contrast, transformants were obtained with pI8 (repU+ ardU-), at a frequency similar to that with pI5 (ardU-). Together, these data demonstrate that the putative repU gene encodes a function necessary for replication, most likely the replication initiation protein. The few transformants obtained with pI6 may be the result of chromosomal integration, conceivably via htrU. This gene which is not present on pI9, is highly similar to the htrA homolog located on chromosome I (see above), which could provide the source of homology for such an event to occur. The highest nucleotide similarity observed was 86.5% in a continuous 52-bp sequence.

                              
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  		TABLE 3.   Transformation frequencies of pI3 and its derivativesa

The minimal origin was further delineated by removal of the putative resolvase gene (resU), resulting in pI10. In a final step, a 0.5-kb fragment was removed from the Cmr-orfA intergenic region of pI10. Both pI10 and the resulting plasmid, pI12, were able to replicate in D. radiodurans R1, producing Cmr transformants at a frequency similar to that for the other ardU- derivatives. Thus, the minimal region required for autonomous replication resides within a 2,624-bp fragment containing the putative repU gene and the A+T-rich sequence downstream of this gene.

Next, these deletion derivatives were isolated from D. radiodurans R1, and their respective restriction patterns were compared to those extracted from E. coli. These patterns were identical (results not shown). Southern hybridization using pI12 as a probe demonstrated that whereas pI3 and pI5 are present as monomers in D. radiodurans R1, pI10 and pI12 (Delta resU) exist predominantly in a multimeric form, suggesting the existence of an active resolution system on the resU+ plasmids (Fig. 3) (see below). Cells harboring pI8 (resU+) also accumulated multimeric forms of the plasmid, albeit at a much lower level than pI10 and pI12. Although the copy numbers of pI3 and its derivatives were not accurately determined, it appears that these are all similar, and judged from the intensity of bands in gel electrophoresis, they were estimated at 5 to 10 copies per cell.


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  		FIG. 3.   Analysis of pI3 and its deletion derivatives. Agarose gel electrophoresis (A) and Southern hybridization (B) of undigested plasmid DNA isolated from D. radiodurans R1 are shown. Lanes: 1 and 7, molecular size marker (1-kb ladder; Gibco BRL); 2 and 8, pI3; 3 and 9, pI5; 4 and 10, pI8; 5 and 11, pI10; 6 and 12, pI12. In each lane, 2 µl of miniprep DNA was loaded. EaeI-digested pI12 was used as a probe. The signal observed with the molecular size marker (arrowhead) is caused by hybridization of the vector containing the 1-kb ladder with the pKK232-8 moiety of pI12. The asterisk indicates the position of multimeric pI10 and pI12; solid arrows mark the mono- and dimeric forms of these two constructs. The additional signal observed in the ethidium bromide stain in panel A (open arrow) most likely represents a chromosomal DNA contamination.

Construction of a shuttle vector for E. coli and D. radiodurans. The pUE10-derived promoter driving expression of the chloramphenicol resistance gene of pI3 was shown previously to be located within an 0.43-kb fragment (26) (GenBank accession no. M94966). In the present work we have further localized this promoter by transformation of D. radiodurans R1 with pI12, carrying a 532-bp AocI deletion. This construct produced Cmr colonies at a frequency comparable to that for pI10 (see above) (Table 3). Thus, it appears that the promoter driving expression of the resistance gene is located within 129 bp between the AocI and SmaI sites of the pI3 insert. Based on these data, the region containing the minimal replicon and Cmr cassette present on pI12 was transferred to a smaller E. coli vector, pMTL23 (9), which contains an extended multiple cloning site and has a much higher copy number in E. coli than the pBR322 origin of pI3. The resulting plasmid, pRAD1 (Fig. 4), is 6.3 kb in size and contains an extended multiple cloning site. It efficiently transformed D. radiodurans R1 to Cmr (Table 3) and could be shuttled back to E. coli.


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  		FIG. 4.   Physical map of the E. coli-D. radiodurans shuttle vector, pRAD1. The positions of the genes and antibiotic resistance markers as well as the unique restriction sites are indicated. The gray and black segments represent pI3- and pMTL23-derived sequences, respectively; the hatched box indicates the A+T-rich region downstream of the repU gene. See text for details on the construction of pRAD1.

Expression of a plasmid-encoded reporter gene and generation of a promoter probe vector. To assess the feasibility of using the newly constructed shuttle vector, a promoter probe vector was generated using the E. coli lacZ gene. First, a BglII-XbaI fragment containing lacZ was ligated to BglII-XbaI-digested pRAD1, yielding pRADZ1. pRADZ1 has cloning sites upstream of lacZ that make it potentially useful for promoter cloning and analysis. To test this function, a putative promoter segment of the D. radiodurans R1 groESL genes (R. Meima and M. Lidstrom, unpublished data) was inserted in both orientations in the unique BglII site of pRADZ1 that is upstream of lacZ, and LacZ expression was analyzed. When the groESL promoter was fused to lacZ in the proper orientation (pRADZ3), beta -galactosidase activity was significant (219 nmol min-1 OD600 unit-1) compared to that for pRADZ1 (50 nmol min-1 OD600 unit-1) and the groESL fragment in the wrong orientation (pRADZ30) (19 nmol min-1 OD600 unit-1). These results demonstrate that a heterologous gene can be expressed efficiently from the shuttle vector and that the promoter probe vector can be used to assess promoter activity.

Functional analysis of resU, the putative resolvase gene. Although the mechanisms underlying the stabilizing effect of plasmid-encoded site-specific recombinases on plasmid maintenance are not fully understood, their role has been suggested in several studies (8, 19, 30, 33, 38). It has long been established that these so-called resolvases contribute to plasmid maintenance by resolving plasmid multimers to monomers, thus increasing the number of segregation units to be distributed during cell division (for a review, see reference 30). Failure to resolve multimers prior to segregation can cause rapid accumulation of plasmid-free cells, even with multicopy plasmids such as pBR322 (dimer catastrophe hypothesis [38]). In addition, these enzymes also contribute to the fidelity of plasmid replication by a variety of mechanisms. The site-specific recombinase of Streptococcus pyogenes plasmid pSM19035 was shown to act not only as a resolvase (8) but also as an efficient DNA invertase ensuring faithful replication of both arms of the inverted repeats present on this plasmid (33). In another inc18 plasmid, pAMbeta 1 of Enterococcus faecalis, Resbeta was postulated to enhance faithful replication by catalyzing D-loop arrest of DNA polymerase I, allowing its replacement by the highly processive DNA polymerase III holoenzyme during the early stages of replication (19).

The possible role of the resU-encoded resolvase homolog in the maintenance of pI3 and its derivatives was analyzed by measuring the segregational stability of these plasmids under nonselective conditions (Fig. 5). The results of both direct- and replica-plating experiments clearly showed that the Delta resU plasmids tested (pI10 and pRAD1) are unstable compared to the resU-containing plasmids pI3, pI5, and pI8, which exhibited a high level of stability (100% Cmr after 75 generations). From the data shown in Fig. 5, the appearances of Cms cells with pI10 and pRAD1 were calculated at 1.22% (regression coefficient [R] = 0.9825) and 1.32% per generation (R = 0.9876), respectively. The structural integrity of the plasmids was verified by isolation of DNA from chloramphenicol-enriched cultures at the end of the assay and transformation of E. coli using these isolates (results not shown). The observation that the Delta resU constructs are unstable is in good agreement with the high level of multimers produced by these derivatives (Fig. 3B). Together, these data suggest that the resU+ plasmids contain both an active resolvase and its cognate resolution site(s) and that maintenance of pUE10 and its derivatives depends, at least in part, on the resolvase system.


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  		FIG. 5.   Maintenance of pI3 and a number of its derivatives in D. radiodurans R1. At the onset of the experiment, cultures were inoculated from overnight cultures grown in TGY broth containing 3 µg of chloramphenicol per ml. Diluted samples were plated at regular intervals on selective and nonselective agar. The stability is shown as the percentage of Cmr colonies of the total viable count, as determined by replica-plating colonies from nonselective to selective agar (closed symbols). Points agreed within ±10%. Plasmids tested were: pI3 (resU+) (), pI5 resU+ (), pI8 (resU+) (×), pI10 (resU-) (black-triangle), and pRAD1 (resU-) (black-lozenge ). For comparison, the open symbols indicate data obtained from direct plating of samples on both selective and nonselective media; shown here are pI3, pI10, and pRAD1.

Biochemical analysis of the RepU protein. To analyze whether the putative replication protein is able to recognize a specific sequence within the minimal replicon, the corresponding gene was fused to a His6 tag, allowing for overexpression and purification of the protein. The resulting plasmids, pQREPU1 through -3, carrying the insert fused in three different reading frames, were maintained in an E. coli host containing a plasmid-encoded copy of lacI (pREP4) to ensure full repression of the lac promoter. Expression was induced by addition of IPTG at various concentrations. Samples were taken at regular intervals and analyzed for protein content. A protein with an apparent molecular mass of about 38 kDa was observed upon induction of cells carrying pQRESU1 (37,579 Da; resU fused to the His6 tag in the proper frame), confirming that resU encoded a protein of the correct size. However, RepU overexpression could not be obtained with any of the fusions tested (Fig. 6A). The same was true when a copy of the repU gene containing a mutation of the ATG start codon was used (primers repU5'Ad and repU3'A). However, when E. coli M15(pREP4) was transformed with a plasmid carrying a copy of the repU gene in which the TTG start codon was mutated (pQREPU1Bd), a product with an apparent molecular mass of 45 kDa was observed upon induction with IPTG (predicted mass of His-RepU fusion, 44,092 Da) (Fig. 6A). These data suggest that the TTG may in fact be the true translation initiation codon (Fig. 2A; Table 2). Using Ni-NTA column chromatography, the His-RepU protein was purified (Fig. 6B). The purified fusion protein was subsequently applied in gel retardation experiments with 32P-labeled MaeI restriction fragments of pRAD1AocI covering the minimal replicon. These analyses indicated that the protein binds to a fragment containing the AT-rich region and DnaA boxes, as well as to sequences upstream of the repU gene, but not to other tested regions (not shown). These initial observations were confirmed by performing retardation assays with (nested) PCR fragments in the regions for which binding was observed (Fig. 7). Under the conditions used, binding was observed with fragments AT5 (containing a portion of the AT-rich region), DnaA1 (containing one of the putative DnaA boxes), and Prep1 (containing the region immediately upstream of repU). As a negative control, a PCR fragment derived from the resU gene (present in pI8) was used. These data suggest that the RepU protein has affinity for both the AT-rich stretch and the upstream region of the repU gene. This property could provide a dual mechanism for copy number control of pUE10, namely, at the levels of (i) replication initiation and (ii) autoregulation of repU expression.


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  		FIG. 6.   Identification and overexpression of the RepU and ResU proteins in E. coli. The corresponding genes were fused to a His6 tag, and the resulting constructs were maintained in strain M15(pREP4) to ensure repression of transcription. (A) Overexpression of His-ResU and His-RepU. Expression was induced by addition of 100 µM IPTG to exponentially growing cells. Samples were prepared by boiling pelleted cells in 1× SDS sample buffer. The arrowheads indicate the positions of the RepU (45-kDa) and ResU (38-kDa) proteins. Lanes: 1, prestained low-molecular-weight marker (Bio-Rad, Hercules, Calif.); 2 and 3, pQRESU1; 4 and 5 pQRESU10; 6 and 7, pQREPU1Bd; 8 and 9, pQREPU10Bd; 10, pQE30. Even-numbered lanes represent uninduced cultures; odd-numbered lanes indicate lysates obtained from cultures induced with IPTG for 2 h. (B) Purification of the His-RepU fusion protein by Ni-NTA chromatography. Total protein of M15(pREP4, pQREPU1Bd) was isolated from a 50 ml of 100 µM IPTG-induced culture and subjected to chromatography as indicated in the supplier's manual. Lanes: 1, prestained low-molecular-weight marker (Bio-Rad); 2, uninduced culture at 2 h; 3, induced culture at 6 h; 4, total lysate; 5, flowthrough (ft) from Ni-NTA column; 6 and 7, wash (W) steps; 8 to 10, elution (e) steps.


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  		FIG. 7.   Binding of the His-RepU fusion protein to pI8- and pRAD1-derived PCR fragments. (A) Purified His-RepU protein was mixed with purified 32P-labeled PCR fragment (±4,000 cpm) and incubated at room temperature for 30 min with the fragments shown in panel B (lanes 1 to 6); Cntrl (lane 7), PCR fragment derived from the resU region of pI8. The reaction mixtures were subsequently electrophoresed on 4% polyacrylamide-TAE gels. (B) Schematic representation of the repU region of pI3, showing the locations of the PCR fragments bound by the His-RepU fusion protein. (C) Specificity of the His-RepU interaction with the repU upstream region (32P-Prep1; see panel B). The specificity of this interaction was assayed by adding increasing amounts of noncompetitive DNA (lanes 2 to 5) and unlabeled (Unlab.) competitive DNA (Prep1) (lanes 6 to 9); the ratio between 32P-labeled probe and poly[d(I-C)] or unlabeled probe, respectively, is shown.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

As a result of its remarkable resistance to ionizing radiation and other DNA-damaging agents, D. radiodurans has become one of the most promising tools for bioremediation of mixed wastes containing radionuclides. To further expand the range of molecular tools for genetic engineering of this bacterium, we set out to construct small, versatile shuttle vectors for E. coli and D. radiodurans. For this purpose, we determined the sequence of a fragment within pI3, which contains the origin of replication of pUE10, a cryptic plasmid from D. radiodurans SARK (25, 26), a strain that is different from the one for which the genome sequence is available (strain R1).

The sequence of the pUE10-derived fragment of pI3 consists of 11,910 bp. The insert contains only three Sau3A sites, which may explain why previous attempts to isolate the minimal replicon of pUE10 using a shotgun approach met with no success (26). The overall percent G+C content of the pUE10 moiety of pI3 is 64.5, which is comparable to that of the D. radiodurans R1 chromosomes I and II and the megaplasmid (44). In contrast, the fourth genetic element found in D. radiodurans R1, a 45.7-kb plasmid, has a considerably lower G+C content (56.1%) than the chromosomes and megaplasmid. Although pUE10 (37 kb) (25) and the 45.7-kb plasmid are of similar size, this difference in percent G+C content suggests that these elements are of different origins, and given the high G+C content of pUE10, it is possible that this plasmid, unlike the 45.7-kb plasmid, was already present prior to the branching of Deinococcus and the closely related Thermus species. Annotation of the nucleotide sequence revealed 12 putative ORFs, all of which are transcribed in the same direction. None of these showed significant similarity to ORFs on the 45.7-kb plasmid of D. radiodurans R1, except for a XerD-type recombinase (44% overall similarity with ResU). Again, these data demonstrate that these Deinococcus plasmids are very different in terms of sequence, structural organization, and, possibly, mode of replication (see below).

Two important characteristics for plasmids used as genetic tools are replication and stability. The two most interesting ORFs with respect to these two characteristics are repU and resU, with products showing similarity to replication and resolvase proteins, respectively. The RepU protein shares similarity with the replication initiation proteins of two different Thermus plasmids (15, 43) (Table 2). The 244-amino-acid product of an ORF present on the D. radiodurans R1 45.7-kb plasmid showed weak similarity with the Rep proteins of gram-positive plasmids pSM19035 (RepS) (7), pIP501 (RepR) (6), and pAMbeta 1 (RepE) (36) but not with RepU. These observations provide further support for our hypothesis that pUE10 and the 45.7-kb plasmid do not have a common ancestry. Downstream of the repU gene, an A+T-rich region (42% G+C) of approximately 500 bp was found. A region similarly rich in A and T was found downstream of the RepA gene of the Thermus sp. strain ATCC 27737 plasmid (15) but not in pTsp45s (43). The presence of an A+T-rich sequence is thought to facilitate strand separation for initiation of replication by RepU and DnaA. DnaA was shown to be crucial for chromosome replication as well as for replication of several theta-replicating plasmids (20). In the latter process, the concerted activity of the plasmid-encoded Rep protein and DnaA is thought to be involved in loading of the replication helicase DnaB by stimulating unwinding of the plasmid origin. In addition to the A+T-rich region and the DnaA-binding sites, classical oriA-containing plasmids such as P1, F, pSC101, R6K, and the broad-host-range plasmid RK2 are characterized by the presence of a series of directly repeated sequences called iterons (for reviews, see references 16 and 20). These repeats constitute the primary binding site for the replication initiation proteins, thereby forming a nucleoprotein complex at the origin of replication. No such repeats are present in the repU region, suggesting that the replication initiation protein of pUE10, as well as those of the Thermus plasmids, recognize a nonrepeated sequence. However, pUE10 replication resembles that of the oriA-containing plasmids in terms of its independence of DNA polymerase I (18). Together, these data show that the pUE10 origin shares some characteristics with class A-type replicons, while the absence of a typical oriA and the locations of the DnaA boxes are features unique to pUE10 and the Thermus plasmids. DNA binding (helix-turn-helix [17]) or other motifs typical of regulatory proteins were not detected in the amino acid sequence of RepU. However, we were able to demonstrate binding of this protein to two separate regions of the minimal replicon, consistent with our hypothesis that the RepU protein may be involved not only in initiation of replication, but also in copy number control through autoregulation of its own expression (for a review, see reference 10). Further work will be required to test this hypothesis. Our present sequence data did not reveal the presence of additional putative copy number control elements in the vicinity of repU, although such functions may be present on the parental plasmid.

Although not strictly required for replication, a plasmid-encoded resolvase (Resbeta ) was shown to enhance the processivity of DNA synthesis during initiation of pAMbeta 1 replication (19, 31). In addition, the presence of an active resB gene greatly enhanced the segregational stability of pAMbeta 1 derivatives by preventing the accumulation of plasmid multimers (39). Likewise, the pSM19035-encoded site-specific recombinase is required for both maintenance and accurate replication, by acting as both a resolvase and a DNA invertase (8, 33). The stability of pI3 and its derivatives also appears to depend on the plasmid-encoded site-specific resolvase system. Whereas the resU+ plasmids were found to be very stable, the Delta resU derivatives analyzed in our studies were lost at a rate of approximately 1% per generation, suggesting that resolution of the multimers observed with these constructs (Fig. 3B) is crucial for their maintenance.

The presence of an ardA homolog is of particular interest since thus far, these antirestriction systems were thought to exist exclusively on bacteriophages and representatives of the F, I, and P incompatibility complexes of enterobacterial plasmids (12). Since these functions were shown to be essential for the survival and establishment of plasmids during conjugation, this finding suggests that pUE10 might be a conjugative plasmid. However, in an earlier study, pUE10 could not be conjugated (37). It is not clear why ArdU appears to be important for high transformation frequencies in our studies, but it may reflect a need to protect incoming transforming DNA from degradation.

Combining the data obtained previously by Masters and Minton (26) (GenBank accession no. M94966) with the sequence presented here, we further narrowed down the fragment containing the promoter that drives expression of the Cmr marker in pI3 and its derivatives. This promoter, the Cmr marker, and the minimal replicon defined in the present work were used to construct a versatile E. coli-D. radiodurans shuttle vector with a large number of unique restriction sites and small size (pRAD1). It efficiently transforms D. radiodurans to Cmr, and the lacZ reporter can be expressed at substantial levels from this vector. Although pRAD1 is poorly maintained in the absence of selective pressure, we have opted not to include the resolvase system in order to limit the size of the vector and increase the number of available restriction sites for cloning. Our present experience with this vector is that instability is not a problem in the presence of antibiotic selection. Likewise, we have not included the ArdU region, since the transformation frequencies in the absence of ardU are sufficiently high for routine work. Therefore, pRAD1 is a convenient and useful general-purpose cloning vector. In addition, pRADZ1, containing a lacZ reporter, is a useful vector for analyzing promoter activity. These vectors are present in the cell at approximately the same copy number as the chromosome, which is present at 7 to 10 copies per cell.

Together with the annotated genome sequence, the availability of these molecular tools will significantly enhance the genetic amenability of this intriguing and potentially useful microorganism.


    ACKNOWLEDGMENTS

We are indebted to Michael J. Daly for generously providing plasmids pI3 and pMD209 and to John Battista for making available D. radiodurans R1. We thank Marion Franke and Khue Quang Trinh for technical assistance.

This work was funded by a grant from the DOE EMSP program (DEFG0797ER20294).


    FOOTNOTES

* Corresponding author. Mailing address: Department of Chemical Engineering, Box 351750, University of Washington, Seattle, WA 98195-1750. Phone: (206) 616-5282. Fax: (206) 616-5721. E-mail: lidstrom{at}u.washington.edu.


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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Applied and Environmental Microbiology, September 2000, p. 3856-3867, Vol. 66, No. 9
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