Utilization of Virus ϕCh1 Elements To Establish a Shuttle Vector System for Halo(alkali)philic Archaea via Transformation of Natrialba magadii M. Mayrhofer-Iro, A. Ladurner, C. Meissner, C. Derntl, M. Reiter, F. Haider, K. Dimmel, N. Rössler, R. Klein, U. Baranyi, H. Scholz and A. Witte Appl. Environ. Microbiol. 2013, 79(8):2741. DOI: 10.1128/AEM.03287-12. Published Ahead of Print 15 February 2013.
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Utilization of Virus Ch1 Elements To Establish a Shuttle Vector System for Halo(alkali)philic Archaea via Transformation of Natrialba magadii M. Mayrhofer-Iro,a A. Ladurner,b C. Meissner,c C. Derntl,d M. Reiter,e F. Haider,a K. Dimmel,a N. Rössler,f R. Klein,g U. Baranyi,h H. Scholz,i A. Wittea Department of Microbiology, Immunobiology and Genetics, Max F. Perutz Laboratories, University of Vienna, Vienna, Austriaa; Department of Pharmacognosy, University of Vienna, Vienna, Austriab; Institute of Virology, Charité University Hospital, Berlin, Germanyc; Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austriad; Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, Austriae; Nycomed Austria GmbH, Linz, Austriaf; Children’s Cancer Research Institute, St. Anna Kinderkrebsforschung, Department of Pediatrics, Medical University of Vienna, Vienna, Austriag; Department of Surgery, Division of Transplantation, Vienna General Hospital, Vienna, Austriah; Bundeswehr Institute of Microbiology, Munich, Germanyi
N
atrialba magadii belongs to the haloalkaliphilic group of the Halobacteriaceae. In contrast to the neutrophilic haloarchaea, the alkaliphilic haloarchaea require a high pH (8.5 to 11) and high salt (4 to 5 M NaCl) for growth and thus are considered a distinct physiological group (1). Although there is limited information on the biology of this group, the extremophilic properties of the haloalkaliphiles with respect to salinity and pH suggest that these microbes and their enzymes represent an underutilized resource for basic research and industrial applications. A wide range of extracellular enzymes, such as alkaline proteases, cellulases, and amylases, have been isolated from the alkaliphilic Bacteria (mostly Bacillus spp.) and used for industrial production (2). The members of these classes of enzymes are also encoded by the haloalkaliphilic Archaea, as exemplified by the haloarchaeal amylase of Natronococcus occultus, which exhibits extracellular activity (2). N. magadii has been phylogenetically classified within the order Halobacteriales, which includes the intensively studied Halobacterium salinarum and Haloferax volcanii (3). For a variety of neutrophilic members of the order Halobacteriales, data about the methods for transformation and genetic manipulation are available (4). To our knowledge, there are no reports about the genetic manipulation of some haloalkaliphilic members of the Archaea. Although a plasmid, pNB101, of the Natronobacterium sp. strain AS7091 (5, 6) was isolated, attempts to transform haloalkaliphilic members of the Archaea with this vector failed (5). Nevertheless, a shuttle vector created by fusion of pNB101 with the Escherichia coli plasmid pKSII⫹ and additional incorporation of a mevinolin resistance cassette was successfully used to transform the halophiles H. salinarum and H. volcanii (6). This indicated that pNB101-based plasmids are, in principle, useful vectors but that standard transformation methods for the halo-
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philic Archaea (7, 8, 9, 10) are not sufficient to efficiently transform the haloalkaliphilic Archaea. Ch1 was the first virus isolated from a member of the haloalkaliphilic branch of the Archaea (11). Ch1 is a head-tail virus belonging to the family Myoviridae. It infects the species N. magadii, thereby eventually causing cell lysis. Two strains of N. magadii are available: the lysogenic strain L11 and strain L13, which has been cured of the virus (11). Ch1 shows remarkable sequence similarity to the halophilic archaeal virus H, which infects H. salinarum (12), and in general the same organization of functional modules, at least for those parts of the H genome that have been sequenced (13, 14). This is evident for the module-containing genes that encode structural proteins. Moreover, the central part of the Ch1 genome, nucleotides (nt) 30,000 to 42,000 (accession no. AF440695), shares sequence identity ranging from 50% to 97% with the so-called L fragment of H. A comparison of the central parts of the 2 viruses revealed that most of the Ch1 open reading frames (ORFs) can also be found on the invertible L segment of H and vice versa. Since the L fragment of H is able to replicate as an autonomous plasmid (pHL) in H. salinarum cells
Received 25 October 2012 Accepted 6 February 2013 Published ahead of print 15 February 2013 Address correspondence to Angela Witte,
[email protected]. M.M.-I. and A.L. contributed equally to this work. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.03287-12. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.03287-12
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In the study described here, we successfully developed a transformation system for halo(alkali)philic members of the Archaea. This transformation system comprises a series of Natrialba magadii/Escherichia coli shuttle vectors based on a modified method to transform halophilic members of the Archaea and genomic elements of the N. magadii virus Ch1. The shuttle vector pRo-5, based on the repH-containing region of Ch1, stably replicated in E. coli and N. magadii and in several halophilic and haloalkaliphilic members of the Archaea not transformable so far. The Ch1 operon ORF53/ORF54 (repH) was essential for pRo-5 replication and was thus identified as the minimal replication origin. The plasmid allowed homologous and heterologous gene expression, as exemplified by the expression of Ch1 ORF3452, which encodes a structural protein, and the reporter gene bgaH of Haloferax lucentense in N. magadii. The new transformation/vector system will facilitate genetic studies within N. magadii and other haloalkaliphilic archaea and will allow the detailed characterization of the gene functions of N. magadii virus Ch1 in their extreme environments.
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(14), it is likely that the respective region of Ch1 harbors the viral origin of replication. Genetic analysis of Ch1 has been hampered so far by the inability to transform its host strain, namely, N. magadii. Here we present a method for the transformation of N. magadii. The method is based on a technique used for the transformation of halophilic members of the Archaea, but it includes additional steps for the efficient generation of spheroplasts. We demonstrate the functionality of the method by employing a series of N. magadii-E. coli shuttle vectors comprising genetic elements of the virus Ch1. Furthermore, we demonstrate the applicability of these vectors for the transformation of not only N. magadii but also a series of other, previously untransformable halo(alkali)philic members of the Archaea. In addition, we also define the origin of replication of Ch1.
Strains, plasmids, and primers. All strains, plasmids, and primers used are listed in Table S1 in the supplemental material. Media and growth conditions. N. magadii and Natronomonas gregoryi were incubated in a nutrient-rich medium (11). Virus titers were determined by plating appropriate dilutions prepared in a nutrient-rich medium as described previously (11). Plates were incubated at 37°C in sealed plastic bags for 1 to 2 weeks. Natrialba asiatica and H. salinarum R1 were grown in 5 g yeast extract, 5 g Casamino Acids, 1 g Na-glutamate, 2 g KCl, 3 g Na3-citrate, 20 g MgSO4 · 7 H2O, 200 g NaCl, 36 mg FeCl2 · 4 H2O, and 0.36 mg MnCl2 · 4 H2O per liter at pH 7. E. coli strains were incubated in Luria-Bertani medium at 37°C as previously described (15). Ampicillin and tetracycline were added to the LB medium to a final concentration of 100 g/ml or 10 g/ml, as required. Halorubrum saccharovorum, Halorubrum coriense, and Halorubrum lacusprofundi were incubated in 18% MGM medium, as described before (16). Transformation of H. volcanii was performed as described previously (7, 9). Novobiocin or mevinolin was added to a final concentration of 3 g/ml or 4 g/ml when required. Transfection and transformation of N. magadii L13. Cells were incubated in nutrient-rich medium containing 75 g/ml bacitracin at 37°C to an optical density of 0.6 (600 nm). The culture was collected by centrifugation (6,000 rpm); the pellet was resuspended in high-salt-buffered solution (http://www.haloarchaea.com/resources/halohandbook/) with 20 g/ml proteinase K and incubated for 48 h at 42°C with agitation. Cells after treatment with bacitracin and/or proteinase K were used to prepare spheroplasts as described previously (7, 9). A 1.5-ml volume of the sample was collected by centrifugation and resuspended in 150 l high-salt solution. After the addition of EDTA (50 mM) and incubation for 10 min at room temperature, DNA was added and incubated for 5 min at room temperature. A 150-l volume of 60% polyethylene glycol (PEG) 600 in high-salt solution was added and incubated for 30 min at room temperature. The cells were washed with nutrient-rich medium. After regeneration of the cells at 37°C for 16 h, the transfection samples were mixed with N. magadii L13 cells and poured on plates with top agar. Transformation samples were plated on agar plates containing novobiocin (3 g/ml) or mevinolin (7.5 g/ml) and incubated for 10 to 15 days at 37°C before determination of plaque forming or numbers of CFU, respectively. DNA isolation from halophilic members of the Archaea and retransformation. Plasmid DNA (50 ng) prepared from N. magadii L13 by a modified alkaline lysis procedure (http://www.haloarchaea.com /resources/halohandbook/) was transformed into E. coli XL1-Blue cells. Plasmid integrity was verified by restriction analysis. Plasmids containing the cloned Ch1 origin of replication were verified by PCR analysis using primers TR-1/TR-4. The integrity of plasmids carrying the origin of replication derived from plasmid pNB101 (5) was assessed using primers NB-3/MevR-4. Chromosomal DNA of N. magadii L13 was isolated as described before (11).
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RESULTS AND DISCUSSION
Spheroplast formation and transfection of N. magadii L13 with Ch1 DNA. Because archaeal genetic markers were unavailable at the time, the first efficient transformations of a halophilic archaeon, namely, Halobacterium salinarum, were performed using DNA isolated from the virus H (7). The polyethylene glycolmediated transformation method described was quickly adapted for the transformation of Haloferax volcanii (8) and various other members of the Archaea, including Methanococcus maripaludis and Pyrococcus abyssi (23, 24). However, this method is effective only in species for which spheroplasts can readily be generated (i.e., H. volcanii and H. salinarum), usually by removing the paracrystalline glycoprotein surface layer (S layer) by EDTA treatment of the cells (4). Using this method, transformation rates as high as 1 ⫻ 106 CFU per microgram of plasmid DNA can be obtained (25). To evaluate the efficacy of N. magadii transformation, we tried to introduce purified Ch1 DNA into N. magadii cells. Since Ch1 virus particles are able to infect the cured N. magadii L13 strain (11), plaque formation on an agar lawn was used as an indication of successful transformation. In a first attempt, the polyethylene glycol-mediated transformation method was used, and the morphology of the cells was monitored by phase-contrast microscopy (Fig. 1). Using this transformation method, neither spheroplast nor plaque formation because of successful transfection was observed (Fig. 2). As shown earlier, the antibiotic bacitracin interferes with the glycosylation of the H. salinarum S layer and thereby inhibits the growth of H. salinarum, but it also causes a morphological change of the cells from rod-shaped to spherical (26, 27). In H. salinarum, bacitracin is thought to interfere with the processing of the dolichol pyrophosphate carrier used for glycosylation of the S-layer glycoprotein (28). Growth of N. magadii L13 with bacitracin (75 g/ml) did not produce spheroplasts but rather cells with pleiotropic morphology (Fig. 1b). Using cells of N. magadii L13 grown in 75 g/ml bacitracin, successful transfection was not observed (Fig. 2). The less-pronounced effect of bac-
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MATERIALS AND METHODS
Plasmid copy number determination. Copy numbers of the different shuttle vector constructs were determined as already described (17). Numbers of CFU were determined by plating. Isolation and purification of virus particles. Virus particles and virus nucleic acids were isolated as previously described (11). Plasmid construction. A description of the constructed plasmids is given in Text S3 of the supplemental material. Hybridization techniques. Southern hybridization was performed as described previously (18) by using the NEBlot Phototope-Star labeling and detection kit (New England BioLabs). Protein manipulations. Western blot analyses were performed as described previously (19), and immune detection was performed using polyclonal antibodies against protein gp36. As a secondary antibody, antirabbit IgG linked to horseradish peroxidase was used. Detection of the antigen-antibody complex was performed using the SuperSignal West Pico chemiluminescent substrate (Pierce). -Galactosidase assays. -Galactosidase (BgaH) activities in N. magadii L13 were determined as described previously (20). The protein concentrations were quantified using the Bradford method (21). Periodic acid-Schiff’s reagent staining. Periodic acid-Schiff’s reagent (PAS) staining was performed as described previously (22). SDS-PAGE gels were incubated in 7.5% acetic acid (30 min, room temperature [RT]) and transferred to 0.2% periodate (1 h, 4°C) and then to Schiff reagent (1 h, 4°C). The stained gels were returned to 7.5% acetic acid (30 min, RT) and extensively washed in water.
Shuttle Vector System for Halo(alkali)philic Archaea
itracin on N. magadii L13 cell morphology can be explained by the lower stability of bacitracin in alkaline solutions (29). Since N. magadii growth requires a high pH of 9.5, bacitracin may quickly become inactivated in the N. magadii growth medium. Methods to transform halophilic members of the Archaea are based on the removal of the outermost S layer, which eventually leads to spheroplast formation and uptake of foreign DNA. As shown by Mescher and Strominger, incubation of H. salinarum cells with insoluble protease changed the morphology of the cells to spheroplasts and led to the removal of the S-layer protein (27). Since the addition of bacitracin or EDTA did not lead to the formation of spheroplasts of N. magadii and therefore did not lead to transfection with Ch1 DNA, we tried to remove the N. magadii S layer by proteinase K treatment. Proteinase K retains activity at high salt concentrations (11). Treatment of N. magadii cells with proteinase K led to the formation of spheroplasts (Fig. 1c), and these cells could be transfected with Ch1 DNA (Fig. 2). Here, a transfection rate of 103 PFU per microgram Ch1 DNA was obtained. Compared to the transfection rates of H. halobium with H DNA (7), this method is significantly less effective. To improve the efficacy of transfection, a combined method was used: cells were grown in nutrient-rich medium in the presence of bacitracin (75 g/ml) to an optical density of 0.6 (measured at 600 nm) before treatment with proteinase K (20 g/ml) for an additional 48 h at 42°C. Treatment of N. magadii L13 with bacitracin/ proteinase K caused a morphological change from the normal rod shape to spherical (Fig. 1a and c). The cells remained viable, and their rod-shaped morphology was obtained by incubation in nutrient-rich medium lacking proteinase K (Fig. 1d). These observations indicated that a cell wall protein, probably the putative Slayer protein, had been removed from the cell surface of N. magadii. SDS-PAGE gels containing extracts of N. magadii cells before and after incubation with proteinase K were stained for glycoproteins with periodic acid-Schiff’s reagent (PAS staining) (22). A specific band of approximately 110 kDa, degraded by the protease after 48 h of incubation, was detected (data not shown). Other detectable proteins did not change (data not shown). Incu-
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bation of the cells with proteinase K led to the formation of spheroplasts concomitant with the disappearance of the major glycosylated protein. These results suggest that the glycoprotein removed by combined treatment with bacitracin/proteinase K is a major envelope structural component responsible for the maintenance of cell shape in N. magadii L13. When bacitracin/proteinase K-treated cells were incubated with Ch1 DNA, we were able to transfect with Ch1 DNA at a rate of 104 PFU/g DNA (Fig. 2). Although the transfection rate was lower than the transfection/transformation rates of other halophilic strains, this method enables genetic manipulation of N. magadii and functional analysis of Ch1 elements in their natural host. We therefore used this method to develop an E. coli/N. magadii shuttle vector system based on elements of the Ch1 genome. Construction and efficiency of a shuttle vector for N. magadii L13. Sequence analysis of the minimal replication origin of the plasmids of halophilic Archaea showed that a unique gene, repH, and an AT-rich region located upstream of the gene were required (accession number AF440695) (30). Elimination of either the ATrich sequence or the repH gene abolished the autonomous replication ability of the plasmids (30). Sequence similarity analysis revealed an open reading frame (ORF54) within the Ch1 genome with similarities to the protein-encoding sequences of halophilic plasmids (12). ORF54 encodes a large 581-amino-acid (aa) protein with a calculated size of 65.2 kDa and an isoelectric point of 4.8. The predicted protein shares similarities (highest similarity using a BLASTP alignment, 5 ⫻ 10⫺46) with archaeal proteins encoded by Haloarcula marismortui plasmid pNRC100, H. salinarum plasmid pHH1, H. salinarum pHL, Halobacterium sp. (strain NRC-1) plasmid pNRC100, and Haloferax volcanii plasmid pHV2. Some of the proteins are essential components of the minimal replicons of the plasmids in halophilic members of the Archaea (30, 31). Analysis of the deduced amino acid sequence of ORF54 using the software program COILS revealed a putative coiled-coil domain located in the central part of the protein (aa 254 to 282; data not shown). However, ORF54 is smaller than its homologs, and sequence similarities were found only in the C-ter-
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FIG 1 Preparation of spheroplasts of N. magadii L13. (a to d) Micrographs of N. magadii. Cells were grown in nutrient-rich medium (a) and in the presence of bacitracin (b) to an optical density of 0.6, followed by incubation of the cells with proteinase K for 48 h at 42°C, and treated with PEG 600 (c). Cells were regenerated (48 h) in nutrient-rich medium and incubated at 37°C (d).
FIG 2 Determination of the transfection efficiency of N. magadii L13 with Ch1 DNA. Ch1 DNA (1 g) was used to transform 1 ⫻ 108 cells after different treatments. No treatment, competent cells were prepared as described before (7); bacitracin, treatment with bacitracin; proteinase K, treatment with proteinase K; bacitracin/proteinase K, treatment with bacitracin and proteinase K as described previously (7). The efficiency is given in PFU per g on the left side. Transfection assays were performed in triplicate. Error bars are indicated, ⫾1 SD.
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Shuttle Vector System for Halo(alkali)philic Archaea
DNA than for methylated DNA was reported for H. volcanii (18). This is due to a restriction-modification system in H. volcanii that seems to degrade DNA methylated in a Dam- and/or Dcm-like fashion. To investigate such a possible effect for N. magadii L13, unmethylated and methylated pRo-5 DNA was used for transformations. Plasmid DNA was isolated from E. coli strain JM110 (dam dcm), and N. magadii L13 was transformed with this DNA. No increase in the transformation rates was detected when unmethylated plasmid DNA was used (Fig. 3b). This suggests that N. magadii lacks or has a restriction-modification system different from that of H. volcanii and that transformation is independent of the methylation status of the plasmid DNA. Palindromic sequences were found adjacent to the AT-rich sequences of the upstream region of ORF53 and the downstream region of ORF54 (Fig. 3a). To analyze the importance of these sequences, one (pRo-7 and pRo-9) or both (pRo-10) of them were deleted. Transformation assays revealed that these palindromic sequences are nonessential for plasmid replication in N. magadii L13 (Fig. 3b). The importance of ORF53 and ORF54 was proven by the introduction of frame shifts into both genes (pRo-8 and pRo-11). This created a stop codon in both cases, and both plasmids were unable to replicate in N. magadii L13. Therefore, both genes, ORF53 and ORF54, seem to be essential for replication in N. magadii. Suitability of the shuttle vector for functional analysis of Ch1 genes or establishment of reporter gene assays. In a former study, we investigated the putative function of ORF48 (32). By similarity searches and by analyzing the mutant strain Ch1-1, 2 genes, ORF48 (rep) and ORF49, were identified as encoding possible regulators of the Ch1 life cycle. Both genes are arranged head-to-head. Promoter consensus sequences (AT-rich sequences typical for halophilic members of the Archaea [33]) are present in the intergenic region of the 2 genes. Investigation of the ORF48ORF49 intergenic region in H. volcanii revealed promoter activity. Data indicated that ORF48 could act as a repressor via binding to 2 direct repeats located in its coding region (32). Expression of the repressor gene rep dramatically decreased the expression level of the reporter gene bgaH when transcribed from the ORF49 promoter. These results suggested that Rep, the gene product of ORF48, shuts down expression of ORF49 and thus probably acts as a transcriptional repressor. This is in accordance with protein structure predictions suggesting that Rep belongs to the family of winged helix repressor proteins (32). To verify the function of ORF48 as a repressor, the rep gene and its upstream region were cloned into pRo-5. N. magadii L13 was transformed with the plasmid and subsequently infected with Ch1. The relative plating efficiency of Ch1 on the strain N. magadii L13 (pRo-5/2) carrying the repressor gene ORF48 was significantly reduced, by 3 or-
FIG 3 Presentation of the putative origin of replication of Ch1. (a) Part of the Ch1 sequence is shown (nt 33701 to 38000) (12). Large arrows indicate ORF53 and ORF54, as well as ORF49 and ORF55. Sequence similarities of ORF53 and ORF54 to the pNRC100 replication protein H-like open reading frame of H. marismortui are indicated (3 ⫻ 10⫺7 and 3 ⫻ 10⫺45, respectively), as well as the open reading frame. The promoter within the 5= region of ORF49 is indicated with an arrow and marked with “P.” Arrows mark the palindrome sequences. Lanes indicate the parts of the sequence cloned, and the names of the different constructs are given on the right. The asterisk and the vertical lane of the clones pRo-8 and pRo-11 indicate the sides where frame shifts were introduced into ORF53 and ORF54, respectively. The interruptions in the lines indicate the introduced deletions. (b) Transformation efficiency of N. magadii L13 with different constructs. The efficiency is given in CFU per microgram of plasmid (CFU/g) on the left side, and the clones used in this study are indicated at the bottom. pRo-5*, plasmid DNA of pRo-5 was isolated from the dam- and dcm-lacking E. coli strain JM110 and transformed into N. magadii L13. Transformation assays were performed in triplicate. Error bars are indicated, ⫾1 SD. (c) Physical map of the shuttle vector pRo-5. Black area shows ColE1 origin of replication; dark gray arrow indicates -lactamase (bla); gray arrow indicates novobiocin resistance cassette (gyrB); light gray arrows indicate putative origin of Ch1. Positions of the restriction sites are (clockwise from the top) NotI (3385), EcoRV (3841), HindIII (7065), and KpnI (7105). The positions of the sites are given in parentheses.
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minal parts of these proteins. ORF53, located upstream of ORF54, had a lower but significant similarity to the RepH protein encoded by H. marismortui, namely, pNRC100 (Fig. 3a). This similarity is restricted to the N-terminal part of RepH. The same arrangement was found in the genome of the closest relative of Ch1, i.e., H. salinarum virus H. Both genes are part of the autonomously replicating plasmid pH-L (13, 14). In analogy to regions found upstream of genes involved in the replication of plasmids of halophilic Archaea, an AT-rich region was also detected upstream of ORF53, and a second one was identified downstream of ORF54. No such area was seen in the 5= region of ORF54 (Fig. 3a). Taken together, it is reasonable that the above-described sequence comprises the minimal replicon of Ch1. Therefore, this region was investigated for its capability to promote autonomous replication in N. magadii and ultimately for its usability to develop a shuttle vector system for E. coli/N. magadii. Because of the lack of a selectable marker for haloalkaliphilic members of the Archaea, the cloned and mutated gyrB gene of halophilic Haloferax alicantei, which confers resistance toward novobiocin (17), was used as a selection marker. The gene was isolated from the plasmid pMDS11 (25) and cloned into the vector pKSII⫹, giving rise to the plasmid pNov-1. In a second step, different parts of the region containing ORF53 and ORF54 (Fig. 3a) were cloned into pNov-1, and the resulting plasmids were tested for their ability to autonomously replicate upon transformation of N. magadii L13. As shown in Fig. 3b, the plasmids pRo-1 and pRo-2, lacking the promoter region upstream of ORF49 as well as the AT-rich region of the ORF53 upstream region, were not able to replicate in N. magadii L13. The AT-rich region downstream of ORF54 (pRo-2) alone was not able to maintain the plasmid in N. magadii L13. In contrast, replication in N. magadii L13 was achieved with the plasmid pRo-4, containing the entire region described above. Only the plasmids pRo-3, pRo-5, pRo-6, pRo-7, and pRo-10 were able to transform N. magadii with satisfactory transformation rates of 103 CFU/g DNA. The transformation rate of the plasmid pRo-9 was only slightly reduced, to 102 CFU/g DNA. The plasmids pRo-3 and pRo-6 contain open reading frames that encode possible transcriptional repressors: ORF49 seems to influence the infection cycle of Ch1, as demonstrated by the earlier onset of lysis in a Ch1 mutant harboring a mutated ORF49 (19), and ORF55 displays similarities to known transcriptional regulators (data not shown). To avoid interference with ORF49 or ORF55 in upcoming genetic studies, all subsequent experiments were performed with pRo-5. It lacks the start codons of ORF49 and ORF55. A schematic representation of pRo-5 is shown in Fig. 3c. A detailed characterization of strain N. magadii (pRo-5) can be found in Text S1 of the supplemental material. Previously, higher transformation efficiency for unmethylated
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FIG 4 Expression of ORF34 in N. magadii L13. Cell extracts of N. magadii
ders of magnitude, from that with the wild-type strain, N. magadii L13 (pRo-5) (1 ⫻ 107 PFU/ml and 1 ⫻ 1010 PFU/ml, respectively). The plaques observed by infection of Ch1 on the strain expressing the rep gene were turbid, as were plaques found on plates with the wild-type strain. No “clear plaque” mutants appeared. These results support the previous findings that Rep acts as a repressor, and they agree with the results obtained for the related Rep protein, H (34). Similarly reduced infectivity was observed upon expression of the rep gene from a plasmid. In both cases, the data suggest the activity of the repressors is weak, not comparable to the activity of strong repressors, such as CI encoded by E. coli phage (35). The bgaH gene of H. lucentense is a suitable reporter gene for the halophilic Archaea (36, 37). It has also been used by us to investigate the promoter activity of the intergenic region between rep and ORF49 of Ch1, as well as the influence of rep on transcription in H. volcanii (31). To establish the bgaH gene as a reporter for gene expression in N. magadii, the gene, including its own promoter region, was cloned into pRo-5, resulting in pRo-5/ BgaH. BgaH activity in N. magadii L13 (pRo-5/BgaH) was low (13 mU/g protein) compared to the activity in H. volcanii (500 mU/g protein) when the gene was expressed from pMLH32 (20). One possible explanation for this result could be that the promoter of bgaH was not as efficiently recognized in N. magadii as in H. volcanii. To circumvent a possible dysfunctionality of the bgaH promoter in N. magadii, we tried to express the bgaH gene from the Ch1 promoter of the ORF48-ORF49 intergenic region (31). The intergenic region of Ch1 and the bgaH gene were isolated from plasmid pMI-1 (31) and introduced into the shuttle vector pRo-5, resulting in pRo-5/1. BgaH activities in N. magadii L13 transformed with pRo-5/1 were in the range of 100 mU/g protein (data not shown), which is comparable to the promoter activity of this region when used to express the bgaH gene in H. volcanii (31). Thus, combining the bgaH gene with the ORF48ORF49 intergenic region of Ch1 allowed us to establish a functional reporter system for future genetic analyses of N. magadii or its virus Ch1. In a third approach to evaluate the efficacy of the N. magadii transformation system, we tried to express the Ch1 tail fiberencoding gene ORF3452 (38, 39) in N. magadii. The gene, as well as its upstream region, was cloned into pRo-5, creating pRo-5/3452.
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TABLE 1 Transformation of halo(alkali)philic members of the Archaea with pRo-5 Strain
Transformation with pRo-5
Plasmid stabilitya
N. magadii L13 H. saccharovorum H. coriense H. lacusprofundi H. volcanii WFD11 H. volcanii WFD11(pMDS24) H. salinarum R1 N. asiatica N. gregoryi
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹/⫺ ND ⫹ ⫹ ⫹
a Plasmid stability was determined by isolation of plasmid DNA from transformed cells, retransformation of the DNA into E. coli cells, reisolation, and restriction of the plasmid DNA with SacI (performed in triplicate). ND, not determined; ⫹/⫺, 50% of the plasmid samples were stable.
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were separated on a 12% PAA gel and subjected to Western blot analyses with anti-ORF 36 antibodies. Lane 1, purified protein gp3452; lane 2, N. magadii L13 (pRo-5); lane 3, N. magadii L13 (pRo-5/3452). Samples were taken in the stationary growth phase of the cultures. Purification of protein gp3452 was performed as described previously (39). The molecular size marker is indicated on the left, and the gp3452 signal is marked with an arrow on the right.
After transformation of N. magadii L13 with the plasmid, crude extracts were prepared and separated by SDS-PAGE. The presence of gp3452, the gene product of ORF ORF3452, was investigated by Western blotting using anti-ORF36 antibodies. The cross-reactivity of the anti-ORF36 antiserum with ORF34 and ORF36 results from the occurrence of the peptide repeats MDAV within both proteins (38). As shown in Fig. 4, a gp3452 signal could be detected only with crude extracts of strain N. magadii L13 (pRo-5/3452) (Fig. 4, lane 3), not with crude extracts of the control strain N. magadii L13 (pRo-5) (Fig. 4, lane 2). The results demonstrate the successful expression of ORF3452 in N. magadii after transformation with the plasmid pRo-5/3452. Transformation of other halo(alkali)philic members of the Archaea. To determine the host range of pRo-5, we attempted to transform other halophilic and haloalkaliphilic Archaea with the plasmid (Table 1). Besides strains commonly used as model organisms for genetic studies, such as H. volcanii and H. salinarum, other halo(alkali)philic Archaea that have not been transformed so far were tested. Successful transformation was achieved with all strains, i.e., Halorubrum saccharovorum, H. coriense, H. lacusprofundi, H. volcanii WFD11, Natronomonas gregoryi, and H. salinarum. Therefore, we demonstrated that the region of Ch1 minimally required for pRo-5 propagation in N. magadii is also sufficient to maintain the plasmid in a variety of other halophilic archaeal strains. To investigate the compatibility of the Ch1 origin of replication with that of other plasmids of halophilic Archaea, we tried to cotransform H. volcanii WFD11 carrying pMDS24 (16) with pRo-5. Successful transformation was feasible in this particular situation, thereby demonstrating that cotransfections with pRo-5 and plasmids containing pHV2-originating origins of replication, such as pMDS24, are achievable (Table 1). Therefore, both origins of replication seem to belong to different incompatibility groups (40). This is supported by the relatively low sequence similarity between the RepH protein encoded by pHV2 and the ORF53/54 proteins of Ch1 (31% identity using a BLAST search) (41; data not shown). In general, we were able to extend the host range for pRo-5 to a variety of other halo(alkali)philic strains, making the plasmid a very valuable tool for simple genetic manipulation of a whole set of archaeal strains. The compatibility with other plasmids commonly used for the transformation of the halophilic Archaea is an additional ben-
Shuttle Vector System for Halo(alkali)philic Archaea
REFERENCES 1. Tindall BJ, Mills AA, Grant WD. 1984. Natronobacterium gen. nov. and Natronococcus gen. nov., two new genera of haloalkaliphilic archaebacteria. Syst. Appl. Microbiol. 5:41–57. 2. Horikoshi K. 1999. Alkaliphiles: some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 63:735–750. 3. Soppa J, Baumann A, Brenneis M, Dambeck M, Hering O, Lange C. 2008. Genomics and functional genomics with haloarchaea. Arch. Microbiol. 190:197–215. 4. Allers T, Mevarech M. 2005. Archaeal genetics—the third way. Nat. Rev. Genet. 6:58 –73. 5. Zhou M, Xiang H, Sun C, Li Y, Liu J, Tan H. 2004. Complete sequence and molecular characterization of pNB101, a rolling-circle replication plasmid from the haloalkaliphilic archaeon Natronobacterium sp. strain AS7091. Extremophiles 8:91–98. 6. Zhou M, Xiang H, Sun C, Tan H. 2004. Construction of a novel shuttle vector based on an RCR-plasmid from a haloalkaliphilic archaeon and transformation into other haloarchaea. Biotechnol. Lett. 26:1107–1113. 7. Cline SW, Doolittle WF. 1987. Efficient transfection of the archaebacterium Halobacterium halobium. J. Bacteriol. 169:1341–1344. 8. Charlebois RL, Lam WL, Cline SW, Doolittle WF. 1987. Characterization of pHV2 from Halobacterium volcanii and its use in demonstrating transformation of an archaebacterium. Proc. Natl. Acad. Sci. U. S. A. 84:8530 – 8534. 9. Cline SW, Schalkwyk LC, Doolittle WF. 1989. Transformation of the archaebacterium Halobacterium volcanii with genomic DNA. J. Bacteriol. 171:4987– 4991. 10. Holmes ML, Dyall-Smith ML. 1991. Mutations in DNA gyrase result in novobiocin resistance in halophilic archaebacteria. J. Bacteriol. 173:642– 648. 11. Witte A, Baranyi U, Klein R, Sulzner M, Luo C, Wanner G, Krüger DH, Lubitz W. 1997. Characterization of Natronobacterium magadii phage Ch1, a unique archaeal phage containing DNA and RNA. Mol. Microbiol. 23:603– 616. 12. Klein R, Baranyi U, Rössler N, Greineder B, Witte A. 2002. Natrialba magadii virus Ch1: first complete nucleotide sequence and functional organization of a virus infecting a haloalkaliphilic archaeon. Mol. Microbiol. 45:851– 863.
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13. Stolt P, Grampp B, Zillig W. 1994. Genes for DNA cytosine methyltransferases and structural proteins, expressed during lytic growth by the phage H of the archaebacterium Halobacterium salinarium. Biol. Chem. Hoppe Seyler 375:747–757. 14. Gropp F, Grampp B, Stolt P, Palm P, Zillig W. 1992. The immunityconferring plasmid pHL from the Halobacterium salinarium phage H: nucleotide sequence and transcription. Virology 190:45–54. 15. Miller JH. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 16. Jolley KA, Rapaport E, Hough DW, Danson MJ, Woods WG, DyallSmith ML. 1996. Dihydrolipoamide dehydrogenase from the halophilic archaeon Haloferax volcanii: homologous overexpression of the cloned gene. J. Bacteriol. 178:3044 –3048. 17. Holmes ML, Dyall-Smith ML. 1990. A plasmid vector with a selectable marker for halophilic archaebacteria. J. Bacteriol. 172:756 –761. 18. Klein R, Greineder B, Baranyi U, Witte A. 2000. The structural protein E of the archaeal virus Ch1: evidence for processing in Natrialba magadii during virus maturation. Virology 276:376 –387. 19. Harlow E, Lane D. 1988. Antibodies. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 20. Holmes ML, Dyall-Smith ML. 2000. Sequence and expression of a halobacterial -galactosidase gene. Mol. Microbiol. 36:114 –122. 21. Bradford MM. 1976. A rapid and sensitive method for quantitation of microgram quantites of protein utilising the principle of protein dyebinding. Anal. Biochem. 72:248 –254. 22. Dubray G, Bezard G. 1982. A highly sensitive periodic acid silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal. Biochem. 119:325–329. 23. Tumbula DL, Makula RA, Whitman WB. 1994. Transformation of Methanococcus maripaludis and identification of a PstI-like restriction system. FEMS Microbiol. Lett. 121:309 –314. 24. Lucas S, Toffin L, Zivanovic Y, Charlier D, Moussard H, Forterre P, Daniel Prieur D, Erauso G. 2002. Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi. Appl. Environ. Microbiol. 68:5528 –5536. 25. Holmes ML, Nuttall SD, Dyall-Smith ML. 1991. Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase. J. Bacteriol. 173:3807–3813. 26. Mescher MF, Strominger JL. 1975. Bacitracin induces sphere formation in Halobacterium species which lack a wall peptidoglycan. J. Gen. Microbiol. 89:375–378. 27. Mescher MF, Strominger JL. 1976. Structural (shape-maintaining) role of the cell surface glycoprotein of Halobacterium salinarum. Proc. Natl. Acad. Sci. U. S. A. 73:2687–2691. 28. Wieland F, Lechner J, Bernhardt G, Sumper M. 1981. Halobacterial glycoprotein saccharides contain covalently-linked sulphate. FEBS Lett. 132:319 –323. 29. Pavli V, Kmetec V. 2006. Pathways of chemical degradation of polypeptide antibiotic bacitracin. Biol. Pharm. Bull. 29:2160 –2167. 30. Ng WL, DasSarma S. 1993. Minimal replication origin of the 200kilobase Halobacterium plasmid pNRC100. J. Bacteriol. 175:4584 – 4596. 31. Pfeifer F, Ghahraman P. 1993. Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas vesicle gene cluster and insertion elements. Mol. Gen. Genet. 238:193–200. 32. Iro M, Klein R, Gálos B, Baranyi U, Rössler N, Witte A. 2007. The lysogenic region of virus Ch1: identification of a repressor-operator system and determination of its activity in halophilic Archaea. Extremophiles 11:383–396. 33. Soppa J. 1999. Normalized nucleotide frequencies allow the definition of archaeal promoter elements for different archaeal groups and reveal basespecific TFB contacts upstream of the TATA box. Mol. Microbiol. 31: 1589 –1592. 34. Stolt P, Zillig W. 1993. In vivo and in vitro analysis of transcription of the L region from the Halobacterium salinarium phage H: definition of a repressor-enhancing gene. Virology 195:649 – 658. 35. Ptashne M. 1987. A genetic switch. Blackwell Scientific Publications and Cell Press, Oxford, United Kingdom. 36. Patenge N, Haase A, Bolhuis H, Oesterhelt D. 2000. The gene for a halophilic -galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum. Mol. Microbiol. 36: 105–113. 37. Gregor D, Pfeifer F. 2001. Use of a halobacterial bgaH reporter gene to
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efit and allows for more complex cotransformation experiments (for details, see Text S2 in the supplemental material). Conclusions. We have developed a method for the transformation of the haloalkaliphilic archaeon N. magadii. Stable plasmid replication was achieved with pNB102, an engineered version of a plasmid originally isolated from the haloalkaliphilic Natronobacterium sp. strain AS7091, but also with a series of plasmids constructed in this study that are based on the genetic elements of N. magadii virus Ch1. Thereby, we were also able to experimentally determine the origin of replication of Ch1. The plasmids were stably maintained within the cells, did not integrate into the host chromosome, and did not interfere with the growth rate of the cells. As demonstrated, the plasmids can be used for expression of homologous and heterologous genes in N. magadii. The Ch1-derived plasmid pRo-5 replicated not only in N. magadii but also in a series of other halo(alkali)philic Archaea, making it a universal tool for simple (comparative) genetic studies in a broad range of the Archaea. The plasmid was compatible with other plasmids containing either a H. volcanii pHV2- or Natronobacterium sp. strain AS7091 pNB101derived origin of replication, thus also allowing cotransformation experiments and simple expression of sets of genes. Taken together, the development of the described transformation method and construction of plasmids for replication in N. magadii make this organism accessible to genetic manipulation and open the path for a detailed characterization of gene functions of N. magadii virus Ch1.
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analyse the regulation of gene expression in halophilic archaea. Microbiology 147:1745–1754. 38. Rössler N, Klein R, Scholz H, Witte A. 2004. Inversion within the haloalkaliphilic virus Ch1 DNA results in differential expression of structural proteins. Mol. Microbiol. 52:413– 426. 39. Klein R, Rössler N, Iro M, Scholz H, Witte A. 2012. Haloarchaeal myovirus Ch1 harbors a phase variation system for the production of
protein variants with distinct cell surface adhesion specificities. Mol. Microbiol. 83:137–150. 40. Rawlings DE, Tietze E. 2001. Comparative biology of IncQ and IncQ-like plasmids. Microbiol. Mol. Biol. Rev. 65:481– 496. 41. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389 –3402.
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Applied and Environmental Microbiology
