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Research into archaea will not achieve its full potential until systems are in place to carry out genetics and biochemistry in the same species. Haloferax volcanii is widely regarded as the best-equipped organism for archaeal genetics, but the development of tools for the expression and purification of H. volcanii proteins has been neglected. We have developed a series of plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature the tryptophan-inducible p.tnaA promoter and a 6×His tag for protein purification by metal affinity chromatography. Purification is facilitated by host strains, where pitA is replaced by the ortholog from Natronomonas pharaonis. The latter lacks the histidine-rich linker region found in H. volcanii PitA and does not copurify with His-tagged recombinant proteins. We also deleted the mrr restriction endonuclease gene, thereby allowing direct transformation without the need to passage DNA through an Escherichia coli dam mutant.
Over the past century, our understanding of fundamental biological processes has grown exponentially, and this would have been impossible without the use of organisms that are amenable to experimental manipulation. Model species, such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Arabidopsis thaliana, have become a byword for scientific progress (15). The rational choice of a model organism is critically important, and certain features are taken for granted, such as ease of cultivation, a short generation time, and systems for genetic manipulation. This list has now grown to include a genome sequence and methods for biochemical analysis of purified proteins in vitro.
Research into archaea has lagged behind work on bacteria and eukaryotes but has nonetheless yielded profound insights (2). One hurdle has been the paucity of archaeal organisms suitable for both biochemistry and genetics. For example, Methanothermobacter thermautotrophicus is a stalwart of archaeal biochemistry but has proved resistant to even the most rudimentary genetic manipulation (2). Progress has recently been made with another biochemical workhorse, Sulfolobus spp., and a few genetic tools are now available (6, 13, 37). Methanosarcina spp. and Thermococcus kodakaraensis offer alternative systems with an increasing array of techniques (16, 35, 36), but sophisticated genetics has traditionally been the preserve of haloarchaea, of which Haloferax volcanii is the organism of choice (39). It is easy to culture, the genome has been sequenced (19), and there are several selectable markers and plasmids for transformation and gene knockout (3, 7, 31), including a Gateway system (14), as well as reporter genes (20, 33) and a tightly controlled inducible promoter (26).
The genetic prowess of H. volcanii is not yet fully matched by corresponding systems for protein overexpression and purification. Like other haloarchaea, H. volcanii grows in high salt concentrations (2 to 5 M NaCl), and to cope with the osmotic potential of such environments, it accumulates high intracellular concentrations of potassium ions (12). Consequently, halophilic proteins are adapted to function at high salt concentrations and commonly feature a large excess of acidic amino acids; the negative surface charge is thought to be critical to solubility (28). This can pose problems for expression in heterologous hosts, such as E. coli, since halophilic proteins can misfold and aggregate under conditions of low ionic strength. The purification of misfolded halophilic enzymes from E. coli has relied on the recovery of insoluble protein from inclusion bodies, followed by denaturation and refolding in hypersaline solutions (8, 11). This approach is feasible only where the protein is well characterized and reconstitution of the active form can be monitored (for example, by an enzymatic assay). Furthermore, archaeal proteins expressed in heterologous bacterial hosts lack posttranslational modifications, such as acetylation or ubiquitination (4, 22), which are critical to understanding their biological function.
Systems for expression of halophilic proteins in a native haloarchaeal host are therefore required. A number of studies have successfully purified recombinant proteins with a variety of affinity tags after overexpression in H. volcanii. For example, Humbard et al. employed tandem affinity tagging to purify 20S proteasomal core particles from the native host (23). However, the protein expression constructs used in these studies were custom made and somewhat tailored to the application in question. We report here the development of “generic” plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature a tryptophan-inducible promoter derived from the tnaA gene of H. volcanii (26). We demonstrate the utility of these vectors by overexpressing a hexahistidine-tagged recombinant version of the H. volcanii RadA protein. Purification was greatly facilitated by a host strain in which the endogenous pitA gene was replaced by an ortholog from Natronomonas pharaonis. The latter protein lacks the histidine-rich linker region found in H. volcanii PitA (5) and therefore does not copurify with His-tagged recombinant proteins. Finally, we deleted the mrr gene of H. volcanii, which encodes a restriction enzyme that cleaves foreign DNA methylated at GATC residues. The mrr deletion strain allows direct transformation of H. volcanii without the need to passage plasmid DNA through an E. coli dam mutant (21).
Unless stated otherwise, chemicals were from Sigma and restriction enzymes were from New England Biolabs. Standard molecular techniques were used (34).
H. volcanii strains (Table (Table1)1) were grown at 45°C on complete (Hv-YPC) or Casamino Acids (Hv-Ca) agar or in Hv-YPC or Hv-Ca broth, as described previously (3, 17). Gene deletion/replacement mutants were constructed using a knockout system described previously (3, 7). The plasmids for gene deletion/replacement and protein overexpression are shown in Table Table22 and were generated by PCR using the primers in Table Table3.3. E. coli strains XL1-Blue MRF′ (ΔmcrA183 ΔmcrCB-hsdSMR-mrr173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacIqZΔM15 Tn10]) and GM121 (F− dam-3 dcm-6 ara-14 fhuA31 galK2 galT22 hdsR3 lacY1 leu-6 thi-1 thr-1 tsx-78) were grown in Luria-Bertani medium with 100 μg/ml ampicillin where appropriate. The latter strain was used to prepare unmethylated DNA for transformation of H. volcanii.
A starter culture was grown overnight in 40 ml Hv-Ca broth to an optical density at 650 nm (OD650) of ~0.6 and used to inoculate 360 ml Hv-YPC broth containing 1 mM tryptophan (Trp) to induce protein expression. The culture was incubated at 42°C with shaking (175 rpm) for 5 to 6 h to an OD650 of ~0.5, when protein expression was further induced by adding 36 ml prewarmed 25 mM Trp dissolved in 18% salt water (SW) (3), and the culture was incubated at 42°C with shaking for a further 1 h. The culture was then centrifuged at 3,300 × g for 10 min at 4°C, and the cells were resuspended in 7 ml ice-cold binding buffer (2 M NaCl, 20 mM HEPES, pH 7.5, 20 mM imidazole, 1 mM phenylmethanesulfonyl fluoride) and lysed by sonication on ice until the suspension was no longer turbid. The cell lysate was clarified by centrifugation at 16,000 × g for 15 min at 4°C and incubated overnight at 4°C with 0.5 ml of IMAC Sepharose 6 FastFlow beads (GE Healthcare) that had been charged with Ni2+ and equilibrated in binding buffer. The slurry was applied to a Poly-Prep column (Bio-Rad), and the flowthrough was collected and reloaded onto the column, followed by three washes with 4 ml of ice-cold binding buffer and one wash with 1 ml ice-cold binding buffer containing 50 mM imidazole. Bound protein was eluted with 1 ml binding buffer containing 500 mM imidazole. Samples were analyzed on 12.5% SDS-PAGE gels with PageRuler size marker (Fermentas), and quantification of protein bands stained with Coomassie blue was carried out using ImageGauge v4.22 (Fuji).
Proteins in gel bands were reduced, carboxyamidomethylated, and digested with Trypsin Gold (Promega) on a robotic platform for protein digestion (MassPREP station; Waters). The resulting peptides were analyzed by electrospray ionization-tandem mass spectrometry (MS/MS) after on-line separation on a PepMap C18 reversed-phase, 75-μm-inner-diameter, 15-cm column (LC Packings) on a CapLC system attached to a Q-TOF2 mass spectrometer equipped with a nanolockspray source (Waters) and operated with MassLynx version 4.0 acquisition software. ProteinLynxGlobalSERVER software version 2.1 (Waters) was used to generate a peak list file of uninterpreted fragment mass data, which was used to search the Swissprot 57.1 and NCBInr databases using the MASCOT search engine (32). Only protein identifications with probability-based MOWSE scores above a threshold of P < 0.05 were accepted. The H. volcanii genome sequence (19) is not currently available for interrogation with uninterpreted mass spectral data; therefore, protein identifications relied on matches to publicly available sequences from related haloarchaea (see Table Table4),4), which were used to find corresponding sequences in the H. volcanii genome at HaloLex (https://www.halolex.mpg.de/public/).
Alignment of the Mrr core domain was carried out in MacVector using ClustalW (BLOSUM; penalty for open gap = 10; extend gap = 2). A neighbor-joining tree was built using Schizosaccharomyces pombe SPAC824.03c as an outgroup.
The sequences of the major plasmid vectors constructed in this study have been deposited in the EMBL nucleotide sequence database under accession numbers FN645893 (pTA927), FN645892 (pTA929), FN645894 (pTA949), FN645891 (pTA962), and FN645890 (pTA963).
A series of plasmid shuttle vectors for conditional protein overexpression that utilized the p.tnaA tryptophanase promoter of H. volcanii were constructed (Fig. (Fig.1).1). Genes under the control of the p.tnaA promoter show rapid and strong induction of expression upon addition of ≥1 mM tryptophan (26). For expression of proteins with an N-terminal hexahistidine (6×His) tag, a (CAC)6 tract was incorporated downstream of the p.tnaA promoter; plasmid variants without the 6×His tag were also generated (Fig. (Fig.1).1). To ensure that the gene was insulated from read-through transcription initiated elsewhere on the plasmid, the expression cassette was flanked by two transcriptional terminators, the L11e rRNA terminator (t.L11e) (38) and a synthetic terminator (t.Syn) comprising a T tract flanked by G/C-rich sequences. The vectors were based on pTA230 (3) and used the pHV2 replication origin, which maintained the plasmid at a copy number of ~6 per genome equivalent (10). Selection was based on the pyrE2 and hdrB markers, which allowed growth on media lacking uracil and thymidine, respectively (3, 7). The former marker complemented the pyrE2 deletion found in almost all laboratory strains of H. volcanii (3), while the latter marker allowed the plasmid to be maintained in rich medium (Hv-YPC); plasmid variants are available with the pyrE2 marker only (Fig. (Fig.11).
The H. volcanii PitA protein is a fusion of chlorite dismutase-like and antibiotic synthesis monooxygenase-like domains within a single open reading frame (5). PitA is unique to haloarchaea and in almost all species features a histidine-rich linker between the conserved N- and C-terminal domains (Fig. (Fig.2A).2A). Owing to the numerous histidines in this region, PitA is a major contaminant of His-tagged recombinant proteins purified from H. volcanii by immobilized metal affinity chromatography (5). We attempted to delete pitA by the pop-in/pop-out technique based on the pyrE2 counterselectable marker (7), using the deletion constructs pMM1231 and pMM1232. In the latter construct, the pitA coding sequence is replaced with an hdrB marker, allowing direct selection for gene deletion events (3). With either construct (in strains WR755 and WR756, respectively), we were unable to recover cells with a pitA deletion (data not shown), indicating that this gene is most likely essential.
The PitA ortholog from the haloalkaliphile Natronomonas pharaonis is unique in that it does not feature a high number of histidines in the central linker region. We reasoned that replacing H. volcanii pitA (pitAHvo) with the N. pharaonis gene (pitANph) would prevent copurification with His-tagged recombinant proteins. Gene replacement with the construct in pTA1106 (Fig. (Fig.2B)2B) was carried out using the pop-in/pop-out technique (3, 7). Successful replacement of pitAHvo with pitANph was established by colony lift and verified by both PCR and Southern blotting of a restriction digest (Fig. 2C to F). The resulting H. volcanii strains, H1154 and H1155, with the pitANph gene replacement showed no obvious growth defects in Hv-YPC and Hv-Ca broth and agar.
6xHis-tagged RadA was used to test the conditional protein overexpression system. RadA is the archaeal RecA family recombinase; it forms a nucleoprotein filament with single-stranded DNA and catalyzes strand exchange during homologous recombination (18, 40). Purification of recombinant 6×His-tagged RadA from E. coli has proved problematic. The protein copurifies with DNA, and removal of the latter requires harsh conditions (denaturation and benzonase treatment) that interfere with RadA activity (K. Bunting, personal communication).
The radA coding sequence was cloned in pTA963 and used to transform H. volcanii strains H98 and H1155. To determine the optimal induction regime for protein overexpression, the conditions shown in Fig. Fig.3A3A were used. Since concentrations of >1 mM tryptophan affect the growth of H. volcanii (even without the RadA expression construct), it proved best to delay full induction (with 3 mM tryptophan) until 1 h before the cells were harvested. Metal affinity chromatography was used to purify 6×His-tagged RadA in the presence of 2 M NaCl, using an IMAC Sepharose column charged with Ni2+. We had previously established that the yield of 6×His-tagged protein from columns charged with Co2+ was very low when the buffers contained >1 M NaCl (data not shown). The 6×His-tagged RadA purified from the pitAHvo strain H1045 showed significant contamination with PitA, while this was not seen when 6×His-tagged RadA was purified from the pitANph strain, H1173 (Fig. (Fig.3B);3B); the identities of both proteins were confirmed by mass spectrometry (Table (Table44).
The absence of PitAHvo in cell lysates of H1173 revealed an additional contaminant, which was identified by mass spectrometry as Cdc48d (HVO_1907); Cdc48d is also present in H1045 but is difficult to distinguish in size from PitA (Fig. (Fig.3B).3B). HVO_1907 is one of four H. volcanii isoforms of a putative AAA+ ATPase that show homology to the yeast Ccd48 and E. coli FtsH proteins. In eukaryotes, Cdc48 is a ubiquitin-dependent chaperone that is involved in protein degradation (29), while bacterial FtsH is a Zn2+ metalloprotease that degrades a set of short-lived proteins (24); both are thought to regulate cell division at the level of protein stability. Notably, H. volcanii Cdc48d features a histidine-rich C terminus (Fig. (Fig.3C)3C) that is almost certainly responsible for its copurification on metal affinity chromatography columns.
We attempted to delete cdc48d by the pop-in/pop-out technique (3, 7), using the deletion construct pTA1180 (in strain H1228). We were unable to recover cells with a cdc48d deletion (data not shown), indicating that the gene is essential. Unfortunately, all known haloarchaeal homologs of Cdc48d feature the conserved histidine-rich C terminus (Fig. (Fig.3C),3C), ruling out the gene replacement strategy that had proved successful in eliminating PitA contamination. Increased concentrations of imidazole in the binding and wash buffers (>20 mM) were not helpful, and the yield of 6×His-tagged RadA was reduced significantly without eliminating Cdc48d contamination. However, growth at lower temperatures was more successful; copurification of Cdc48d was reduced by 50% when cultures were grown at 40°C or 42°C, rather than the standard cultivation temperature of 45°C (Fig. (Fig.3D).3D). Contamination by Cdc48d was also less pronounced during the purification of other recombinant proteins, such as 6×His-tagged RadB (Fig. (Fig.3E3E).
It has long been suspected that H. volcanii encodes a methylation-sensitive restriction enzyme that targets 5′-GATC-3′ sequences. Transformation of H. volcanii is much more efficient when the DNA is purified from E. coli dam mutants, which are unable to methylate 5′-GATC-3′, than from dam+ strains (21). Furthermore, transformation of H. volcanii with DNA methylated at 5′-GATC-3′ sequences leads to frequent plasmid loss, presumably due to cutting by restriction enzymes followed by recombination with chromosomal sequences (21). We hypothesized that the H. volcanii restriction endonuclease might be HVO_0682, which belongs to the Mrr family of enzymes that recognize and cleave N6-methyladenine- and 5-methylcytosine-containing DNA (9). The conserved core of HVO_0682 shows homology to other Mrr family members from archaea, bacteria, and yeast (Fig. (Fig.4A).4A). However, in a phylogenetic tree, the H. volcanii Mrr protein does not group with homologs from other haloarchaea (Fig. (Fig.4B4B).
We successfully deleted the mrr gene (from both pitAHvo and pitANph strains) by the pop-in/pop-out technique (3, 7), using the construct pTA1150. Successful deletion of mrr was established by colony lift and verified by Southern blotting of a restriction digest (Fig. 4C to E). The H. volcanii Δmrr mutants H1206 to H1209 showed no obvious growth defects in Hv-YPC and Hv-Ca broth and agar. To test the methylation-dependent restriction barrier, mrr+ and Δmrr strains (and the pitANph equivalents) were transformed with the shuttle vector pTA354 (31), which had been purified from either an E. coli dam mutant or a dam+ strain. The number of transformants obtained in an mrr+ strain was ~50-fold higher with DNA from the E. coli dam mutant than with DNA from the dam+ strain, while the Δmrr mutants were transformed efficiently regardless of the source of the DNA (Fig. (Fig.4F).4F). Therefore, deletion of the mrr gene removed the need to passage DNA through an E. coli dam mutant prior to transformation of H. volcanii. Furthermore, it allowed direct transformation of H. volcanii with a DNA ligation, which might be useful for cloning of protein overexpression constructs that are not tolerated by E. coli.
To test this possibility, we PCR amplified a mutant allele of radA [radA(A196V)], ligated the cut product with pTA963, and transformed the H. volcanii Δmrr strain H1209. Although only ~200 transformants were obtained (from ~1 μg vector DNA), 3 out of the 6 tested contained the correct plasmid construct (pTA1182). In contrast, we had failed on six previous occasions to construct this plasmid in E. coli, even though the PCR primers and restriction digests used were identical to those that had been used successfully to construct the radA+ overexpression plasmid pTA1041. This suggests that in E. coli, the p.tnaA promoter is leaky and RadA(A196V) is toxic. To confirm that 6×His-tagged RadA(A196V) could be purified from H. volcanii, we induced overexpression with tryptophan and carried out metal affinity chromatography as usual using strain H1227 (Fig. (Fig.4G).4G). There was no significant difference in the level of expression of mutant RadA(A196V) compared to the wild-type protein.
Existing methods for expression of halophilic enzymes in E. coli are far from ideal; the protein is often insoluble and must be reactivated by denaturation and refolding, which is not always successful (11). Even when the protein is soluble, problems can arise. For example, H. volcanii RadA expressed in E. coli is bound to host DNA that cannot be removed easily. Purification from the native host is the obvious solution, but until now, systems for overexpression in H. volcanii have failed to capitalize on recent advances in haloarchaeal genetics (2, 39). For example, existing vectors feature constitutive gene promoters and rely on mevinolin resistance or novobiocin resistance markers for selection (30), which are far from ideal, since the use of these antibiotics impairs cell growth.
We have harnessed the conditional p.tnaA promoter to develop a series of plasmid vectors (Fig. (Fig.1)1) for rapid and strong induction of protein expression upon addition of tryptophan (26). Our constructs use pyrE2 and hdrB markers that maintain plasmids in rich medium (Hv-YPC) without the use of antibiotics (3); they are available with and without an in-frame 6×His tag for protein purification by metal affinity chromatography. This technique is ideal for purification of halophilic proteins, since it is compatible with the high salt concentrations used. However, it has until now been problematic in H. volcanii due to contamination by PitA, a protein with a histidine-rich linker region. We have replaced pitA with the gene from N. pharaonis, which encodes an ortholog lacking the histidine-rich region (Fig. (Fig.2).2). Proteins purified from the pitANph gene replacement strain are free of PitA contamination (Fig. (Fig.3).3). Interestingly, contamination by H. volcanii PitA is more pronounced in strains overexpressing 6×His-tagged RadA than in strains containing the empty vector (Fig. (Fig.3B).3B). This is also true for Cdc48d, another histidine-rich contaminant that we identified. PitA and Cdc48d might be upregulated in response to cell stress, in this case due to overexpression of a recombinant protein, which is consistent with our observation that contamination by Cdc48d is reduced by growth at lower temperatures (Fig. (Fig.3D).3D). Furthermore, proteins that are expressed at lower levels than 6×His-tagged RadA, such as 6×His-tagged RadB, exhibit less contamination by Cdc48d (Fig. (Fig.3E3E).
Finally, we have deleted the mrr gene, which encodes a homolog of the methylation-sensitive restriction enzyme Mrr (9). In contrast to the wild type, H. volcanii Δmrr strains exhibit high transformation efficiencies regardless of whether the plasmid DNA is methylated at 5′-GATC-3′ sites by E. coli Dam methylase (Fig. (Fig.4F).4F). Therefore, Δmrr strains remove the need to passage plasmid DNA through an E. coli dam mutant before transformation (21). Not only does this save time, it also reduces the risk of incurring mutations during growth in E. coli dam mutants, which are deficient in DNA mismatch repair (27). Furthermore, H. volcanii Δmrr strains may be transformed directly with a DNA ligation, which permits the cloning of protein overexpression constructs that are toxic to E. coli.
We urge caution regarding the use of Δmrr strains in routine genetic experiments, since they have not been phenotyped extensively and deletion of the mrr gene might have pleiotropic effects. For example, Salmonella enterica serovar Typhimurium mutants with a constitutively active mrr gene display increased basal SOS induction (1). Some restriction modification systems act as selfish mobile genetic elements that resist their loss from the host, leading to cell death (25). However, mrr is unlikely to have been acquired by lateral gene transfer, since the synonymous codon usage of this gene is identical to the genome average (data not shown). We consider it more probable that Mrr acts in cellular defense against foreign DNA. Furthermore, we have demonstrated that Δmrr strains are fit for purpose when used in protein overexpression (Fig. (Fig.4G),4G), thereby bringing closer the goal of an archaeal model organism that is suitable for both biochemistry and genetics.
We are grateful to the BBSRC, Wellcome Trust, Leverhulme Trust, and MRC for funding and to the Royal Society for a University Research Fellowship awarded to T.A.
We thank Stéphane Delmas, Bob Lloyd, Ed Bolt, Geoff Briggs, and Karen Bunting for helpful advice and Charles Daniels for the t.Syn sequence.
S.L., K.W., M.M., and T.A. wrote the paper; M.M. and T.A. designed the experiments; S.B., K.W., and T.A. performed the microbiological and biochemical experiments; S.L. carried out the mass spectrometry; and S.L. and T.A. analyzed the data.
Published ahead of print on 22 January 2010.