Strains and plasmids used in this study are listed in Table 1. All Hfx. volcanii strains were grown in either Hv-YPC or Hv-CA medium at 42°C while shaking at 200 rpm. Hv-YPC and Hv-CA media were produced using the formulas outlined in The Halohandbook (Dyall-Smith, 2009). Hv-min medium used in growth experiments was modified from the formula in The Halohandbook to exclude a carbon source (Hv-min -C). Media were supplemented with uracil (50 μg/mL) and 5-fluoroorotic acid (50 μg/mL) as needed. For growth on Petri plates, 2% agar (w/v) was added to the media.

All Escherichia coli strains were grown in either S.O.C. media or LB-media at 37°C while shaking at 200 rpm. S.O.C. media was provided by Clontech (Cat. # 636763) and New England BioLabs (Cat. # B9020S). LB medium was produced by adding 5 g NaCl, 5 g tryptone, and 2.5 g of yeast extract to deionized water to a final volume of 500 mL and pH set to 7.0. LB was supplemented with ampicillin (100 μg/mL) as needed. When LB cell culture plates were produced, 1.5% agar (w/v) was added. LB plates were supplemented with 40 μL of X-gal (20 mg/mL) as needed.

All primers used in this study are listed in Table 2. DNA used for plasmid construction and screening was amplified via PCR. Reactions for PCR were assembled as 10 μL volumes and contained the following reagents: 5.9 μL of deionized water, 2 μL of 5x GC Phusion buffer (Thermo Scientific, Cat. # F-519), 1 μL of 100% DMSO (Thermo Scientific, Cat. # TS-20684), 0.4 μL of 10 mM dNTP (Promega, Cat. # U1511), 0.2 μL of 10 μM forward primer, 0.2 μL of 10 μM reverse primer, 0.2 μL of template DNA, and 0.1 μL of Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Cat. # F-530S). When needed, water was substituted with 20% acetamide. The reactions were performed in a Mastercycler EP Gradient (Eppendorf) with the following cycle: a DNA melting step at 94°C for 22 s, an annealing step at 58.1°C for 35 s, and an extension step at 72°C for 90 s. This cycle was repeated 40 times, after which a final annealing step at 72°C for 5 min was performed. Template DNA included Hfx. volcanii DS2 genomic DNA (20 ng/μL), plasmid DNA listed in Table 1, and DNA from E. coli and Hfx. volcanii colonies.

Gel electrophoresis was performed to separate and analyze the PCR products using 0.8% (w/v) agarose in 1 × TAE buffer (40 mM Tris acetate, 2 mM EDTA). After gel electrophoresis, PCR products were excised from the gel and purified using the Wizard SV Gel and PCR Clean-Up System (Promega). Plasmids from E. coli strains were extracted and purified using the PureYield Plasmid Miniprep System (Promega). Plasmids linearized via digestion with restriction enzymes (BamHI, HindIII, XhoI, or XbaI) were also purified using the Wizard SV Gel and PCR Clean-Up System.

Three Hfx. volcanii genes (dhaKLM; HVO_1544, HVO_1545, and HVO_1546), which encode homologs to the putative DHA kinase genes in Hqm. walsbyi, and a glycerol kinase gene (glpK; HVO_1541), were targeted for deletion in Hfx. volcanii strain H26 using the In-Fusion HD Cloning Kit (Clontech). The strategy for gene deletion was based on the methodology outlined in a study by Blaby et al. (2010) with a few modifications. Flanking regions of the targeted genes were developed to be between 800 and 1000 bp in length. The 15-bp linker used to combine the flanking regions was altered to so that EcoRI and BstOI sites were included for the dhaKLM deletion linker and BglI and BstOI sites were included for the glpK deletion linker. The pTA131 was linearized with HindIII and BamHI for the dhaKLM deletion and XhoI and XbaI for the glpK deletion. Constructed plasmids were transformed into Stellar Competent Cells (Clontech, Cat. # 636763), according to the directions of the provider, and were plated on LB-amp plates with X-gal. White colonies were screened via colony PCR using the external primers of the target gene flanking regions. Confirmed deletion plasmids (listed in Table 1) were subcloned in dam⁻/dcm⁻ Competent E. coli (New England BioLabs, Cat. # C2925H) to produce demethylated plasmids for transformation of Hfx. volcanii. Hfx. volcanii H26 colonies were screened for deleted genes via PCR using the external primers of the target gene flanking regions. The size of PCR products of screened cells were compared to those produced with wild-type DNA (Figure 1). Smaller product size indicated that the gene had been deleted. The Hfx. volcanii H26 deletion strains produced by this process are listed in Table 1.

The dhaKLM and glpK genes deleted in Hfx. volcanii H26 were resuscitated by constructing complementation plasmids. Primers were designed which amplified the upstream native promoter and the coding region of the targeted genes in Hfx. volcanii. The primers were also designed to have 15 bp of homology with pTA409. Restriction digestion of pTA409 was performed using BamHI and XhoI to linearize the plasmid. After the linearized pTA409 and gene fragments were gel-purified, the DNA fragments were combined together using the In-Fusion HD Cloning Kit according to the instructions of the provider. The constructed plasmids were cloned, screened, and demethylated as described in the above gene deletion protocol. Purified constructed plasmids (listed in Table 1) were then transformed into the Hfx. volcanii H26 deletion strains using the PEG mediated transformation of Haloarchaea protocol from The Halohandbook. PCR was used to confirm transformation success. The Hfx. volcanii complementation strains produced by this process are listed in Table 1.

Hfx. volcanii strains listed in Table 1 were grown to late-exponential phase (OD₆₀₀ = ~ 0.6−0.8) in Hv-YPC medium. The cell cultures were then centrifuged at 3220 RCF for 15 min and resuspended in Hv-min -C media supplemented with uracil. Centrifugation was repeated a total of three times to wash the cells of residual Hv-YPC media. During the final resuspension of the cells in Hv-min -C media, the cell cultures were diluted to OD₆₀₀ ~0.01. Each cell culture was then distributed into the wells of a 96-well plate, with each well receiving 190 μL of cell culture. Also, 200 μL of Hv-min -C was added to the plate to be used as a blank. Three wells of each culture were treated with 10 μL of either 0.1 M DHA (final concentration of 5 mM DHA), 0.05 M DHA (final concentration of 2.5 mM DHA), 0.02 M DHA (final concentration of 1 mM DHA), or deionized water (negative control). The 96-well plate was then placed into a Multiscan FC plate reader (Fisher Scientific), which incubated the plate at 42°C while shaking it at low speed. The plate reader measured the OD₆₂₀ of each well every hour for 72 h.

The amino acid sequences of the Hfx. volcanii putative DHA kinase gene dhaK (HVO_1546) and glycerol kinase gene glpK were used to perform BLASTp (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches of the NCBI database to determine other halobacterial species with DHA kinase and glycerol kinase genes. The amino acid sequences were retrieved from the NCBI database (dhaK GI number 292655696; glpK GI number 292655691). The search was restricted to the Halobacteriales (taxid 2235) with an E-value cut-off of 1e-20. Reciprocal BLASTp was performed to analyze only orthologous genes. The halobacterial genomes queried in this BLASTp search are listed in Table 3.

Three DHA kinase genes (HQ2672A, HQ2673A, and HQ2674A) have been annotated in the genome of Hqm. walsbyi (Bolhuis et al., 2006), a halobacterial species which is able to metabolize external DHA (Elevi Bardavid and Oren, 2008). Homologs of these three genes are also annotated in Hfx. volcanii (HVO_1544, HVO_1545, and HVO_1546). In order to determine the prevalence of DHA kinase genes among the Halobacteria, the Hfx. volcanii dhaK gene (HVO_1546) was used to perform a BLASTp search against the database of Halobacteria genomes available on NCBI. The search yielded significant hits among 31 different halobacterial species (Table 4). Except for Haloferax larsenii and Haloferax elongans, all queried Haloferax species yielded significant hits in the BLASTp search. Species from the Halobiforma, Halococcus, Halorubrum, and Natronococcus genera also yielded significant hits, but not all queried species from these genera produced results. All representatives from the genera Haladaptatus, Halalkalicoccus, Halarchaeum, Haloquadratum, Halosarcina, and Salinarchaeum yielded significant hits. Halobacteria genera that did not yield significant hits in the BLASTp search (E-value cut-off of 1e-20) include Haloarcula, Halobacterium, Halobaculum, Halogeometricum, Halogranum, Halomicrobium, Halopiger, Haloplanus, Halorhabdus, Halosimplex, Halostagnicola, Haloterrigena, Halovivax, Natrialba, Natrinema, Natronobacterium, Natronolimnobius, Natronomonas, and Natronorubrum.

Although putative DHA kinase genes are present in Hfx. volcanii, no previous research has demonstrated that Hfx. volcanii is able to grow on DHA as a carbon source. Therefore, experiments were performed to test the growth of Hfx. volcanii strain H26 on 5 mM, 2.5 mM, and 1 mM DHA. The results indicated that H26 was capable of growth on DHA as the sole carbon source. The cell density at which H26 reached stationary phase was also dependent on the initial concentration of DHA provided to the cells (Figure 2). H26 cells grown in medium supplemented with 1 mM DHA reached stationary phase at the lowest cell density, whereas cells grown with the highest tested concentration of 5 mM DHA reached stationary phase at the highest cell density. These data indicate that growth of Hfx. volcanii on DHA as a carbon source is concentration dependent.

Evidence indicates that Hfx. volcanii, like Hqm. walsbyi, can use DHA as a carbon source. Although both organisms have DHA kinase genes, no previous studies demonstrated these putative DHA kinase genes have a role in DHA metabolism. In order to determine that DHA metabolism in Hfx. volcanii utilizes the annotated DHA kinase, the operon dhaKLM (HVO_1544—HVO_1546) was deleted in Hfx. volcanii strain H26. The growth of this deletion strain (ΔdhaKLM) on 5 mM DHA was then tested in comparison to the parent strain H26 as well as a complementation strain (ΔdhaKLM + pdhaKLM). The results indicate that the deletion of dhaKLM causes a reduction in growth on DHA, and that complementation of the deleted genes negates this growth deficiency (Figure 3). However, the ΔdhaKLM was still capable of growth on DHA, exhibiting a 33% decrease in growth compared to H26. These results indicate that the dhaKLM genes are used by Hfx. volcanii in DHA metabolism, most likely for the phosphorylation of DHA to DHA phosphate, and that the genes are apparently not essential. Since it is still capable of growth on DHA there must be additional genes involved in the phosphorylation step.

In other organisms, glycerol kinase is also capable of phosphorylating DHA (Hayashi and Lin, 1967; Weinhouse and Benziman, 1976; Jin et al., 1982). Therefore, the other gene involved DHA metabolism in Hfx. volcanii was hypothesized to be the glycerol kinase gene glpK (HVO_1542). In order to test this hypothesis, the glpK gene was deleted in H26. The deletion strain (ΔglpK), and its complementation strain (ΔglpK + pglpK), were both grown on 5 mM DHA along with the parent strain H26. The results indicate that the deletion of glpK caused a reduction in growth on DHA even greater than deletion of dhaKLM, and that complementation of the glpK gene restores growth to normal levels (Figure 4). In comparison to the parent strain H26, ΔglpK strain demonstrated an 83% decrease in growth. This decrease is far greater than the 33% decrease exhibited by the ΔdhaKLM deletion mutant. These results indicate that the glpK gene is used by Hfx. volcanii in DHA metabolism, and that its role is potentially greater than that of the dhaKLM operon.

In order to further test the roles of the DHA kinase and glycerol kinase in DHA metabolism in Hfx. volcanii, the dhaKLM operon and glpK gene were both deleted in H26. This double deletion mutant (ΔdhaKLM ΔglpK), along with a DHA kinase complementation strain (ΔdhaKLM ΔglpK + pdhaKLM), a glycerol kinase complementation strain (ΔdhaKLM ΔglpK + pglpK), and the parent strain H26, were then grown on 5 mM DHA. The results indicate that the deletion of both kinases abolishes growth on DHA, and that complementation with glycerol kinase restores growth to a greater degree than complementation with DHA kinase (Figure 5). The ΔdhaKLM ΔglpK strain did not exhibit any growth, remaining at the initial OD₆₂₀ of 0.0035. The ΔdhaKLM ΔglpK + pdhaKLM strain was able to grow on DHA, but demonstrated an 84% decrease compared to the H26 parent strain. The ΔdhaKLM ΔglpK + pglpK was also capable of limited growth on DHA, but demonstrated a 39% growth decrease from H26 and a 390% growth increase compared with ΔdhaKLM ΔglpK + pdhaKLM. Overall, these data confirm that glycerol kinase is more important for DHA metabolism in Hfx. volcanii than DHA kinase.

Since growth experiments indicated that glycerol kinase has a significant role in DHA metabolism, the presence of this gene in halobacterial species could potentially be a determinant of DHA metabolism in those species. Although the distribution of glpK homologs has been examined in previous studies (Sherwood et al., 2009; Anderson et al., 2011), a greater number of halobacterial genomes have become available since those studies. Therefore, the glpK gene in Hfx. volcanii was used to perform a BLASTp search against the halobacterial genomes available on NCBI. The search yielded 90 significant hits among 82 different species of Halobacteria (Table 5), indicating a much wider distribution of glycerol kinase compared to DHA kinase among the Halobacteria. Six species yielded more than one significant hit: Halogeometricum borinquense (3 hits), Haladaptatus paucihalophilus (3 hits), Haloferax prahovense (2 hits), Haloferax mucosum (2 hits), Haloferax gibbonsii (2 hits), and Natronomonas moolapensis (2 hits). The multiple hits indicate the presence of glpK paralogs in these species. Only 18 of the 100 queried halobacterial species did not yield significant hits: Haloarcula sp. AS7094, Halobacterium sp. DL1, Halobacterium sp. GN101, Halobaculum gomorrense, Halococcus sp. 197A, Halopiger sp. IIH2, Halopiger sp. IIH3, Haloplanus natans, Halorubrum ezzemoulense, Halosarcina pallida, Halostagnicola larsenii, Halovivax asiaticus, Halovivax ruber, Natrinema sp. CX2021, Natrinema sp. J7-1, Natronobacterium gregoryi, Natronobacterium sp. AS-7091, and Natronomonas pharaonis. It should be noted, however, that only the genomes of Halovivax ruber, Natronobacterium gregoryi, and Natronomonas pharaonis are completely sequenced, whereas the other genomes without significant hits are incomplete, leaving open the possibility that these species might have glpK homologs. With the exception of Halosarcina pallida, which has an incompletely sequenced genome, all halobacterial species that yielded significant hits in the dhaK BLASTp search also yielded significant hits in the glpK BLASTp search.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.