An improved genetic system for bioengineering buoyant gas vesicle nanoparticles from Haloarchaea
© DasSarma et al.; licensee BioMed Central Ltd. 2013
Received: 4 November 2013
Accepted: 17 December 2013
Published: 21 December 2013
Gas vesicles are hollow, buoyant organelles bounded by a thin and extremely stable protein membrane. They are coded by a cluster of gvp genes in the halophilic archaeon, Halobacterium sp. NRC-1. Using an expression vector containing the entire gvp gene cluster, gas vesicle nanoparticles (GVNPs) have been successfully bioengineered for antigen display by constructing gene fusions between the gvpC gene and coding sequences from bacterial and viral pathogens.
To improve and streamline the genetic system for bioengineering of GVNPs, we first constructed a strain of Halobacterium sp. NRC-1 deleted solely for the gvpC gene. The deleted strain contained smaller, more spindle-shaped nanoparticles observable by transmission electron microscopy, confirming a shape-determining role for GvpC in gas vesicle biogenesis. Next, we constructed expression plasmids containing N-terminal coding portions or the complete gvpC gene. After introducing the expression plasmids into the Halobacterium sp. NRC-1 ΔgvpC strain, GvpC protein and variants were localized to the GVNPs by Western blotting analysis and their effects on increasing the size and shape of nanoparticles established by electron microscopy. Finally, a synthetic gene coding for Gaussia princeps luciferase was fused to the gvpC gene fragments on expression plasmids, resulting in an enzymatically active GvpC-luciferase fusion protein bound to the buoyant nanoparticles from Halobacterium.
GvpC protein and its N-terminal fragments expressed from plasmid constructs complemented a Halobacterium sp. NRC-1 ΔgvpC strain and bound to buoyant GVNPs. Fusion of the luciferase reporter gene from Gaussia princeps to the gvpC gene derivatives in expression plasmids produced GVNPs with enzymatically active luciferase bound. These results establish a significantly improved genetic system for displaying foreign proteins on Halobacterium gas vesicles and extend the bioengineering potential of these novel nanoparticles to catalytically active enzymes.
Buoyant gas vesicles are prokaryotic organelles that are widely distributed among bacterial and archaeal microorganisms and constitute protein nanoparticles (GVNPs) that may be engineered for biotechnological applications [1–3]. These organelles naturally promote flotation and increase the availability of light and oxygen to many aquatic microorganisms, especially those with photosynthetic or phototrophic capabilities. Water is excluded from the interior, a property that is thought to be a consequence of the hydrophobicity of the interior surface of the proteinaceous membrane. While the exact protein composition of the membrane has been difficult to ascertain due to its extreme stability against solubilization, production of these structures is easily scaled-up and they are simple to purify by hypotonic lysis of the host and concentrate by flotation, enhancing their intrinsic value for biotechnological applications [4, 5].
The protein composition of gas vesicle nanoparticles has been studied primarily by Western blotting analysis using antisera directed against individual gvp gene products . Initially, only GvpA and GvpC proteins were found , but further analysis showed the presence of five additional proteins, GvpF, GvpG, GvpJ, GvpL, and GvpM . GvpA, J, and M constitute a small family of proteins (Pfam 741) likely involved in gas vesicle membrane formation, while GvpF and L are coiled-coil proteins (Pfam 6386) with self-associative properties thought to be important for nucleation or growth of the nanoparticles [9, 15]. Most of these proteins (GvpA, GvpC, GvpF, GvpJ, and GvpL) were also identified in a recent proteomic study . In genome sequencing studies, genes corresponding to these same proteins were also found in other gas vesicle-forming microbes . An exception was the gvpC gene, which was reported only in the haloarchaeal and cyanobacterial gas vesicle producers.
In Halobacterium sp. NRC-1, the gvpC gene encodes a hydrophilic protein with a predicted molecular weight of 42,391 and a highly acidic pI of only 3.57 [8, 9]. In this haloarchaeon, the GvpC protein sequence contains 8 imperfect repeats and an extremely acidic stretch located near the C-terminus. The slight similarity of the haloarchaeal repeats to the repeats in the cyanobacteria suggested that the GvpC proteins play similar roles in both haloarchaea and cyanobacteria . In the cyanobacterium, Anabaena flos-aquae, GvpC has been shown to serve a strengthening role in gas vesicles , while in Halobacterium sp. NRC-1, insertion mutations in the gvpC gene generated vesicles with altered shape and size . These findings suggested that GvpC proteins facilitate gas vesicles’ growth and enhance stability in strains which produce them.
The potential value of GvpC protein for bioengineering floating GVNPs was established during mutagenesis of the gvp gene cluster from Halobacterium sp. NRC-1. A gvpC::κ insertion mutant was found to produce primarily spindle-shaped gas vesicles with smaller than wild-type size (Figure 1B & C) and excision of most of the κ insert resulted in the production of vesicles with a peptide fused to GvpC protein that was antigenically displayed and immunologically accessible on the surface [7, 11]. Further studies with SIV and chlamydial proteins have shown that bioengineered GVNPs may be used for antigen display and elicit both humoral and cellular responses in mice [4, 5, 20–22].
The genetic system currently in use for bioengineering of gas vesicle nanoparticles is technically challenging due to the large size and complexity of the gvp gene cluster [7, 8]. In order to facilitate bioengineering of nanoparticles, we constructed a new Halobacterium sp. NRC-1 derived host strain and a series of smaller, more versatile plasmid expression vectors. The work documented in this report establishes a significantly improved genetic system for expression of GvpC-fusion proteins, including an active luciferase enzyme from Gaussia princeps.
Construction of a Halobacterium ΔgvpC strain and gvpCexpression vectors
In order to improve the genetic system for bioengineering of GVNPs [7, 8], our first goal was the construction of a gvpC deletion strain, via the ura3-based gene deletion method for Halobacterium sp. NRC-1 [24, 25]. Approximately 500-bp flanking regions of gvpC, including the first and last few codons of gvpC, were cloned into the suicide vector, pBB400 , and the resulting plasmid, pBB400ΔgvpC, was used to transform Halobacterium sp. NRC-1Δura3. After selecting sequentially for integration and excision (see Methods), the resulting Halobacterium sp. NRC-1Δura3ΔgvpC deletion strain (referred henceforth as ΔgvpC deletion strain) (Figure 1A) showed a partially gas vesicle-deficient phenotype with small, largely spindle-shaped gas vesicles, similar to that reported for a gvpC::κ insertion mutant (cf. Figure 1B and C).
Strains and plasmids used in this study
Source or reference
Halobacterium sp. strain NRC-1
Sequenced wild-type strain
Laboratory collection 
Halobacterium sp. strain SD109
Strain NRC-1 deleted for the gvp gene cluster of pNRC100
Laboratory collection 
Halobacterium sp. strain NRC-1Δura3
Strain NRC-1 deleted for ura3 gene coding orotidine-5′-monophosphate
Halobacterium sp. strain NRC-1Δura3ΔgvpC
Strain NRC-1Δura3 deleted for the gvpC gene
Halobacterium-E. coli shuttle plasmid containing entire gvp gene cluster with κ insertion in the gvpC gene
Laboratory collection 
Suicide plasmid capable of replication in E. coli but not Halobacterium, containing the ura3 gene
Laboratory collection 
pBB400 plasmid containing gvpC gene-flanking regions for deletion construction
Halobacterium expression vector with cspD2 promoter and Halorubrum lacusprofundi β-galactosidase gene
Laboratory collection 
Halobacterium sp. NRC-1 expression vector with cspD2 promoter and adapter containing a start codon, His-tag, and restriction sites for insertion of the gvpC gene fragments
pARK derivative with gvpC gene C1 fragment
pARK derivative with gvpC gene C2 fragment
pARK derivative with gvpC gene C3 fragment
pARK derivative with gvpC complete gene (C4)
Halobacterium sp. NRC-1 expression vector with gvpA promoter and adapter containing a start codon, His-tag, and restriction sites used for insertion of the gvpC gene fragments
pDRK derivative containing gvpC gene C1 fragment fused to codon-optimized Gaussia princeps luciferase gene
pDRK derivative containing gvpC gene C2 fragment fused to codon-optimized Gaussia princeps luciferase gene
pDRK derivative containing gvpC gene C3 fragment fused to codon-optimized Gaussia princeps luciferase gene
pDRK derivative containing gvpC complete gene (C4) fused to codon-optimized Gaussia princeps luciferase gene
Engineering of the gvpC gene and expression of GvpC fragments
Luciferase expression and display on gas vesicles
Interestingly, when members of the pDRK-C-L plasmid series were transformed into Halobacterium sp. NRC-1, which contains a wild-type gvpC gene, nanoparticles containing engineered GvpC-luciferase proteins were also detectable by luciferase activity (Figure 8, pink bars) and Western blotting assays (data not shown). Although higher levels of luciferase activity were observed bound to floating gas vesicle nanoparticles in the ΔgvpC (pDRK-C1-L to C4-L) strains compared to the NRC-1 (pDRK-C1-L to C4-L) strains, luciferase activity was clearly measurable in nanoparticles in the transformed wild-type strain (Figure 8). Moreover, the wild-type GvpC protein was also detected bound to GVNPs in the NRC-1 (pDRK-C1-L to C4-L) strains (data not shown), indicating that two different GvpC forms may be simultaneously bound to the nanoparticles. These results extend the possible biotechnological uses of GVNPs to other applications requiring nanoparticle-bound enzymes and multivalency.
We have established an improved genetic system for bioengineering of GVNPs in the model halophilic archaeon, Halobacterium sp. NRC-1. A strain deleted for the gvpC gene and plasmid vectors containing highly active promoters for producing GvpC-fusion proteins were constructed. The system was tested by expressing the entire gvpC gene, N-terminal portions of gvpC gene fragments, and GvpC-luciferase fusion proteins, all of which bound to the buoyant nanoparticles. The improved genetic engineering system provides the opportunity for insertion of multiple foreign sequences and the potential for production of GVNPs displaying multiple antigens. The work reported here represents a significant step forward in demonstrating the bioengineering capabilities of GVNPs, including their application to antigen display and vaccine development.
The current work has capitalized on the Halobacterium sp. NRC-1 genetic system and recently constructed expression plasmids [25–27, 31]. These biotechnological tools have been used to overexpress, purify, and characterize a polyextremophilic β-galactosidase enzyme from an Antarctic haloarchaeon, and bioengineer resistance of haloarchaeal cells to ionizing radiation by overexpression of a mammalian-type RPA protein [27, 31]. The constructed expression plasmids (pARK and pDRK) contain the high-copy number Halobacterium sp. pGRB miniplasmid for replication and the mevinolin resistance gene for selection in haloarchaea, as well as the plasmid pUC18 vector for replication and selection in the E. coli host. The pARK expression plasmids contain the cspD2 promoter while the pDRK expression plasmids contain the gvpA promoter. Both of these promoters were reported to drive expression of genes inducible under cold temperatures . The pARK and pDRK plasmids were tailored for expression of GvpC fusion proteins and represent convenient vectors for production of bioengineered GVNPs.
Our GVNP-bioengineering and expression system exploits genetic properties of the Halobacterium sp. NRC-1ΔgvpC deletion strain and recombinant capabilities of the gvpC gene from Halobacterium sp. NRC-1 pNRC100 plasmid [8, 9]. The ΔgvpC deletion strain, constructed using our ura3-deletion method [24, 25], contained gas vesicles with smaller, more spindle-shaped vesicles observable by transmission electron microscopy. This finding is consistent with earlier observations suggesting a key role for GvpC protein in shape determination of gas vesicles in haloarchaea [11, 32]. As larger GvpC protein variants were supplied via expression plasmids, we observed generally longer and wider vesicles, suggesting that the nanoparticles were increasingly strengthened. Similar results were also obtained for some cyanobacteria, where a strengthening role for the GvpC protein was reported [19, 33]. In one study, A. flos-aquae GvpC protein produced in E. coli could bind and strengthen the structures after native GvpC protein had been removed by urea treatment. GvpC genes have been reported in most if not all gas vesicle-containing haloarchaea and cyanobacteria, indicating that the protein may serve similar functions in these two groups of aquatic microorganisms. However, gvpC is reportedly absent in other gvp gene-containing species, a finding suggesting that it may not be absolutely essential for biosynthesis of gas vesicles [1, 15].
An interesting feature of the GvpC protein is the presence of internal repeats (8 in Halobacterium sp. NRC-1) . Our results show that even a small subset of these repeats in truncated variants of GvpC proteins is sufficient to permit binding to GVNPs. In the pARK-C1 construct, only 3 copies are present, while in pARK-C2, there are 5. Both of these plasmids produced proteins that bound to the vesicles. The longer GvpC variants produced from pARK-C3 and C4 (7 or 8 repeat copies, respectively), complemented production of the nanoparticles considerably better than the smaller GvpC proteins, based on both colony phenotype and vesicle morphology. Similar conclusions were previously reported for A. flos-aquae GvpC protein variants containing three or four repeats (out of 5 in the full-length protein) in in vitro experiments . In this cyanobacterial system, GvpC depleted vesicles had their strength better restored with proteins containing larger numbers of repeats. In Halobacterium sp. NRC-1, the presence of a highly acidic C-terminal region suggests a further role for this feature in stabilizing gas vesicles, likely reflecting the high salinity found in the cytoplasm.
We used a synthetic Gaussia princeps luciferase gene to further assess the binding of GvpC fusion proteins to gas vesicles. Initially, we found that the luciferase protein was active when produced in Halobacterium via expression vectors alone (our unpublished results) or as a fusion with the GvpC fragments or full-length protein, demonstrating that the marine enzyme was capable of adopting an active structure even after exposure to the hypersaline cytoplasm of Halobacterium. Further investigation showed that the GvpC-luciferase fusion proteins were bound to buoyant gas vesicles, confirming that the enzyme is likely displayed on the surface of nanoparticles. Although antigenic proteins and protein fragments have been previously found to be displayed on gas vesicles, these findings now show that an enzyme may also decorate the nanoparticles while retaining its catalytic activity. Moreover, when two different gvpC genes (wild-type and shortened/fused to luciferase) were present, we found that both GvpC forms were bound to the nanoparticles. These results extend the possible biotechnological uses of GVNPs to applications requiring multivalency.
All together, our results provide improved genetic and plasmid resources for engineering of GVNPs for biotechnological applications. The original system described required the incorporation of target genes into a large plasmid containing the entire gvp gene cluster, pFM104d, and a natural mutant strain deleted for the gene cluster, SD109 [7, 8, 11–13, 35]. The newly described system utilizes the much smaller and more versatile plasmid series, pARK and pDRK, containing a relatively small portion of the gvp gene region. The new system allows more facile cloning of genes of interest into the smaller expression vectors and replacement of only a single deleted gene (ΔgvpC) in the gvp gene cluster. These features will greatly facilitate expression of foreign proteins in GVNPs, including antigenic proteins from pathogenic microorganisms for vaccine development.
Gas vesicle nanoparticles (GVNPs) in the halophilic archaeon, Halobacterium sp. NRC-1, are successfully being used for antigen display and vaccine development. The genetic tools for bioengineering GVNPs have now been greatly improved through construction of a Halobacterium strain deleted for the gvpC gene and smaller plasmids for expression of foreign proteins fused to GvpC proteins. The utility of the improved system has been demonstrated by expression of an active Gaussia princeps luciferase enzyme fused to GvpC and bound to buoyant gas vesicles. These results establish a significantly improved genetic system for displaying foreign proteins on GVNPs and extend the bioengineering potential of these novel nanoparticles to catalytically active enzymes.
Culturing and nanoparticle preparation
Halobacterium strains used for this study (Table 1) included NRC-1, the wild-type (ATCC 700922/JCM11081) , SD109, with deletion of the entire gas vesicle gene cluster [13, 35], SD109 (pFM104gvpC::κ1), with insertions of a kanamycin (κ) cassette in the gvpC gene , NRC-1Δura3 [24–26] for gene knockouts, and NRC-1Δura3ΔgvpC constructed in this study. These strains were grown in CM+ media, as previously described, with the addition of mevinolin (20 μg/ml) (generously provided by Merck, Sharp, and Dohme, Rahway, NJ) when transformed with expression plasmids [26, 27].
For preparation of nanoparticles, lawns of Halobacterium cells were collected by washing with 5 ml of PBS solution [137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, and 2 mM potassium phosphate monobasic (pH 7.4)] containing 1.0 mM MgSO4. Ten μg/ml of DNase I (Roche Diagnostics, Indianapolis, IN) was added and the cell lysate suspension was incubated for 3 hours at 37°C. Lysates were centrifuged at 60 × g overnight in a swinging bucket rotor using a Jouan CR412 centrifuge (Thermo Scientific, Rockford, IL) to accelerate flotation of the gas-filled nanoparticles. Next, intact buoyant nanoparticles were carefully collected into a clean tube and resuspended in PBS solution, floated by overnight centrifugation, as above, and re-collected. The flotation procedure described above was repeated until a milky white suspension of GVNPs was obtained.
For preparation of whole cell extracts, liquid cultures of Halobacterium strains were grown in an illuminated Innova 44 incubator shaker (New Brunswick Scientific, Enfield, CT) at 42°C with shaking at 220 rpm. Ten ml cultures (OD 1.2 at 600 nm) were harvested by centrifugation (8000 rpm × 10 min at 4°C) in a Sorvall RC-5B centrifuge. Pellets were resuspended in 0.5 ml of sterile distilled water containing freshly prepared 1 mM phenylmethylsulfonyl fluoride (Sigma Corporation, St. Louis, MO), 10 μg/ml DNase I was added, and the lysates incubated at 37°C for 30 minutes and dialyzed against 4 liters of distilled water at 4°C overnight. Protein concentrations were determined by the Bradford dye (Bio-Rad Laboratories, Hercules, CA) binding method  using bovine serum albumin (BSA, Sigma Corporation) as a standard.
Construction of Halobacterium ΔgvpC strain
Oligonucleotides used in this study
gvpC deletion construction
Amplification of gvpA promoter
gvpC gene segment amplification
His-tag adapter for gvpC and antigen fusion expression plasmid construction
Sequencing of promoters and inserts in pARK and pDRK series plasmids
Universal F 20mer
Sequencing across gvpC deletion
Universal R 20mer
Luci Int R
Sequencing and determination of luciferase gene orientation
pBB400ΔgvpC transformants were selected by plating on CM+ media lacking uracil (HURA), colonies picked and grown in liquid HURA media, and genomic DNA extracted, as previously described [26, 37]. Integrant candidates were screened by PCR using the flanking primers and genomic DNA as template, and integrants were plated on CM+ plates containing 250 μg/ml 5-fluorouracil (5-FOA) (Toronto Research Chemicals, North York, Canada). Excisant colonies were picked and grown in liquid CM+ media containing 5-FOA, genomic DNA was extracted, and PCR reactions were used to screen for knockout mutants using primers flanking the gvpC gene (Table 2).
Construction of the pARK and pDRK expression plasmids
For construction of the Halobacterium sp. pARK and pDRK expression plasmids, pMC2 expression plasmid was used as the backbone [26, 27]. The β-galactosidase gene was excised and replaced with an adapter (see Table 2) containing a start codon, hexahistidine-tag (His-tag), and AflII, AvrII, and AfeI restriction sites. The C1-C4 GvpC fragments were PCR amplified and inserted via the AflII and AvrII sites, and the synthetic Gaussia princeps luciferase gene (LifeTechnologies, Grand Island, NY) was inserted via the AfeI site . The promoter region was replaced via the KpnI and NdeI sites. The constructs were validated by DNA sequencing.
For thin-sectioning, cells were fixed in 3% glutaraldehyde-20% NaCl, postfixed in 2% OsO4-20% NaCl for 4 hours, rinsed with 20% NaCl, stained with 5% uranyl acetate in 20% NaCl-20% acetone for 1 hour, and then dehydrated by immersion in a series of isotonic acetone solutions. Samples were then embedded in Spurr medium which was polymerized at 70°C for 8 hours . Thin sections of 600 Å (60 nm) were examined on copper grids stained with lead .
For negative staining, purified nanoparticles were adsorbed to glow discharged 400 mesh carbon coated parlodion copper grids for 30 seconds. Grids were then rinsed in distilled deionized water, 3 times for 30 seconds each. Nanoparticles were negatively stained two times for 30 seconds each in 1% uranyl acetate with 0.04% tylose. Grids were blot dried with Whatman #1 filter paper and samples imaged on a Hitachi 7600 TEM at 80 kV. Images were captured with an AMT CCD (1 K × 1 K) camera at 8,000× and 30,000× magnifications. Fifty representative gas vesicle nanoparticles from each strain were measured and average values and standard deviations calculated.
Western blotting analysis
The methods used were similar to those previously described . Briefly, cell lysates containing 50 μg of protein or purified gas vesicle nanoparticle preparations containing 2 μg of protein were electrophoresed on 12% polyacrylamide-SDS gels, for 90 minutes at 100 volts using a Bio-Rad vertical gel electrophoresis unit. Proteins were transferred to 0.45 μm Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Boston, MA) for 1 hour at 100 volts using a Bio-Rad gel blotter. The membranes were washed twice for 5 minutes with TBS buffer [20 mM Tris–HCl (pH 7.6), 137 mM NaCl], blocked for 1 hour with 5% BSA in TBS buffer, incubated overnight at 4°C with affinity column purified rabbit GvpC antibodies (Thermo Scientific) diluted 1:500  or rabbit anti-His-tag antibody (Cell Signaling Technology, Beverly, MA) diluted 1:750. Membranes were then washed 5 times each for 5 minutes with TBS buffer containing 0.1% Tween 20, and incubated with goat anti-rabbit secondary antibodies labeled with alkaline phosphatase (Sigma Corporation), diluted (1:2500) in a solution containing 5% BSA in TBS buffer. For detection of the protein bands, the membrane was incubated in 1-Step NBT/BCIP Substrate (Thermo Scientific) according to the manufacturer’s specification.
Whole cell lysates or purified gas vesicle nanoparticles prepared as described above were assayed for Gaussia princeps luciferase activity using the Glow Assay system (Thermo Scientific) according to the manufacturer’s specification. Assays were conducted in 96-well plates using a SpectraMax M5 luminometer (Molecular Devices, Sunnyvale, CA). Induction was calculated in relative light units of the treated sample/average relative light units of the untreated samples.
This work was supported by Bill & Melinda Gates Foundation grant OPP1061509 and National Institutes of Health grant R03 AI107634. We thank Mr. Abish Regmi and Ms. Heather McDaniel for expert experimental assistance and Dr. Sook Chung for generously providing the luminometer for luciferase assays.
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