Gas vesicles are organelles of bacteria and archaea that constitute protein nanoparticles. Gas vesicles are known to provide flotation to the microorganisms, so that oxygen and light are properly available to them especially in phototrophic organisms. Gas vesicles are useful in biotechnological applications as their protein membrane is stable against solubility and they are produced in large scale. The hypotonic lysis of the host can lead to the purified gas vesicles.
The gas vesicles are identified by genetic analysis of halophilic Archaea bacteria, where the gene cluster responsible for gas vesicle formation is established in the plasmid of the bacterium. The gene cluster representing gas vesicles was found to be present in 191 kb plasmid called pNRC100 in the Halobacterium sp.NRC-1. The gene cluster constitutes transcription of gvpACNO directed towards right, while the transcription of gvpDEFGHIJKLM is directed towards left. The promoters are situated in the range of gvp A to D region that is of 210bp length. Interruption in any one of these gvp genes would result in the phenotype that is gas vesicle deficient. Hence, all the genes in the cluster are found to be essential for the formation of gas vesicle.
The proteins available in the gas vesicle nanoparticles are identified by western blotting analysis with the help of antisera single gvp gene products. The proteins are named as GvpA, GvpC, GvpF, GvpG, GvpJ, GvpL, and GvpM. Gvp A, J and M are found to be useful in the formation of gas vesicle membrane. The Gvp F and Gvp L are coiled-coil proteins useful for growth or nucleation of nanoparticles. These proteins are also identified in genomic analysis of other microbes having gas vesicles. The gvpC gene was found to be present in the cyanobacterial and haloarchaeal microbes that can produce gas vesicles.
GvpC gene codes for protein sequence that is known to consist of eight repeats present in both cyanobacteria and haloarchaea. Mutations in the GvpC gene caused due to the insertion in Halobacterium sp. NRC-1, could generate gas vesicles with altered shape. The stability of the bacterial strain is enhanced and growth of gas vesicles was facilitated by GvpC protein. For bio-engineering of nanoparticles, a new strain of Halobacterium sp. NRC-1 was constructed with plasmid expression vectors.
Results of the study
Construction of gvpC expression vectors
Bio-engineering of gas vesicle nanoparticles was improved in a study by creating a GvpC deletion strain of Halobacterium sp. NRC-1 through ura3 based gene deletion method. A suicide vector pBB400ΔgvpC was generated with the flanking regions of gvpC cloned into it. This plasmid was transformed into Halobacterium sp. NRC-1 Δura3. The integration of the genes was detected by specific selection of Halobacterium sp.NRC-1 Δura3 ΔgvpC deletion strain. This strain had partial gas vesicle deficient phenotype with small and spindle shaped vesicles.
To complement ΔgvpC strain with the expression system, gvpC expression vector series were constructed. To carry out this expression, pMC2 plasmid backbone was constructed with cspD2 promoter that is cold inducible. This plasmid was known to have replication and selection capacity in E.coli as well as in Halobacterium constructed basically in another research study for investigating beta-galactosidase protein of a haloarchaeon. In this study, beta-galactosidase of pMC2 was replaced with a start codon followed by a hexahistidine tag and restriction sites to allow the insertion of foreign genes all together in an adapter. The resultant plasmid was called as pARK. The two restriction sites of pARK adapter region were AflII and AvrII and gvpC gene fragments were inserted in between them after PCR amplification of the fragments. The resultant plasmid construct consisting of gvpC regions was pARK-C.
Engineering of gvpC gene and GvpC expression
The impact of GvpC length on the bio-engineering and production of gas vesicle nanoparticles was determined by transforming the pARK-C plasmids coding N-terminal regions of GvpC into ΔgvpC strain. The lengths of GvpC in the pARK-C plasmid series differed. pARK-C1 consisted of 130AAs, pARK-C2-200AAs, pARK-C3-280AAs, and wild type as well as pARK-C4 consisted of 382AAs in the GvpC sequence. The phenotype with elevated opacity due to increased gas vesicles was observed in the strains containing the plasmids in the given order. ΔgvpC (pARK-C) < ΔgvpC (pARK-C1) < ΔgvpC (pARK-C2) < ΔgvpC (pARK-C3) ~ ΔgvpC (pARK-C4) ~ Δura3. Therefore, lesser quantities of gas vesicles were formed in the strains that are expressing small GvpC fragments while higher quantities are formed in strains expressing large fragments of GvpC protein.
The morphology of gas vesicle nanoparticles in ΔgvpC derivative strains and in ΔgvpC wild type strain was examined by purification of nano particles through centrifugally accelerated flotation. The mean lengths and widths of gas vesicle nanoparticles was between 386 - 432 nm and 163 - 202 nm respectively of pARK-C1, C2 and C3. The lengths and widths were 344 nm and 158 nm respectively of ΔgvpC wild type strain; 458 nm and 256 nm respectively of Δura3 strain. The localization of GvpC protein and N-terminal fragments expressed by pARK-C plasmids in ΔgvpC transformants was done by western blotting. The various sizes that were expected of GvpC proteins (fragments and full protein) present in the cell lysates and floating gas vesicle nanoparticles were observed in the western blot.
Shiladitya DasSarma, Ram Karan, Priya DasSarma, Susan Barnes, Folasade Ekulona and Barbara Smith. An improved genetic system for bioengineering buoyant gas vesicle nanoparticles from Haloarchaea. BMC Biotechnology 2013, 13:112.
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