The Study On Temperate Halophilic Archaea Virus And Its Losogenic Host From The Genera Natrinema

Plenty of bioinformatics analysis revealed that the proviruses and their sequence segments existed extensively in genomes of all microorganisms. Because it is difficult to find the corresponding sensitive strains, these temperate viruses were often neglected. At present, the majority of the archaea viruses are isolated from the natural environments, only few of them are found in lysogenic hosts. In this study, temperate haloarchaeal virus SNJ1was induced from the lysogenic host Natrinema sp. J7-1with mitomycin C, and it could produce plaques on lawns of Natrinema sp. J7-2. It is the first identified Natrinema infecting virus and the third isolated provirus of halophilic archaea. We found that the host Natrinema sp. J7-1can release virus particles spontaneously under the normal conditions, but the titer of self-released virus is not high. We optimized the induction conditions of mitomycin C to increase the titer from-104PFU/ml to-1011PFU/ml. Single-step growth curve gave a latent period of4-5h and a burst size of-100PFU/cell on J7-2. Results showed that SNJ1was strongly dependent on the NaCl concentration and sensitive to the chloroform, which had a great impact on activity maintaining, adsorption rate and plaque formation of the virus. The morphology of highly purified viruses particles are spherical with an internal membrane, which was previously described in haloviruses (SHI).The sequenced genome of SNJ1is a circular, double-stranded DNA (dsDNA) molecule (16341bp). Interestingly, it is identical to a previously described plasmid pHH205, indicating that this plasmid is the provirus of SNJ1. Ten structural protein-encoding genes were identified in the viral genome including two major capsid proteins (MCPs) through MS. The putative large and small MCPs coding genes were amplified from SNJ1genome, heterogeneously expressed and purified. The purified proteins were used as immunogens to produce polyclonal antisera against these proteins. SNJ1from different sources could be detected by western blotting and immune electron microscopy using these antisera, which is conceivable that the plasmid pHH205is the pro virus of SNJ1in lysogenic host. A putative ATPase in SNJ1genome was found through33ORFs blasting the protein database sequences with PSI-BLAST, which revealed poor similarities to previously identified virus genome sequences. It was multiple aligned with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses, and it harbors a P9specific motif in addition to the Walker A and B ones. This P9motif is found only in icosahedral dsDNA viruses with an internal membrane and having a similar coat protein fold. The type virus for such a lineage is bacteriophage PRD1. The presence of lipid constituents from the host phospholipid pool strongly suggests that SNJ1belongs to the PRD1-type lineage of dsDNA viruses with an internal membrane.In the exploration of the relationship between SNJ1and host, sensitive strain, the strain J7-1and J7-2sample were subjected whole genome sequencing, respectively. Surprisingly, the results showed that the genome sequence of J7-1and J7-2are almost completely identical to each other. The differences between the two strains are the plasmids. While strain J7-1harbors a plasmid, named pHH205with16.3Kbp, another plasmid pJ7-I, about95.9Kbp, was found in J7-2, but not in J7-1. We infected J7-2with SNJ1and recovered the center of the plaque in the culture. After being cultured in the liquid culture with high concentrition of sodium chloride, we obtained lysogenic strain J7(LJ7). After PCR amplification and pulsed field gel electrophoresis (PFGE) detection, we certify that both plasmid pHH205and pJ7-I exist in the cell of LJ7. Lysogenic strain LJ7can release SNJ1and be immune to the infection of SNJ1. The acquisition of LJ7can help us better understand the correlation between halo archaea virus and their hosts. The16S rRNA gene of strain J7-1and J7-2were closest to haloarchaea Natrinema gari. Several haloarchaea closely related in genera Natrinema and Natrinema gari14663T were tested for their plasmid and sensitivity to SNJ1. They did not contain similar pJ7-I plasmid, and SNJ1was unable to produce plaques on lawns of these strains.Research into extreme Haloarchaea is largely impeded due to scarcity of selective markers, restriction of celluar structures, prohibitions of genetic properties, and limitation of extremely culture condition. This study is mainly aiming at a general study on developing a genetic transformation system for the haloarchaea, Natrinema sp. J7-2, which is isolated from the salt mines of Yingcheng City. Experiments have been carried on in the following fields, generation and regeneration of protoplasts, tests on inhibitor candidates and screen recombinants through two layer plating method. In addition, a vector has been designed based on the genome of SNJ1(plasmid pHH205). By transferring this vector into H. volcanii DS52, hopefully this approach will let us to circumnavigate constrains of genetic transformation laid by J7-2.