| Extremely abundant diversity was shown in archaeal viruses, though the umber of which is still very limited. The study on them let us forms a new knowledge of the origin and classification of viruses and their influence on the evolution of life. Comparing with around 6200 bacteriophages, only about 120 haloarchaea viruses have been reported so far. Restricted by the lack of stable and tractable virus-host genetic system, further study is limited to a few of viruses. Most of our knowledge of the mechanism of their life cycle, such as viral entry, genome replication, assembly and release is derived from bio informatics analyses of their genome sequence. Further studying on molecular biology mechanism of more archaeal viruses and developing virus-host genetic system are undoubtedly significant for further revealing the biological characteristic of archaeal viruses. In our study involving haloarchaeal virus SNJ1 of which the pro-virus resides in extremely halophilic archaea Natrinema sp. J7 as a plasmid, the genes and regions closely related to virus/plasmid replication, the stable segregation of plasmid and the copy number control was determined and E.coli-Natrinema sp. shuttle vector was constructed. Based on the genetic tool of SNJ1 constructed in this study, the crucial protein responsible for the establishment of superinfection immunity of SNJ1 was identified, the model of the super infect ion immunity was built and lysogenic-lytic switch of SNJ1 was predicted.Halophilic archaea Natrinema sp. J7 was isolated in Yingcheng salt mine, Hubei province by our lab. According to the different containing plasmid, its two kinds of derivatives were named as J7-1 and CJ7. J7-1 contains circular SNJ1 pro-viral genome pHH205 and CJ7 contains no plasmid and can be infected by SNJ1 to form plagues. Based on the arrangement and conservativeness of large and small capsid protein and its ATPase, SNJ1 was categorized to Sphaerolipovirus. To date, our study on SNJ1 is limited on revealing the biological characteristic, and there is no research on its replication model and the regulatory mechanism of its life cycle. In addition, since Nnm sp. J7, the host of SNJ1, has been isolated, no mature genetic system has been established which greatly limits the research on J7 and its viruses.In our study, by constructing a number of is. coli-Natrinema sp. shuttle vector containing different regions of SNJ1, a 3.9 kb region was identified as the minimal replication region of SNJ1. It contains seven predicted ORFs (ORF5 to ORF11-12) which are organized into two operons (ORF5 to ORF6, ORF7 to ORF11-12). Among them, only one protein, the product of ORF11-12, is essential for the replication of SNJ1, thus designated as RepA. Western blot analysis showed that the expression of RepA increased during the lytic cycle of SNJ1. Bioinformatics analysis showed that RepA is a distant homolog of rolling circle replication protein which belongs to the HUH endonucleases superfamily and it shares three conserved motif with these protein speculating that RepA probably mediates the rolling-circle replication of SNJ1. In addition to RepA, two genetic elements located within both termini of the minimal replication region were also found to be required for SNJ1 replication. The copy number of SNJ1 pro-viral genome as well as the shuttle vectors was determined to be 1 to 3 copies per chromosome. Mutating the start codon of ORF4 increases the copy number to about 30 copies per chromosome suggesting that gp4 is a negative regulator of the copy number of SNJ1. Besides, in the absent of antibiotic, the lack of gp4 could seriously impact on the stable maintenance of SNJ1 pro-viral genome in J7. In addition to gp4,294-401 and 4481-6482 region also affect the stable segregation of shuttle vectors to some extent.Based on the stable replication region of SNJ1,1-4481 region, the first E. coli-Natrinema sp. expression shuttle vector pYCJ-HH was constructed. The 9.9 kb pYCJ-HH shuttle vector contains a multiple cloning site, histone coding sequences and the promoter of Haloferax volcanii DS52 hsp70. Using pYCJ-HH, the stable expression of amyH form Haloarcula hispanica 33960 and the purification of amylase were firstly realized in J7. In addition, pYCJ-HH could also stably replicate in Natrinema sp. JCM8980, implying that it may be broadly applied to the genetic manipulation for Natrinema species.In the previous study we found that SNJ1 could infect CJ7, which did not contain SNJ1 pro-viral genome, to form plagues whereas could not infect J7-1, which contains SNJ1 pro-viral genome implying that the lysogenic SNJ1 established superinfection immunity to prevent a secondary infection by SNJ1. In order to determine the region responsible for the establishment of superinfection immunity, shuttle vectors which contains different region of the replication region of SNJ1 were transformed into CJ7. As a result, CJ7 containing pYCJ was found to be immuned to the infection of SNJ1 implying that the crucial region involved in the establishment of superinfection immunity was located in 1-4481 fragment. For the lack of genetic manuscript system for the study on the gene within the replication region of SNJ1, based on the replication origin of the chromosome of J7, shuttle vector pFJ6 which could stably replicate in J7 was constructed. The copy number of the 5.7 kb shuttle vector pFJ6 was 1 copy per chromosome and it could be stably segregated into daughter cell during the replication of J7 and no recombination between it and the chromosome was detected during the experiment. Using pFJ6 shuttle vector, different deletion regions of 1-4481 fragment were expressed and CJ7 containing pFJ-1-656 region was found to be immuned to the infection of SNJ1. The further deletion and mutation of 1-656 region showed that the product of ORF4, gp4, is the key factor for the establishment of superinfection immunity of SNJ1. The same amount of SNJ1 was used to infect CJ7 and CJ7 containing pFJ6-1-656, after 10 hours, all most all the cell of CJ7 could be detected to contain the pro-viral genome of SNJ1 whereas only 10% cells of CJ7 containing pFJ6-1-656 could be detected. It implied that the establishment of superinfection immunity of SNJ1 may due to gp4 that blocks the entry of secondary viral genome into host cells or affects the stable replication and segregation of the secondary viruses.Bio informatics analysis showed that gp4 contains AbrB-like swapped-hairpin domain which is conserved in the transcription regulators in bacterial and archaea inferring that it may be the transcriptional regulator of SNJ1. Besides, our study showed that gp4 plays a vital role in the stable segregation of SNJ1 pro-viral genome, the maintenance of low copy number of SNJ1 and the establishment of superinfection immunity. Thus, gp4 probably be the global regular of SNJ1, and, by regulating the transcription of multiple related genes, controls the transition between lysogenic and lytic state of SNJ1.The identification of the replication region of haloarchaeal virus SNJ1 and study on the functions of the genes within the replication region deepens our understanding of the vital process, such as the replication of genome, copy number control, stable maintenance and the transition between lysogenic and lytic state, in the life cycle of haloarchaeal temperate viruses. The genetic systems based on the replication region of SNJ1 and the chromosome of J7 laid a solid foundation for the further study on Natrinema species and its viruses. The study on the superinfection immunity of SNJ1 deepens our understandinng of the interaction relationship between viruses and its host in extreme environment and make up the lack of the study on the super infection immunity of archaeal viruses. In this study, many regulatory genes and regions are identified to be important for the life cycle of SNJ1, the further study on them will help us deeply understand survival strategy of virus in extreme environment and reveals the evolution relationship of virus and its host... |
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