Preliminary Construction Of Genetic System And Study On The Universality Of Promoters Conferring Cross-domain Activity For Halopilic Archaea Natrinema Genera

In1998, Terry J. McGenity et al proposed Natrinema gen. nov. according to phylogenetic analysis of16S rRNA gene sequences and the taxonomic properties, and studies of genetic system for this genus have not been reported. Halophilic archaea Natrinema sp. strain J7-1and J7-2were conserved in our lab, the result of genome sequencing showed that the chromosome of these two strains were almost similar except that J7-1contains pHH205and J7-2contains pJ7-I. In this dissertioin, some genetic elements of Natrinema sp. J7serial strains were carried out. Genetic systems were constructed using Natrinema sp. J7-2and CJ7as the representative of Natrinema gen. nov.. The universality of archaeal promoters conferring cross-domain acitivty was preliminary studied using Natrinema sp. J7-2as the representative of archaea.As genetic systems for genus Natrinema have not been rep orted yet, and Natrinema sp. J7-1, J7-2from our lab have been extensively investigated in which some of the reported studies provide us useful information to the construction of genetic systems for Natrinema sp. J7-2and CJ7.After J7-2being mutagenized by NTG and UV rays, four auxotrophic mutants of J7-2were isolated and named as J7-2-1, J7-2-22, J7-2-26and J7-2-52. The identification results of auxotrophic phenotype were showed that J7-2-1was leucine auxotrophic, J7-2-26was lysine auxotrophic, J7-2-22and J7-2-52both were arginine auxotrophic. The mutant genes and mutant sites were determined and it showed that in J7-2-1, the mutant gene was leuB, coding3-isopropyl malate dehydrogenase, having a12bp deletion mutant from690bp to701bp, in J7-2-26, the mutant gene was dapD, coding 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase, having a18bp deletion mutant from583bp to600bp and in J7-2-52, the mutant gene was argC, coding N-acetyl-gamma-glutamyl-phosphate reductase, having a point mutation at1051bp (from G to A). With wild type J7-2genome, prototrophs could be acquired from J7-2-1or J7-2-26transformation. The same results could be obtained by J7-2-1or J7-2-26transformed with amplification fragments of leuB gene and dapD gene, or integrative plasmids carrying leuB gene and dapD gene, respectively. The results showed that linear fragments and circular plasmids could be transformed into auxotrophs by using PEG-mediated spheroplasting transformation method which was available in Natrinema genus. In addition, exogenous gene, amyH gene from Haloarcula hispanica DSM4426, was integrated into the auxotrophic genome with the selectable marker genes of the integrative plasmid, and expressed actively in some transformants, which demonstrated that exogenous genes could be expressed in auxotrophs by transformation with integrative plasmids. These results proved that genetic transformation and manipulation are feasible for Natrinema genus.At the same time, we tried to construct a host/shuttle vector genetic system. pHH205was transformed into J7-2and CJ7free of this plasmid, the transformation mixture recovered overnight was induced with MMC the result of which was that the supernatant from the induced mixture could infect sensitive strain J7-2and produce plaques. This result confirmed again the feasibility of PEG-mediate spheroplating transformation in Natrinema sp. J7-2, and confirm the feasibility in CJ7. And the formation of plaque from pHH205transformation was the first time using the experiments to confirm the point of view that plasmid pHH205is the provirus of virus SNJ1.Based on the Mev selection marker and the full length of pHH205, the shuttle vector pUC19-Mev-pHH205i8was constructed and could be transformed into J7-2and CJ7in which this plasmid could replicate. The shuttle vector containing No.4fragment (8.715Kb) also could replicate but with a lower transformation efficiency. Transformants have been screen from No.8fragment (7.047Kb) or No.16fragment (2.706Kb) transformation, but their transformation efficiency were very low even with only one transformant. Obvious plasmid were not extracted which showed that the copy number of the plasmid maybe low. But the correct plasmid was detected after that the extraction samples of plasmid DNA were transformed in to Escherichia coli. The result demonstrated that No.8fragment (7.047Kb) or No.16fragment (2.706Kb) contained the replication region. We also found the transformation efficiency in CJ7was higher, compared with J7-2, maybe the reason was that there is a about95Kb plasmid pJ7-I in J7-2which could influence the exogenous DNA transformation. The experimental results of plasmid stability showed that in case of continuous culture without antibiotic for ten days, the plasmid stability of pUC19-Mev-pHH205which was almost no loss was higher than pUC19-Mev-pHH2054, whereas pUC19-Mev-pHH205has loss situations at a various degree, maybe the reason was that the elements affecting plasmid stability had been cut off from No.4fragment, so in case of strain culturing without antibiotic, the pUC19-Mev-pHH205plasmid was lost at various degree without any pressure from antibiotic selection.The work of producing plaques and SNJ1infecting experiments was also carried on at the same time. The results showed that the supernatant from the strain containing pUC19-Mev-pHH205i8induced by MMC give the formation of plaques, while the supernatant from the strain pUC19-Mev-pHH2054no. On another aspect, SNJ1could not infect the strain containing pUC19-Mev-pHH205i8, but could infect strain with pUC19-Mev-pHH2054, which demonstrated that No.4fragment maybe lost the function elements that made the strains immunity to virus. All these results laid a solid foundation for further work on the investigation of genetics of virus and the mechanism of infection and lysis in virus.In the early studies of our lab, we found that promoter of a-amylase encoding gene in Haloarcular hispanica had cross-domain promoter activity both in Escherichia coli and halophic archaea. This promoter was analysed and found containing'-10Box'similar to the conserved element'-10Box'of Escherichia coli promoter, which maybe the reason of cross-domain activity. Whether the phenomenon of archaeal promoters conferring cross-domain activity has a certain universality, we took genome of Natrinema sp. J7-2as representative for the further investigation. Two vectors were needed to constructed which were used to detect the promoter activity in bacteria and archaea separately. By improving the Escherichia coli-Haloferax volcanii shuttle vector pSYl, the Escherichia coli-Haloferax volcanii shuttle expression vector pAJ was constructed and its ability for shuttle expression and molecular cloning sites was identified by expression of two different proteins in the two domains separately. In bacteria, by improving the promoter probe vector pKK232-8, the new promoter probe vector pUKM was constructed, and studies were done to validate its function of molecular cloning sites and promoter probing. The activity of presumed promoters by directly or random selection from Natrinema sp. J7-2was investigated in bacteria and archaea domains using the pUKM vectors and pAJ vectors separately. It shown that16presumed promoters from the17directly selection based on "-10Box" had promoter activities in bacteria, while only a few of the presumed promoters from the34random selection had promoter activities in bacteria and in these presumed promoters conferring activity in bacteria, we found some motifs which were similar to the "-10Box" in the17directly selection promoters. The results of these archaeal promoters showed that the motifs in them were different from the conserved element such as BRE element, TATA box and INR, in most of archaeal promoters. According to the corresponding experimental results, we speculate that in archaea there is a certain number of promoters conferring cross-domain promoter activity and there is some degree of evolutionary relationship of promoters among archaea, bacteria and eukaryotes.