Characterizations Of A Novel Non-specific Nuclease And Lysogeny-related Proteins From Marine Thermophilic Bacteriophages

Thermostable enzymes from thermophiles have attracted extensive studies. In this investigation, a nuclease-encoding gene (designated as GBSV1-NSN) was obtained from thermophilic bacteriophage GBSV1 for the first time and it shared no homology with any known nucleases. After recombinant expression in Escherichia coli, the purified GBSV1-NSN exhibited non-specific nuclease activity, being able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. The characterization of the recombinant GBSV1-NSN showed that its optimal temperature and pH were 60°C and 7.5, respectively. The results indicated that the enzymatic activity was inhibited by enzyme inhibitors or detergents, such as ethylene diamine tetraacetic acid, citrate, dithiothreitol,β-mercaptoethanol, guanidine hydrochloride, urea and SDS. In contrast, the nuclease activity was enhanced by TritonX-100, Tween-20 or chaps to approximately 124.5% - 141.6%. The Km of GBSV1-NSN nuclease was 231, 61 and 92μmol/L for the cleavage of dsDNA, ssDNA and RNA, respectively. Our study, therefore, presented a novel thermostable non-specific nuclease from thermophilic bacteriophage and its overexpression and purification for scientific research and applications.During the study on the lysogeny of a deep-sea thermophilic bacteriophage GVE2, it was found that the gene organization of GVE2 was different from that ofλphage in the location of the lysis-related proteins. The distance between cⅠand vp462 genes was shorter in GVE2 than that inλphage. In order to further investigate the lysogeny of bacteriophage GVE2, the genes encoding CⅠprotein (VP228) and site-specific recombinase (VP462) were cloned and expressed in E. coli. The CⅠprotein can maintain the lysogenic state by itself and the recombinase plays an important role in establishing the lysogenic state.Temporal analysis showed that the cⅠand vp462 genes were transcribed and expressed at various infection stages. VP462 existed as dimer in vivo and as monomer in vitro. CⅠexisted as dimer and tetramer before 6 hours after the GVE2 infection and as tetramer after 8 hours postinfection, suggesting that the lysogeny was increased.CⅠcould form oligomer if the temperature was above 50℃in vivo and in vitro, showing that the CⅠprotein functioned at high temperatures. CⅠprotein could degrade itself as a peptidase in vitro, which was similar to that ofλphage. Based on ChIP and EMSA assays, it was found that there was only one site for CⅠbinding on the genome of the phage GVE2, which was different from that ofλphage. The binding site was located in the promoter of cⅠgene. These data demonstrated that the original transcription of GVE2 might begin at this site.It is well known that the Cro protein is a regulator that promotes the lytic development and operates on the same site as CⅠin different ways. In this investigation, the vp64, encoding a hypothetic Cro protein, was cloned. But the recombinant VP64 protein could not interact with the CⅠ-interacted site, showing that it was not Cro. Using the DNA of CⅠ-interacted site, the Cro could not be obtained. The CⅡprotein, which plays a key role in the lysis-lysogeny decision, can activate the promoter of recombinase inλphage. However the CⅡprotein was not available using the DNA of the promoter of vp462. Therefore, it might be proposed that the lysogenic regulation of GVE2 was different fromλphage.Based on the attP site ofλphage, it was found that the site-specific recombinase VP462 could bind to the DNA located near its gene by EMSA. This was the attP site of GVE2.In this study, the results displayed that the original transcription and the lysogeny establishment of deep-sea thermophilic bacteriophage GVE2 was different from those of theλphage. The lifestyle of GVE2 was simpler than that ofλphage. This merited to be further studied.