Low-temperature Induction Mechanism Of Deep-sea Shewanella Piezotolerans WP3 Filamentous Phage SW1 & Physiological Functions Of Dna Phosphorothioate Modification

As the most abundant biological entities in deep sea,viruses influence the life activities of bacteria and archaea directly or indirectly,and they play an important role in the overall functioning of the deep-sea ecosystem.Nev-ertheless,most studies are focused on the ecological investigation of benthic phages,and little is known about how they are regulated and how they affect the extreme environmental adaptation of their bacterial hosts,because of the rarity of phage-host systems isolated from deep-sea environments.It is reported that Inoviridae are the main active vrial groups in deep-sea sediments.Previously,we isolated the filamentous phage SW1 from a deep-sea bacterium,Shewanella piezotolerans WP3,which was isolated from west Pacific deep-sea sediment at a water depth of 1914 m in 2007.SW1 can be induced by low temperature,and the induction is independent on the clas-sic SOS response,but the underlying molecular regulatory mechanism has not yet been elucidated.In this study,we first characterized the function of phage-encoded regulator FpsR.Deletion of fpsR led to a significant increase of phage gene transcription level,and phage genome single strand DNA could be detected at 20?.At 4?,the transcription of phage genes could be still induced,which indicated that other regulators were involved in phage gene regulation.FpsR functions as tetramers,and the N terminal and the C terminal is responsible for DNA-binding and polymerization,respectively.C terminal deletion led to the loss of function.EMSA and DNase I Foot-printing showed that FpsR had three binding sites within the promoter region of SW1 structure gene operon,and FpsR could not bind to the fpsR promoter region.However,FpsR carried out a feedback inhibition because of the reverse cross arrangement of SW1 promoters.Biolayer interferometry results showed that FpsR could bind to three sites independently and the binding affinity of FpsR was decreased at low temperature.H-NS is another key regulator which is responsible for phage SW1 gene transcription.Temperature significantly affected H-NS binding affinity,and the binding affinity of H-NS decreased at low temperature.Deletion of hns gene led to a significant increase of transcription level of SW1 structure gene,such as fpsA.However,the transcription level of fpsR decreased sig-nificantly in hns deletion mutant.In vitro experiments showed that H-NS could directly bind to the promoter regions of fpsA and fpsR at one site,re-spectively.H-NS also played an important role in phage DNA replication,and deletion of hns led to SW1 replication defects.In fpsR and hns double deletion mutant,the low-temperature induction effect of SW1 gene expres-sion decreased significantly,for the transcription level of SW1 genes in-creased by only 10%at 4?.Accordingly,we proposed a low-temperature induction regulation model of SW1,in which FpsR and H-NS are the core regulatory factors.We use phage SW1 as a model to study the influence of PT on phage gene expression.DNA phosphorothioate(PT)modification is a novel modi-fication on the DNA backbone,in which a non-bridging oxygen atom is swapped with a sulfur atom by the proteins DndA-E.PT modification exists widely in bacteria;however,the functional landscape of PT modifications has not been firmly established,especially the possible gene regulation func-tion.There are 8 PT sites in SW1 regulatory region.PT led to a significant increase of fpsA and fpsB transcription,but a decrease in fpsR transcription,which indicated that PT influenced both repression and activation of SW1genes.After the 8 PT sites were mutated,there was no significant difference in SW1 gene transcription level between PT strains and control strains.After we deleted the key repressor gene fpsR,there was no significant difference in fpsA and fpsB transcription level between PT strains and control strains,but the transcription level of fpsR was significantly less in PT strains.Transfer-ring a PT system to a SW1-contained WP3 strain led to a significant increase in fpsA and fpsB transcription level,a decrease in fpsR transcription level,and the phages were induced at 20?.EMSA and Isothermal titration calorimetry experiments showed that PT could reduce the binding affinity of FpsR.Antioxidation capablility of phosphorothioate DNA has been proved in some bacteria,so we propose that PT modification may play a role in micro-bial adaptation to many extreme conditions.After obtaining a PT system,WP3 PT strains exhibited superior survival rates/growth in response to X-ray,UV,low temperatures,high pressures,low salinity levels and heavy-metal stresses,but demonstrated a growth defect in stationary phase at high tem-peratures.WP3 PT strains didn't show advantages in response to salinity lev-els,acid,alkali,hydrogen peroxide,and antibiotics stresses.After transform-ing PT system into an E.coli catalase/peroxidase disruption strain Hpx~—,Hpx~—PT strains exhibited superior survival rates/proliferation under almost all stress conditions except antibiotics stresses.PT modification gene expression was relatively constant under different stresses,and redox monitoring sensor results and hydrogen peroxide scavenging experiments indicated that PT sys-tem functioned as an antioxidation system inside cells.WP3 PT strain growth defects under high temperature stress is due to that PT affected the normal induction of heat shock proteins.In PT strains,the expression amounts of heat shock proteins were insufficient,which led to the growth defects.Based on the distribution patterns of dnd and dpt gene clusters among bacterial phylogenic groups,it is evident that the dnd gene clusters located in deeper branches of the Bacteria domain,and are more widespread than is the case for genomes harboring both the dnd and dpt gene clusters.This observa-tion suggests that the PT modification system originated as an antioxidation system and later developed into an atypical restriction-modification system.