| As the most abundant living form in marine ecosystems,virus impacts on the diversity,community structure and function of microbes through both lytic and lysogenic infections.It also regulates the biogeochemical cycle and energy flow in the global ocean,known as a nanoscale driver of global-scale processes.During infection,temperate phage can decide between either lytic or lysogenic cycle.Once establishing lysogeny,phage DNA integrates into the host chromosome to become a prophage.Lysogenic infections are in fact widespread in marine ecosystems.Multiple investigations have shown that about 40-50%of microbial genomes contain prophage fragment(s).However,temperate phages or prophages of many important marine bacterial groups have not been explored yet.Meanwhile,as for the ecological role of marine viruses,there is a long-standing dispute between "killing-the-winner" and"piggyback-the-winner" hypotheses.It seems that varied spatiotemporal patterns of lysogenic infection as well as the complex and diverse temperate phage-bacterium interactions may be the reasons for the coexistence of the two seemingly contradictory models in marine ecosystems.Therefore,it is necessary to investigate life strategies of viruses targeting typical marine microbial taxa,characterize the features and properties of marine temperate phages,and further explore how temperate phages affect the function and dynamics of marine ecosystem by affecting their host bacteria.Here,the typical marine photoheterotrophic bacterium Citromicrobium bathyomarinum and its temperate phages were studied.During studying for master degree,I focused on prophage morphology and genetic diversity,its potential impacts on shaping the geographical distribution of the host species and its ecological roles of"killing-the-relatives" and "invading-the-relatives".During the doctoral study,the author continued with more in-depth investigations on the transformation of phage life strategies,within-host competition and prophage-bacterium interactions.The main findings are as follows:(1)A temperate phage vB_CbaS-RXM(RXM)carrying a tRNATrp(CCA)gene was isolated,which can lyse C.bathyomarinum strains isolated from the surface,subsurface and deep-sea environments.Within the trapping host JL1366,the burst size of RXM is about 75 plaque-forming unit(PFU)per cell.Intriguingly,when infecting one of the susceptible lysogenic hosts,RXM can induce the release of the intracellular prophage,suggesting its role as a natural inducing agent.In order to measure the contribution of host tRNA pool and phage tRNA to viral gene translation,the cosine correlation distance of codon usage pattern of each phage gene and the host genome,as well as the contribution index tRCI of phage tRNA to each phage gene were calculated.It was estimated that the expression of genes related to DNA replication and metabolism together with auxiliary metabolic genes involved in phage-bacterium interactions were significantly dependent on host translation system,while phage tRNA significantly promoted the expression of some hypothetical genes of low host dependence.Such a translation strategy may confer phages encoding tRNA with more fitness during host jumps or habitat changes.(2)Temperate phage RXM belongs to the phiCbK-like subfamily;phiCbK,isolated in the 1965,is a model phage that has contributed to numerous fundamental researches of virology and bacteriology.All members of this subfamily carry conserved lysogeny-related genes.However,no prophage similar to phiCbK has been reported,and two lysogeny tests have failed to isolate any colony harboring phiCbK(-like)prophage.Thus,the life strategy of this subfamily has been unclear so far.In this section,four full-length phiCbK-like prophage genomes were identified from different alphaproteobacterial genomes for the first time,with both lysogenic and pseudolysogenic life styles being observed.For each known phiCbK-like isolate,the alignment of attachment sites on the phage and bacterial genomes(attP-attB)pairing was conducted,so that the corresponding integration site can be determined.The author improved the traditional lysogeny test and confirmed that phage RXM can integrate into the host chromosome,supporting our point that the shared ancestry of phiCbK-like members employed a temperate life strategy.The domain analysis of phage integrase/recombinase indicated that clade I members have lost the Cre-like recombinase of predicted integration activity together with the coupled attP,and thus transforming from temperate into obligate lytic.The genomic length of the temperate phage and the physiological state of the host bacterium collectively account for the transformation of life strategies observed in group Ⅰ members.This work is instructive for future efforts exploring temperate phages in bacterial genomes for therapeutic purposes.(3)Members of several subgroups of C.bathyomarinum are most different in the presence of prophage or the genotype of prophage they harbor,which suggests the high activity of lysogenic infection within this species.Statistics showed that the prophage genome accounts for 5.53%of C bathyomarinum pan genome.Meanwhile,prophage-free and lysogenic lineages of the C3 subgroup have begun to differentiate in their aligned core genome,suggesting that prophages not only contribute to the expansion of the host pan-genome,but also have potential impacts on the evolution of host core genome.The well adaption of prophage codon usage bias to that of the C.bathyomarinum host was noticed,and this reflects the high degree of coevolution between the two.A number of prophage auxiliary metabolic genes(AMGs)have been annotated,some of which may regulate host response to commmon stresses in marine ecosystems,such as low temperature,osmotic stress,starvation and pollutants.These key auxiliary metabolic genes are widely distributed,not only within C.bathyomarinum prophages of different genera,but also from full-length and defective prophages of α-proteobacterial species.Combined results of the in-silico analysis and host-range test,there are a total of 17 combinations of co-infection among 15 different genotypes of C.bathyomarinum prophage,indicating that frequent lateral transmissions during temperatephage co-infection could be a significant source of these auxiliary metabolic genes.(4)C.bathyomarinum prophages display two different t mechanisms for integration and excision;for example,Mu-like members depend on the transposase-mediated integrative and replicative transpositions,while non-Mu-like members undergo integrase-mediated site-specific recombination.It was observed that the latter can horizontally transfer the integrase-attP module,so that homologous phages can exhibit different integration sites.This strategy is helpful for temperate phages to avoid homologous competition and explore new host resources.Moreover,accommodating heterologous prophages could be conducive to the heterogeneity and fitness of the host population.Within-host prophage competition showed that the release of Mu-like members was always higher than that of its non-Mu-like competitive prophage in both Mitomycin C(MmC)-induction and spontaneous induction.The sequencing data provided evidence that Mu-like embers have completed the replicative transposition and DNA capsulation before the competitive prophage initiates the lytic cycle.Statistics also showed that Mu-like members prefer replicative transposition into the competitive prophage genome at a frequency of 1.38-2.81 times higher than expected,which interfere the replication of the competitive prophage. |
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