Prophages In Marine Citromicrobium Bathyomarinum:Diversity,Activity And Interaction With Hosts

Viruses are submicroscopic and the most abundant biological entities with high genetic diversity in the marine ecosystem.They can participate in regulating community structure and composition of marine microbes and influencing the metabolism and genetic evolution of infected host bacteria through both lytic and lysogenic infections.Hence,phages are considered as nanoscale drivers associated with global-scale processes.Prophages,which are lysogenic phage genomes integrated into the genomes of their hosts,were considered as dangerous time bombs inside host cells before.It was until recently that people come to realize prophages can confer various advantage fitness to their infected hosts.However,researches about marine lysogenic phages are still somewhat limited.In order to get a better understanding of the properties and functions of marine lysogenic phages,also to explore how they influence their infected hosts and then further impact the ecology of marine microbes,this study chose 38 typical marine bacteria,which belong to Citromicrobium bathyomarinum,as research objects and analyzed how the environmental factors act on the lysogenic phage-host interactions.Besides,we put extensive efforts on studying the morphologies,genetic diversity,integration and excision properties,infection activities of the induced prophages and phage-host interactions.Here we put the main conclusions below:(1)After scanning through 38 cultivable C.bathyomarinum isolates with different marine habitats and geographical origins,we found that C.bathyomarinum strains have a wide distribution of both horizontal and vertical sides,which indicates that C.bathyomarinum strains can adapt well to various marine habitats.A relatively high proportion of lysogens(60-100%)was observed in isolates originated from the deep sea and sediments,which suggests prophages may contribute to the host survival under adverse environments.Besides,comparative genomic analysis reveals that a high genetic diversity existed within intraspecific individuals sharing 100%16S identity.It is noteworthy that prophage sequences are proven as major contributors of genetic differentiations between these highly similar intraspecific individuals in this study.(2)C.bathyomarinum lysogenic phages exhibit various morphologies and diverse genetic compositions.Altogether 30 intact prophages,which belonged to 14 genotypes and can be grouped into five clusters,were induced from 38 C.bathyomarinum strains through induction experiments.Prophages in Cluster A,B and E belong to siphoviruses and prophages in Cluster C and D were classified as myoviruses according to their morphologies obtained by TEM.Our study uncovers,for the first time,several different hybrid head-tail types of Mu-like prophages existing in C.bathyomarinum genomes probably resulted from genetic exchanges and recombinations.Furthermore,our work is probably the first report of P2-like prophages identified in alpha-proteobacteria.We also find several auxiliary metabolic genes with functions of influencing bacterial chromosome replication and phosphorus and sulfur metabolism encoded by C.bathyomarinum prophages,which are presumably involved in regulating the metabolism of hosts under nutrient-limited environments.(3)We found that different strategies of integration and excision were adopted by C.bathyomarinum prophages through the comparison between phage genomes under integration and capsid-packaged statuses.They can integrate into or excise from their host genomes either through transpose-mediated or integrase-mediated process.In the first case,Prophages in Cluster A exhibit site-preferential transpositions,while prophages in Cluster B show random transpositions.During excision,Mu-like prophages will randomly package and then attached host sequences to their two ends just as the behavior of Mu phage.A duplication of 5 bp of the target DNA flanking the transposed Mu-like prophage after integration and the first 5 host nucleotides which are attached to the two ends of Mu-like prophage after excision both exhibit typical N-Y-S-R-N bias.In the second case,homologous integrases and attP sequences are the major reasons of a similar pattern observed during prophage integration and excision.That's why two different clusters of C.bathyomarinum prophages,i.e.prophages in Cluster D and E,with homologous integrases and attP sequences can site-specific integrate into the same tRNA-Phe-GAA gene and can be precisely excised from host genomes.(4)We compared the lytic activities of all C.bathyomarinum induced phages and the host-range results confirmed the existences of superinfection immunity and 'kill the relatives' effect,which suggests that prophages may play roles as biological weapons in their host bacteria.And then,we screened out the C.batyomarinum prophage-harbouring mutant strains and applied whole-genome sequencing towards them.Genome analysis verified again the integration properties of Mu-like prophages.We also performed SNP analysis and results suggested different SNP patterns were observed when prophages inserted at different sites.Finally,we compared the growth curves of wild-type and the mutant strains under two different nutrient statuses.An evident survival advantage of prophage-harbouring strain was observed compared with the wild-type strain during nutrient-limited stationary and decline phases,which probably suggests prophage can confer a fitness advantage to their infected host to cope with tough environments.