Summary The Pelagibacterales order (SAR11) in Alphaproteobacteria dominates marine surface bacterioplankton communities, where it plays a key role in carbon and nutrient cycling. SAR11 phages, known as pelagiphages, are among the most abundant phages in the ocean. Four pelagiphages that infect Pelagibacter HTCC1062 have been reported. Here, we report 11 new pelagiphages in the Podoviridae family. Comparative genomics classified these pelagiphages into the HTVC019Pvirus genus, which includes the previously reported pelagiphages HTVC011P and HTVC019P. Phylogenomic analysis clustered HTVC019Pvirus pelagiphages into three subgroups. Integrases were identified in all but one HTVC019Pvirus genome. Site‐specific integration of HTVC019Pvirus pelagiphages into host tRNA genes was verified experimentally, demonstrating the capacity of these pelagiphages to propagate by both lytic and lysogenic infection. Evidence of pelagiphage integration was also retrieved from the Global Ocean Survey database, showing that prophages are found in natural SAR11 populations. HTVC019Pvirus pelagiphages could impact SAR11 populations by a variety of mechanisms, including mortality, genetic transduction and prophage‐induced viral immunity. HTVC019Pvirus pelagiphages are a rare example of cultured lysogenic phage that can be implicated in ecological processes on broad scales. These pelagiphages have the potential to become a useful model for investigating strategies of host infection and phage‐dependent horizontal gene transfer.

Summary Viruses play a key role in biogeochemical cycling and host mortality, metabolism, physiology and evolution in the ocean. Viruses that infect the globally abundant SAR11 bacteria (pelagiphages) were reported to be an important component of the marine viral communities. Our current knowledge of pelagiphages is based on a few studies and therefore is limited. In this study, 10 new pelagiphages were isolated and genomically characterized. These pelagiphages represent the first cultivated representatives of four viral lineages only found in metagenomic sequencing datasets previously. Many abundant environmental viral sequences, i.e., single‐virus vSAG 37‐F6 and several Global Ocean Viromes (GOV) viral populations, are now further confirmed with these pelagiphages. Viromic read mapping reveals that these new pelagiphages are globally distributed in the ocean and can be detected throughout the water column. Remarkably, isolation of these pelagiphages contributed up to 12% of all viromic reads annotated in the analysed viromes. Altogether, this study has greatly broadened our understanding of pelagiphages regarding their morphology, genetic diversity, infection strategies, and distribution pattern. The availability of these newly isolated pelagiphages and their genome sequences will allow us to further explore their infectivities and ecological strategies.

17The Pelagibacterales order (SAR11) in Alphaproteobacteria dominates marine surface 18 bacterioplankton communities, where it plays a key role in carbon and nutrient cycling. 19SAR11 phages, known as pelagiphages, are among the most abundant phages in the 20 ocean. Four pelagiphages that infect Pelagibacter HTCC1062 have been reported. Here 21 we report 11 new pelagiphages in the Podoviridae family. Comparative genomic analysis 22 revealed that they are all closely related to previously reported pelagiphages HTVC011P 23 and HTVC019P, in the HTVC019Pvirus genus. HTVC019Pvirus pelagiphages share a 24 core genome of 15 genes, with a pan-genome of 234 genes. Phylogenomic analysis 25 clustered these pelagiphages into three subgroups. Integrases were identified in all but 26 one pelagiphage genomes. Evidence of site-specific integration was obtained by 27 high-throughput sequencing and sequencing PCR amplicons containing predicted 28 integration sites, demonstrating the capacity of these pelagiphages to propagate by both 29 lytic and lysogenic infection. HTVC019P, HTVC021P, HTVC022P, HTVC201P and 30 HTVC121P integrate into tRNA-Cys genes. HTVC011P, HTVC025P, HTVC105P, 31 HTVC109P, HTVC119P and HTVC200P target tRNA-Leu genes, while HTVC120P 32 integrates into the tRNA-Arg. Evidence of pelagiphage integration was also retrieved 33 from Global Ocean Survey (GOS) database, suggesting the occurrence of pelagiphage 34 integration in situ. The capacity of HTVC019Pvirus pelagiphages to integrate into host 35 genomes suggests they could impact SAR11 populations by a variety of mechanisms, 36 including mortality, genetic transduction, and prophage-induced viral immunity. 37HTVC019Pvirus pelagiphages are a rare example of a lysogenic phage that can be 38 implicated in ecological processes on broad scales, and thus have potential to become a 39 3 useful model for investigating strategies of host infection and phage-dependent horizontal 40 gene transfer. 41 42 IMPORTANCE 43 Pelagiphages are ecologically important because of their extraordinarily high census 44 numbers, which makes them potentially significant agents in the viral shunt, a concept 45 that links viral predation to the recycling of dissolved organic matter released from lysing 46 plankton cells. Lysogenic Pelagiphages, such as the HTVC019Pvirus pelagiphages we 47 investigate here, are also important because of their potential to contribute to the 48 hypothesized processes such as the "Piggy-Back-the-Winner" and 49 "King-of-the-Mountain". The former explains nonlinearities in virus to host ratios by 50 postulating increased lysogenization of successful host cells, while the latter postulates 51 host-density dependent propagation of defensive alleles. Here we report multiple 52 Pelagiphage isolates, and provided detailed evidence of their integration into SAR11 53 genomes. The development of this ecologically significant experimental system for 54 studying phage-dependent processes is progress towards the validation of broad 55 hypotheses about phage ecology with spe...

SAR11 bacteria dominate ocean surface bacterioplankton communities, and play an important role in marine carbon and nutrient cycling. The biology and ecology of SAR11 are impacted by SAR11 phages (pelagiphages) that are highly diverse and abundant in the ocean. Among the currently known pelagiphages, HTVC010P represents an extremely abundant but under-studied phage group in the ocean. In this study, we have isolated seven new HTVC010P-type pelagiphages, and recovered 77 nearly full-length HTVC010P-type metagenomic viral genomes from marine metagenomes. Comparative genomic and phylogenomic analyses showed that HTVC010P-type pelagiphages display genome synteny and can be clustered into two major subgroups, with subgroup I consisting of strictly lytic phages and subgroup II mostly consisting of phages with potential lysogenic life cycles. All but one member of the subgroup II contain an integrase gene. Site-specific integration of subgroup II HTVC010P-type pelagiphage was either verified experimentally or identified by in silico genomic sequence analyses, which revealed that various SAR11 tRNA genes can serve as the integration sites of HTVC010P-type pelagiphages. Moreover, HTVC010P-type pelagiphage integration was confirmed by the detection of several Global Ocean Survey (GOS) fragments that contain hybrid phage–host integration sites. Metagenomic recruitment analysis revealed that these HTVC010P-type phages were globally distributed and most lytic subgroup I members exhibited higher relative abundance. Altogether, this study significantly expands our knowledge about the genetic diversity, life strategies and ecology of HTVC010P-type pelagiphages.

Viruses play critical roles in influencing biogeochemical cycles and adjusting host mortality, population structure, physiology, and evolution in the ocean. Marine viral communities are composed of numerous genetically distinct subfamily/genus-level viral groups. Among currently identified viral groups, the HMO-2011-type group is known to be dominant and broadly distributed. However, only four HMO-2011-type cultivated representatives that infect marine SAR116 and Roseobacter strains have been reported to date, and the genetic diversity, potential hosts, and ecology of this group remain poorly elucidated. Here, we present the genomes of seven HMO-2011-type phages that were isolated using four Roseobacter strains and one SAR11 strain, as well as additional 207 HMO-2011-type metagenomic viral genomes (MVGs) identified from various marine viromes. Phylogenomic and shared-gene analyses revealed that the HMO-2011-type group is a subfamily-level group comprising at least 10 discernible genus-level subgroups. Moreover, >2000 HMO-2011-type DNA polymerase sequences were identified, and the DNA polymerase phylogeny also revealed that the HMO-2011-type group contains diverse subgroups and is globally distributed. Metagenomic read-mapping results further showed that most HMO-2011-type phages are prevalent in global oceans and display distinct geographic distributions, with the distribution of most HMO-2011-type phages being associated with temperature. Lastly, we found that members in subgroup IX, represented by pelagiphage HTVC033P, were among the most abundant HMO-2011-type phages, which implies that SAR11 bacteria are crucial hosts for this viral group. In summary, our findings substantially expand current knowledge regarding the phylogenetic diversity, evolution, and distribution of HMO-2011-type phages, highlighting HMO-2011-type phages as major ecological agents that can infect certain key bacterial groups.