Lysogenic host–virus interactions in SAR11 marine bacteria

Abstract

Host–virus interactions structure microbial communities, drive biogeochemical cycles and enhance genetic diversity in nature1,2. Hypotheses proposed to explain the range of interactions that mediate these processes often invoke lysogeny3,4,5,6, a latent infection strategy used by temperate bacterial viruses to replicate in host cells until an induction event triggers the production and lytic release of free viruses. Most cultured bacteria harbour temperate viruses in their genomes (prophage)7. The absence of prophages in cultures of the dominant lineages of marine bacteria has contributed to an ongoing debate over the ecological significance of lysogeny and other viral life strategies in nature6,8,9,10,11,12,13,14,15. Here, we report the discovery of prophages in cultured SAR11, the ocean’s most abundant clade of heterotrophic bacteria16,17. We show the concurrent production of cells and viruses, with enhanced virus production under carbon-limiting growth conditions. Evidence that related prophages are broadly distributed in the oceans suggests that similar interactions have contributed to the evolutionary success of SAR11 in nutrient-limited systems.

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Fig. 1: Genome alignment of Ca. P. giovannonii strain NP1 to Ca. P. ubique HTCC1062.
Fig. 2: Growth characteristics of Ca. P. giovannonii strain NP1 with phage PNP1.
Fig. 3: Micrographs of Ca. P. spp. NP1 and NP2 and PNP1 and PNP2 virions.
Fig. 4: Sequence alignment of PNP1 and related prophages recovered from seawater.

Data availability

Ca. P. giovannonii strain NP1 and associated data have been deposited under GenBank accession number CP038852 and BioProject number PRJNA531001. The strain NP1 16S rRNA sequence has been deposited under GenBank accession number MH923014. Original negative-stain micrographs will be made available on request. Ca. P. giovannonii strain NP1 cultures have unique growth requirements and cannot be maintained in standard culture collections. Requests for bacterial cultures or DNA from the University of Washington Culture Collection should be addressed to the corresponding author. On receiving a request for materials, we will assist the requesting institution in completing a Uniform Biological Material Transfer Agreement. There is no transmittal fee for academic institutions.

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Acknowledgements

This work was funded by a grant from the National Science Foundation awarded to R.M.M. and A. Ingalls (OCE-15584830). K.L.H. was supported by the National Institute of Allergy and Infectious Disease, award number F32AI14511. We thank members of the Center for Environmental Genomics at the University of Washington for their support and valuable feedback. We especially thank C. Rathwell for her valuable advice and thoughtful insights into marine virus ecology, and B. Durham and M. A. Moran for their help with experimental design leading to the cultivation of lysogenic strains of SAR11.

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Contributions

R.M.M. led the research effort, advised K.R.C. on all research activities, conducted direct cell and virus counts, and wrote the paper with support from K.R.C., K.L.H. and J.M.K. K.R.C. isolated SAR11 strains NP1 and NP2, sequenced the genome of strain NP1, verified the PNP1 phage integration site, maintained cultures and conducted growth experiments as part of her undergraduate research thesis. J.M.K. advised K.L.H. and contributed to the interpretation of TEM and associated data. K.L.H. prepared samples for TEM analyses, took images, and quantified virus and vesicle-like particle sizes.

Corresponding author

Correspondence to Robert M. Morris.

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Extended data

Extended Data Fig. 1 Sequence coverage across the Ca. P. Giovannonii strain NP1 genome.

A total of 5346 reads. Mean coverage is 696 with 0 missing bases.

Extended Data Fig. 2 PCR primers used to amplify NP1 and PNP1 DNA.

PCR primers were designed to connect genomic contigs, to validate genomic assemblies, and to identify phage insertion sites.

Extended Data Fig. 3 Sequences associated with phage and host attachment sites verified by PCR.

a, The bacterial chromosomal sequence (attB), phage sequences (attP), and core sequences in blue, black, and red, respectively. b, PCR reactions verifying sequences associated with phage integration and excision, with bacterial (16S rRNA gene) and phage (internal) controls. All PCR products were sequenced to verify phage integration (attL and attR sites), a circular phage genome with an attachment site (attP), and phage excision from the bacterial chromosome with an attachment site (attB). PCR reactions were verified with DNA extracted from two or more cultures.

Extended Data Fig. 4 Core PNP1 integration sequences identified in public databases.

40 bp core sequences identified in PNP1 (attP) and exact matches to tRNA sequences in SAR11 (attB) or to sequences in the NCBI genomic survey database.

Extended Data Fig. 5 Ca. P. Giovannonii strain NP1 growth initiated from cultures with PNP1 virions added at different ratios.

Ratio of 1:1 (black) and 10:1 (red). Data points are the mean of n=3 biologically independent samples and the error bars are the standard deviation.

Extended Data Fig. 6 Characteristics of PNP1 and PNP2.

Size and sequence characteristics of PNP1 and PNP2 phage relative to other Pelagibacter phages. Measurements are the mean from 2 independent biological samples of NP1 and 1 sample of NP2. Images were acquired from n=2-3 distinct regions on n=2-3 grids. Errors are the standard deviation.

Extended Data Fig. 7 Micrographs of strains NP1 and NP2, virions, and vesicle like particles.

a, Image of a Ca. P. giovannonii NP1 host cell with an elongated morphology, evidence of budding, and possible virion attachment. Image acquired at 8,900x. Arrow mark budding and asterisks mark virions; not all budding and viruses have been marked. b–d, Representative images of vesicle-like particles found in strain NP1, independently acquired at 22,000x; asterisks mark virions. e, Image of strain NP2 host cell showing evidence of budding, acquired at 14,000x. Arrow marks budding. f–h, Representative images of vesicle-like particles found in strain NP2, independently acquired at 22,000x. Scale Bars: A, E = 500 nm; B-D, F-H = 100 nm. Panels A-D, images from 2 separate cultures of NP1. Panels E-H, images from 1 culture of NP2. Images for cultures were acquired from 2–3 distinct regions on 2–3 grids.

Extended Data Fig. 8 AMS1 base media.

Defined media used to grow SAR11 strain NP1 and strain NP2.

Extended Data Fig. 9 Host and virus size exclusion of SYBR Green 1 stained particles.

a, Raw image of cells and free viruses stained, mounted, and viewed by epifluorescence microscopy. b, Cells (purple) and viruses (green) separated and enumerated by size exclusion (for example insert). All counts were determined by taking the average number of cells and viruses from 15–20 images at each time point and in biological triplicate. Direct cell and virus counts were repeated weekly on biologically independent samples and to verify continuous virus production in transfer cultures.

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Morris, R.M., Cain, K.R., Hvorecny, K.L. et al. Lysogenic host–virus interactions in SAR11 marine bacteria. Nat Microbiol 5, 1011–1015 (2020). https://doi.org/10.1038/s41564-020-0725-x

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