Lysogenic host-virus interactions in SAR11 marine bacteria

Abstract

Host-virus interactions structure microbial communities, drive biogeochemical cycles and enhance genetic diversity in nature^1,2. Hypotheses proposed to explain the range of interactions that mediate these processes often invoke lysogeny^3-6, 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 nature^6,8-15. Here, we report the discovery of prophages in cultured SAR11, the ocean's most abundant clade of heterotrophic bacteria^16,17. 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.

Extended Data Figure 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 Figure 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 Figure 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 Figure 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 Figure 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 Figure 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 Figure 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 Figure 8.. AMS1 base media.

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

Extended Data Figure 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 (e.g. 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.

Figure 1.

Genome alignment of Ca. P. giovannonii strain NP1 to Ca. P. ubique HTCC1062. Inner ring, complete genome sequence of strain NP1 mapped to itself (green). Outer ring, complete genome of HTCC1062 (blue). Red border marks the location of the prophage. Black lines mark the location of the 40bp core nucleotide sequences (attL and attR) flanking the prophage genome.

Figure 2.

Growth characteristics of Ca. P. giovannonii strain NP1 with phage PNP1. A,C) Strain NP1 growth and new virus production over the bacterial growth cycle on carbon-replete and carbon-deplete media, respectively. B) Prophage induction with mitomycin C (dark grey bars) and UV radiation (light grey bars). D) Virus decay over time in defined media. Data points in panels A-C are the mean of n=3 biologically independent samples and the error bars are the standard deviation. Bars in panel D are the mean of n=3 biologically independent samples (dot plot). Evidence of concurrent bacterial and virus production was repeatedly verified in transfer cultures.

Figure 3.

Micrographs of Ca. P. spp. NP1 and NP2 and PNP1 and PNP2 virions. A) Image of a representative NP1 cell acquired at 14,000x. B,C) Images of PNP1 virions independently acquired at 44,000x. D) Image of NP2 cell acquired at 14,000x. E,F) Images of PNP2 virions independently acquired at 22,000x. Scale Bars: A, D = 500 nm; B, E = 25 nm. Panels A-C, cells and viruses from n=4 strain NP1 cultures. Panels D-F, cells and viruses from 1 culture of NP2. Images were acquired from n=2–3 individual regions on n=2–3 individual grids.

Figure 4.

Sequence alignment of PNP1 and related prophage recovered from seawater. Sequences derive from the Mediterranean Sea and from 250 m in the North Pacific. Black lines mark the location of 40bp core sequences associated with the attP site.