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Single-cell genomics-based analysis of virus-host interactions in marine surface bacterioplankton - PubMed

Single-cell genomics-based analysis of virus-host interactions in marine surface bacterioplankton

Jessica M Labonté et al. ISME J. 2015 Nov.

Abstract

Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus-host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. A combination of comparative genomics, metagenomic fragment recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus-host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage-host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. Our study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host-virus interactions in complex microbial communities.

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Figures

Figure 1
Figure 1

Phage associated with the SAR116 SAG AAA160-J14 and its genetic potential for integration at the bacterial tRNA-Met-CAT site. (a) Phage genome organization with the attP site represented by an asterisk. (b) Bacterial tRNA-Met-CAT, with the attB region that is identical to phage's recognition site highlighted in yellow.

Figure 2
Figure 2

Single-cell whole-genome MDA kinetics. (a) SAG reaction kinetics, where dots indicate actual measurements, the red curve indicates a logistic model fit, and the blue line indicates the critical point (Cp). The Cp is the time required to reach half of the maximal fluorescence of DNA labeled with SYTO-9. (b) Correlation between the MDA Cp and the estimated genome recovery. Each data point represents a SAG. The solid line indicates a linear regression fit. Dotted lines represent 95% confidence intervals for the linear regression model prediction. Yellow shading indicates SAGs inferred to undergo late lytic infections. The r2 of this regression increases to 0.48 when six outlier SAGs are excluded from the analysis.

Figure 3
Figure 3

Phylogenetic analysis of the Podoviridae-like DNA polymerase A from SAGs (underlined), isolates (labeled) and metagenomes (not labeled). The grey shading indicates the pre-defined groups from Schmidt et al. (2014) and Labonté et al. (2009). The tree was generated using maximum likelihood, with 100 bootstrap replicates, using the LG model with a gamma distribution (+G), estimated rates of variation among sites and a proportion of invariable sites (+I). Bootstrap replicates are not shown for clarity. Scale bar represents the number of amino acid substitution per site.

Figure 4
Figure 4

Synteny between Podoviridae genomes that are most closely related to SAG-associated Podoviridae, based on DNA pol A phylogeny. Each arrow represents a gene: DNA pol A (red), major capsid protein (blue), tail fibers (yellow), terminase (purple) and others (black). tBLASTx was used to identify homologous regions. Color legend indicates DNA sequence identity.

Figure 5
Figure 5

Fragment recruitment of microbial metagenomic reads from the Line P metagenome (Bm) and viral metagenomic reads from the Pacific Ocean Virome (Vm) on the viral (Vc) and bacterial contigs (Bc). The scale bar indicates the fraction of metagenomic reads aligning to each reference with ⩾70% nucleotide identity, normalized by the length of the genome. The right column indicates whether the virus belongs to the Podoviridae (P), Myoviridae (M), Siphoviridae (S) or Phycodnaviridae (Ph) families.

Figure 6
Figure 6

Phylogenetic analysis of Myoviridae phages recovered from SAGs, isolates and environmental sequences. (a) Phylogenetic tree of 13 concatenated conserved proteins within the large Myoviridae family, showing that phages found in Verrucomicrobia SAG AAA164-P11 and Thaumarchaeota SAG AAA160-J20 form a novel phylogenetic group, and that Roseobacter phages AAA076-E06 and AAA160-J18 are most similar to cyanophages. The following parameters were used: maximum likelihood, 100 bootstrap replicates, LG model with a gamma distribution (+G), estimated rates of variation among sites and a proportion of invariable sites (+I). Bootstrap replicates >95% and 75–95% are represented by black and gray circles, respectively. (b) Phylogenetic tree of the major capsid protein gp23 of Myoviridae phages from SAGs (red), isolates (blue) and PCR amplicons from environmental samples (black). The following parameters were used: Neighbor Joining, Jukes Cantor model, 1000 bootstrap replicates. Both trees are rooted with the divergent Rhodothermus phage RM378. Scale bar represents the number of amino acid substitution per site.

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