April 21, 2020  |  

Virus-host coexistence in phytoplankton through the genomic lens

Phytoplankton-virus interactions are major determinants of geochemical cycles in the oceans. Viruses are responsible for the redirection of carbon and nutrients away from larger organisms back towards microorganisms via the lysis of microalgae in a process coined the “viral shunt”. Virus-host interactions are generally expected to follow “boom and bust” dynamics, whereby a numerically dominant strain is lysed and replaced by a virus resistant strain. Here, we isolated a microalga and its infective nucleo-cytoplasmic large DNA virus (NCLDV) concomitantly from the environment in the surface NW Mediterranean Sea, Ostreococcus mediterraneus, and show continuous growth in culture of both the microalga and the virus. Evolution experiments through single cell bottlenecks demonstrate that, in the absence of the virus, susceptible cells evolve from one ancestral resistant single cell, and vice-versa; that is that resistant cells evolve from one ancestral susceptible cell. This provides evidence that the observed sustained viral production is the consequence of a minority of virus-susceptible cells. The emergence of these cells is explained by low-level phase switching between virus-resistant and virus-susceptible phenotypes, akin to a bet hedging strategy. Whole genome sequencing and analysis of the ~14 Mb microalga and the ~200 kb virus points towards ancient speciation of the microalga within the Ostreococcus species complex and frequent gene exchanges between prasinoviruses infecting Ostreococcus species. Re-sequencing of one susceptible strain demonstrated that the phase switch involved a large 60 Kb deletion of one chromosome. This chromosome is an outlier chromosome compared to the streamlined, gene dense, GC-rich standard chromosomes, as it contains many repeats and few orthologous genes. While this chromosome has been described in three different genera, its size increments have been previously associated to antiviral immunity and resistance in another species from the same genus. Mathematical modelling of this mechanism predicts microalga-virus population dynamics consistent with the observation of continuous growth of both virus and microalga. Altogether, our results suggest a previously overlooked strategy in phytoplankton-virus interactions.

April 21, 2020  |  

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race.

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

April 21, 2020  |  

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

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