More than 30 years ago, three papers were published in Nature^1,2,3 showing the enormous abundance of viruses in seawater and their role as mortality agents of heterotrophic bacteria and phytoplankton (Fig. 1); thus, setting the stage for a new paradigm in biological oceanography, and more broadly, for the role of viruses in ecosystems. Since then, thousands of papers have been published on viruses in the sea, documenting their abundance, diversity and ecosystem functions. Yet, despite widely accepted estimates of virus-mediated microbial mortality, indicating that viruses kill about 20 to 30% of the living biomass in the oceans each day^4,5, these are bulk estimates that do not provide information on how lysis is distributed among thousands of microbial taxa. Identifying the taxa undergoing lysis has been one of the most important outstanding questions since viruses were recognized as being major agents of microbial mortality. We have tackled this problem by establishing the use of extracellular ribosomal RNA (rRNA_ext) to estimate taxon-specific cell lysis of microbes.

The rRNA_ext is the result of cell lysis

For years, we were surprised by large amounts of ribosomal RNA (rRNA) “contamination” that was present in 0.22-µm filtered seawater collected to investigate the diversity of RNA viruses. We hypothesized that rRNA was released by cell lysis (Fig. 2), and that this rRNA_ext could be sequenced to reveal the taxonomic composition of the cells that had died.

Preliminary investigations using quantitative reverse-transcription PCR (qRT-PCR) revealed that there were typically millions of copies of rRNA_ext in each mL of 0.22-µm-filtered seawater, and that it was stable for days when incubated on the benchtop. This indicated that the rRNA_ext was likely in the form of ribosome complexes that prevent degradation of the RNA, consistent with observations that rRNA complexed with ribonucleoproteins is protected from nucleases^6,7.

            To confirm the origin of rRNA_ext, we conducted laboratory experiments with marine bacteria, their viruses and grazers. We showed that rRNA_ext is produced when bacteria are infected by viruses, and that grazing by protists does not contribute to rRNA_ext production; in fact, protistan grazing was associated with a decrease in rRNA_ext. Given that viral lysis accounts for approximately half of the mortality of prokaryotes in seawater^2,8,9 and kills approximately 20 to 30% of the standing stock of bacteria each day^4,5, viral lysis is likely responsible for most of the rRNA_ext production. However, other causes of cell lysis such as programmed cell death¹⁰, predation by Bdellovibrio and like organisms (BALOs)¹¹, and bacteriocins^12,13, may also contribute to the production of rRNA_ext, although there is no evidence that these processes are quantitatively significant relative to viral lysis.

rRNA_ext provides a window on taxon-specific microbial cell lysis

In addition to the rRNA_ext, we also sequenced the cellular rRNA genes (rRNAgene_cell) and cellular rRNA (rRNA_cell) (Fig. 3). The taxa in the rRNA_ext pool reflect cell lysis, while taxa in the rRNAgene_cell and rRNA_cell pools indicate taxa for which intact cells are present in the water. By comparing the taxa that are detected in each of the three pools (rRNAgene_cell, rRNA_cell, and rRNA_ext), we can distinguish among taxa undergoing lysis (ongoing-lysis or recent-lysis), taxa in which lysis has already occurred (prior-lysis), and cells in which lysis has not occurred (no-lysis). MoRS allowed us to

• detect lysis in thousands of different prokaryotic taxa
• show that most lysis occurred prior to the time of sampling
• detect lysis in up to 34% of the taxa detected in coastal seawater samples
• detect lysis in 31 out of 36 prokaryotic phyla, including 19 phyla from which viruses have not been reported, indicating that lysis is widespread across prokaryotic phyla
• show that the taxa in which lysis occurred varied greatly among seawater samples (Fig. 4).
• show that of the taxa in which lysis was detectable, only a maximum of 8% were present in the cellular fraction, indicating that in most cases lysis occurred prior to sampling
• show that lysis was undetectable in most taxa at the time of sampling

Cell lysis is uneven among microbial taxa

Sequencing of the rRNA_ext reveals the taxa in which lysis has occurred; if the rRNA_ext is corrected for differences in “rRNA copy number per cell” that occur among taxa and with physiological state, the ratio of free rRNA (rRNA_ext) to cellular rRNA (rRNA_cell) provides an index of the relative amount of lysis in each taxon. Thus, a cell-lysis index (CLI) can be calculated for individual taxa from the ratio of the relative abundance of rRNA_ext to rRNA_cell. Our results revealed ongoing lysis in 128 taxa, with the CLI varying across depths and time. The results also showed that the CLI is highly uneven among taxa within a seawater sample. This is important, as most current models of microbial mortality assume lysis is evenly distributed among taxa¹⁴.

Rare species are associated with high lysis while dominant taxa are associated with low lysis.

An enduring puzzle in marine microbial ecology is the relationship between viral infection and the structure of prokaryotic communities, and in particular the relationship between the abundance of specific taxa and viral lysis. It has been proposed that lysis rates are relatively low for cells in the most abundant taxa, and higher in relatively rare bacteria that are capable of fast growth¹⁵. This view is congruent with “Kill the Winner”, a model, in which viral lysis prevents the most ecologically “fit” cells from dominating the community^16,17. Conversely, cells associated with rare taxa may be subject to significant viral lysis¹⁸. Here, by using MoRS to estimate taxon-specific cell lysis, we demonstrated that high lysis was associated with taxa that were relatively rare (such as copiotrophs), while low lysis was often associated with taxa that were abundant, such as those in the SAR 11 group. Therefore, it is likely that rare species are the result of high lysis, whereas dominants are likely to be successful in part due to relatively low rates of lysis.

In summary, MoRS offers a powerful new approach for understanding how taxon-specific lysis shapes microbial communities and processes, and provides an important tool in our efforts to explain the distribution and abundance of specific microbial taxa in nature.

Publication:

Zhong, K. X., Wirth, J. F., Chan, A. M. & Suttle, C. A. Mortality by ribosomal sequencing (MoRS) provides a window into taxon-specific cell lysis.  ISME J (2022). https://doi.org/10.1038/s41396-022-01327-3.

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