Guegler, C. K. | bioRxiv Science

bioRxiv2022

The evolution of a counter-defense mechanism in a virus constrains its host range

Bacteria use diverse immunity mechanisms to defend themselves against their viral predators, bacteriophages. In turn, phages can acquire counter-defense systems, but it remains unclear how such mechanisms arise and what factors constrain viral evolution. Here, we experimentally evolved T4 phage to overcome a phage-defensive toxin-antitoxin system, toxIN, in E. coli. Through recombination, T4 rapidly acquires segmental amplifications of a previously uncharacterized gene, now named tifA, encoding an inhibitor of the toxin, ToxN. These amplifications subsequently drive large deletions elsewhere in T4s genome to maintain a genome size compatible with capsid packaging. The deleted regions include accessory genes that help T4 overcome defense systems in alternative hosts. Thus, our results reveal a trade-off in viral evolution; the emergence of one counter-defense mechanism can lead to loss of other such mechanisms, thereby constraining host range. We propose that the accessory genomes of viruses reflect the integrated evolutionary history of the hosts they infected. HighlightsO_LIExperimentally evolved T4 to overcome E. coli toxIN, a phage-defensive TA system C_LIO_LIDiscovered TifA, a phage-encoded protein inhibitor of ToxN C_LIO_LIAmplification of the tifA locus drives large deletions elsewhere in the T4 genome C_LIO_LIDeleted genes in evolved T4 clones include those necessary to infect alternative hosts C_LI

microbiology↗

bioRxiv2020

A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

The profiling of gene expression by RNA-sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is ribosomal RNA (rRNA), it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued. Here, we report the development a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin beads then depletes rRNA from a complex, total RNA sample such that ~75-80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold-change measurements in RNA-seq experiments between our method and the previously available Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or metagenomic sample of interest. ImportanceThe ability to examine global patterns of gene expression in microbes through RNA-sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of ribosomal RNA from total RNA samples. Otherwise, rRNA would comprise upwards of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from messenger RNA or requiring high total read depth. A commonly used, kit for rRNA subtraction from Illumina was recently discontinued. Here, we report the development of a do-it-yourself kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligos enable sufficient rRNA depletion to produce RNA-seq data with 75-80% of reads comming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

microbiology↗