Functional Metagenomics of a Replicase from a Novel Hyperthermophilic Aquificales Virus

Abstract

Bacteriophage and viral replisomes typically require fewer proteins to replicate their genome compared to their cellular counterparts and are therefore model systems for studying this fundamental and ubiquitous process. Replication elements also tend to be arranged in a cluster or operon, as opposed to the distributed replication genes found in Bacteria and Archaea. A gene encoding a DNA polymerase with innate reverse transcriptase activity was previously isolated from an uncultivated Octopus hot spring viral metagenome sample collected in Yellowstone National Park. This report describes the complete metagenomic sequence of Octopus Spring OS3173 virus, novel structural variants, and new functionally active polymerase derivatives. The 37,256 bp dsDNA circular viral genome contains 48 open reading frames, with numerous genes associated with replication, including a full-length, polyprotein-like variant of the polymerase. OS3173 is predicted to infect an Aquificales host, as multiple clustered regularly interspaced short palindromic repeat (CRISPR) sequences matching seven locations in the virus genome are found within a pink filament streamer microbial community metagenome from Octopus Spring. Bioinformatic analysis of the DNA surrounding the CRISPR spacer region matches portions of a cultivated Thermocrinis ruber genome from the same location with high sequence identity. Enzymatic screening of large-insert clones yielded numerous polyprotein-containing genes encoding active thermostable variants from this virus, confirming the functional diversity of the polymerase in its native habitat. One variant demonstrated robust PCR capabilities compared to the original “wild-type” enzyme.