Diversity-generating retroelements (DGRs) are novel genetic elements that use reverse transcription to generate vast numbers of sequence variants in specific target genes. Here, we present a detailed comparative bioinformatic analysis that depicts the landscape of DGR sequences in nature as represented by data in GenBank. Over 350 unique DGRs are identified, which together form a curated reference set of putatively functional DGRs. We classify target genes, variable repeats and DGR cassette architectures, and identify two new accessory genes. The great variability of target genes implies roles of DGRs in many undiscovered biological processes. There is much evidence for horizontal transfers of DGRs, and we identify lineages of DGRs that appear to have specialized properties. Because GenBank contains data from only 10% of described species, the compilation may not be wholly representative of DGRs present in nature. Indeed, many DGR subtypes are present only once in the set and DGRs of the candidate phylum radiation bacteria, and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea archaea, are exceptionally diverse in sequence, with little information available about functions of their target genes. Nonetheless, this study provides a detailed framework for classifying and studying DGRs as they are uncovered and studied in the future.

Major radiations of enigmatic bacteria and archaea with large inventories of uncharacterized proteins are a striking feature of the Tree of Life1,2,3,4,5. The processes that led to functional diversity in these lineages, which may contribute to a host-dependent lifestyle, are poorly understood. Here we show that diversity-generating retroelements (DGRs), which guide site-specific protein hypervariability6,7,8, are prominent features of genomically-reduced organisms from the bacterial candidate phyla radiation (CPR) and yet uncultivated phyla belonging to the DPANN archaeal superphylum. From reconstructed genomes we defined monophyletic bacterial and archaeal DGR lineages that expand known DGR range by 120% and reveal a history of horizontal retroelement transfer. Retroelement-guided diversification is further shown to be active in current CPR and DPANN populations, with an assortment of protein targets potentially involved in attachment, defense, and regulation. Based on observations of DGR abundance, function, and evolutionary history, we find that targeted protein diversification is a pronounced trait of CPR and DPANN phyla compared to other bacterial and archaeal phyla. This diversification mechanism may provide CPR and DPANN organisms a versatile tool that could be used for adaptation to a dynamic, host-dependent, existence.

In the evolutionary arms race between microbes, their parasites, and their neighbours, the capacity for rapid protein diversification is a potent weapon. Diversity-generating retroelements (DGRs) use mutagenic reverse transcription and retrohoming to generate myriad variants of a target gene. Originally discovered in pathogens, these retroelements have been identified in bacteria and their viruses, but never in archaea. Here we report the discovery of intact DGRs in two distinct intraterrestrial archaeal systems: a novel virus that appears to infect archaea in the marine subsurface, and, separately, two uncultivated nanoarchaea from the terrestrial subsurface. The viral DGR system targets putative tail fibre ligand-binding domains, potentially generating >10 18 protein variants. The two single-cell nanoarchaeal genomes each possess ≥4 distinct DGRs. Against an expected background of low genome-wide mutation rates, these results demonstrate a previously unsuspected potential for rapid, targeted sequence diversification in intraterrestrial archaea and their viruses.

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA–cDNA molecules, as observed in vivo. We identified RNA–cDNA molecules to be branched and most plausibly linked through 2′-5′ phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.

1-Deoxy-D-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4′-amino-pyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4′-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ~10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes induced by conformational variations in the active site. Additionally, mutagenesis studies reveal DXS to be an evolutionarily resilient enzyme. Particularly, we hypothesize that residues H82 and H304 may act in a compensatory fashion if the other is lost due to mutation. Further, nucleus-independent chemical shifts (NICSs) and aromatic stabilization energy (ASE) calculations suggest that reduction in TDP aromaticity also serves as a factor for regulating ylide formation and controlling reactivity.