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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2014)

Diversity-generating retroelements (DGRs) are distinguished by their ability to iteratively diversify defined DNA sequences which encode the ligand binding domains of target proteins (TP). Diversification occurs through a template-dependent, error prone reverse-transcriptase mediated process, termed mutagenic homing, which introduces nucleotide substitutions into a variable repeat (VR) while preserving cis- and trans- acting elements needed for future rounds of diversification. The process of DNA diversification requires a DGR-encoded reverse transcriptase (RT), an accessory variability determinant (Avd), and a template repeat (TR)-derived RNA intermediate. The archetype DGR is found within the Bordetella bacteriophage BPP however, over 300 putative DGRs have been identified within the bacterial domain as well as within a species of archaea. We have identified DGRs within the opportunistic human pathogen Legionella pneumophila (Lp) as well as within Legionella tunisiensis which encode nearly identical diversification machinery, yet each has a VR with a unique pattern of adenine mutagenesis suggesting individualized diversification in response to selection. We analyzed the DGR within Lp strain Corby and identified that the genetic requirements for mutagenic homing were similar to those of BPP, suggesting all DGRs might function through a conserved mechanism. Using in vitro growth conditions, we observed elevated levels of diversification during Lp transition from exponential to stationary growth phase suggesting that DGR mutagenic homing might be modulated by host regulatory networks. To investigate this hypothesis we generated deletions of key factors, relA and spoT, which are critical for coordinating phenotypic differentiation of Lp. PCR analysis to detect levels of mutagenic homing in wt and mutant Lp cells revealed an increase in relA spoT double deletion mutants. qRT-PCR analysis of wt and mutant cells showed that double deletion mutants had an increase in TR-RNA transcripts, while expression of avd and RT was unchanged. Over-expression of TR-RNA increased levels of mutagenic homing above those observed in wt cells, but not to the extent observed with the overexpression of avd, TR, and RT. Cumulatively these data suggest that in Lp, the abundance of TR-RNA transcripts partially controls rates of mutagenic homing, indicating regulation of these elements on multiple levels. Lp DGRs have the potential to generate ~1019 distinct polypeptide sequences within C-type lectin (CLec) domains of their TPs. The Lp Corby TP, ldtA, expresses a surface displayed outer membrane (OM) lipoprotein whose CLec domain is exposed to the extra-cellular milieu. Translocation of LdtA across the inner membrane requires the twin-arginine translocation (TAT) machinery where we hypothesize it is recognized, modified, and transported to the outer membrane by the localization of lipoproteins (Lol) system. To fully understand the pathways required for surface display of a protein, we identified and analyzed the contribution of a non-canonical lipobox with conserved targeting residues at +2/+3 positions in LdtA. Mutagenesis of the lipobox conserved cysteine as well as replacement of targeting residues with amino acids shown to result in sorting by Lol to the OM all resulted in retention of LdtA in the inner membrane. Furthermore, cleavage of LdtA from pro- to mature-peptide was found to depend on the characteristic of the +2 residue suggesting that maturation and subsequent translocation of LdtA does not follow the established convention for lipoproteins. Furthermore, we demonstrated surface display of LdtA in several species of gram negative bacteria suggesting its localization requires conserved pathways common to bacteria. These results suggest that DGR TPs in Lp are diversified in response to the physiological state of the host and trafficked to the surface by an unusual Lol-related mechanism.

    Cover page: Legionella Diversity Generating Retroelements: Creating massively variable repertoires of surface displayed proteins
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2014)
    Diversity-generating retroelements (DGRs) are DNA diversification machines found in diverse bacterial and bacteriophage genomes that accelerate the evolution of ligand-receptor interactions. Diversification results from a unidirectional transfer of sequence information from an invariant template repeat (TR) to a variable repeat (VR) located in a protein-encoding gene. Information transfer is coupled to site-specific mutagenesis in a process called mutagenic homing, which occurs through an RNA intermediate and is catalyzed by a unique, DGR-encoded reverse transcriptase that converts adenine residues in the TR into random nucleotides in the VR. In the prototype DGR found in the Bordetella bacteriophage BPP-1, the variable protein Mtd is responsible for phage receptor recognition. VR diversification enables progeny phage to switch tropism, accelerating their adaptation to changes in sequence or availability of host cell-surface molecules for infection. Since their discovery, hundreds of DGRs have been identified, and their functions are just beginning to be understood. VR-encoded residues of many DGR-diversified proteins are displayed in the context of a C-type lectin fold, although other scaffolds, including the immunoglobulin fold, may also be used. DGR homing is postulated to occur through a specialized target DNA-primed reverse transcription mechanism that allows repeated rounds of diversification and selection, and the ability to engineer DGRs to target heterologous genes suggests applications for bioengineering. This chapter provides a comprehensive review of our current understanding of this newly discovered family of beneficial retroelements.
      Cover page: Diversity-generating Retroelements in Phage and Bacterial Genomes
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2015)
      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.
        Cover page: Targeted diversity generation by intraterrestrial archaea and archaeal viruses
        • Article
        • Peer Reviewed
        UC Santa Barbara Previously Published Works (2017)
        Major radiations of enigmatic Bacteria and Archaea with large inventories of uncharacterized proteins are a striking feature of the Tree of Life1-5. 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 hypervariability6-8, are prominent features of genomically reduced organisms from the bacterial candidate phyla radiation (CPR) and as yet uncultivated phyla belonging to the DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaea) archaeal superphylum. From reconstructed genomes we have defined monophyletic bacterial and archaeal DGR lineages that expand the 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, defence 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 with a versatile tool that could be used for adaptation to a dynamic, host-dependent existence.
          Cover page: Retroelement-guided protein diversification abounds in vast lineages of Bacteria and Archaea
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2018)
          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.
            Cover page: Diversity-generating retroelements: natural variation, classification and evolution inferred from a large-scale genomic survey
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