DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA.

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation

The Bordetella bacteriophage DGR employs similar mechanisms for retrotransposition in heterologous species

Diversity-generating retroelements (DGRs) are a unique group of retroelements found in bacteria, archaea and their viruses. They mediate hyperdiversification of protein-encoding DNA sequences in facilitate the adaptation of their hosts to changing environments. The prototype DGR was discovered in the Bordetella bacteriophage BPP-1 and consists of three genes, mtd (major tropism determinant), avd (accessory variability determinant) and brt (Bordetella reverse transcriptase), and two imperfect repeats, variable repeat (VR) and template repeat (TR). VR is located at the 3' end of mtd, which encodes the phage distal tail fiber protein responsible for receptor recognition. Diversification of mtd results from unidirectional transfer of sequence information from TR to VR during which adenine residues in TR are converted into random nucleotides in VR, leading to phage tropic variants that recognize different receptor molecules. Here, we show that the BPP-1 DGR is also functional in heterologous bacterial species - Escherichia coli and Pseudomonas aeruginosa, and uses a similar mechanism for cDNA synthesis. However, efficiency of DGR mutagenic homing is affected by target sequence orientation in plasmids. Interestingly, overexpression of Avd and bRT has differential effects on DGR homing into targets inserted in different vectors. Surprisingly, homing into plasmid targets in E. coli is found to be largely independent of IMH (initiation of mutagenic homing) and the DNA stem-loop, elements important for its homing into native phage targets.Santa S. Naorem, Jin Han, Christa Jackson, Bingyue Zhang, James Guo and Huatao Guo ; Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, Missouri 6521

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA.

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation