Diversity-generating retroelements: natural variation, classification and evolution inferred from a large-scale genomic survey.

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.

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.

Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid.

BACKGROUND: Transposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species. RESULTS: We have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported. CONCLUSION: We have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.

Diversity-generating retroelement homing regenerates target sequences for repeated rounds of codon rewriting and protein diversification.

Diversity-generating retroelements (DGRs) introduce vast amounts of sequence diversity into target genes. During mutagenic homing, adenine residues are converted to random nucleotides in a unidirectional, reverse transcriptase-dependent transposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using a Bordetella bacteriophage DGR as a model, we demonstrate that homing occurs through a TR-containing RNA intermediate and is RecA independent. Marker transfer studies show that cDNA integration at the 3' end of VR occurs within a (G/C)(14) element, and deletion analysis demonstrates that the reaction is independent of 5' end cDNA integration. cDNA integration at the 5' end of VR requires only short stretches of sequence homology. We propose that homing occurs through a unique target DNA-primed reverse transcription mechanism that precisely regenerates target sequences. This nonproliferative "copy and replace" mechanism enables repeated rounds of protein diversification and optimization of ligand-receptor interactions.

Surface display of a massively variable lipoprotein by a Legionella diversity-generating retroelement.

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by facilitating localized DNA sequence evolution through a specialized error-prone reverse transcription process. We characterized a DGR in Legionella pneumophila, an opportunistic human pathogen that causes Legionnaires disease. The L. pneumophila DGR is found within a horizontally acquired genomic island, and it can theoretically generate 10(26) unique nucleotide sequences in its target gene, legionella determinent target A (ldtA), creating a repertoire of 10(19) distinct proteins. Expression of the L. pneumophila DGR resulted in transfer of DNA sequence information from a template repeat to a variable repeat (VR) accompanied by adenine-specific mutagenesis of progeny VRs at the 3'end of ldtA. ldtA encodes a twin-arginine translocated lipoprotein that is anchored in the outer leaflet of the outer membrane, with its C-terminal variable region surface exposed. Related DGRs were identified in L. pneumophila clinical isolates that encode unique target proteins with homologous VRs, demonstrating the adaptability of DGR components. This work characterizes a DGR that diversifies a bacterial protein and confirms the hypothesis that DGR-mediated mutagenic homing occurs through a conserved mechanism. Comparative bioinformatics predicts that surface display of massively variable proteins is a defining feature of a subset of bacterial DGRs.

Target site recognition by a diversity-generating retroelement.

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.

Salidroside attenuates oxidized low­density lipoprotein­induced endothelial cell injury via promotion of the AMPK/SIRT1 pathway.

Oxidized low­density lipoprotein (ox­LDL)­induced endothelial damage contributes to the initiation and pathogenesis of atherosclerosis. Salidroside can alleviate atherosclerosis and attenuate endothelial cell injury induced by ox­LDL. However, the mechanisms involved in this process are not fully understood. Therefore, the purpose of the present study was to investigate the role of the adenosine monophosphate­activated protein kinase (AMPK)/sirtuin (SIRT)1 pathway in the protection of salidroside against ox­LDL­induced human umbilical vein endothelial cells (HUVECs) injuries. The results revealed that salidroside reverses ox­LDL­induced HUVECs injury as demonstrated by the upregulation of cell viability and downregulation of LDH release. In addition, salidroside increased the expression of the SIRT1 protein in ox­LDL­treated HUVECs. Next, it was demonstrated that SIRT1 knockdown induced by transfection with small interfering (si)RNA targeting SIRT1 (siSRT1) abolished the protection of salidroside against ox­LDL­induced HUVECs injuries. This was illustrated by a decrease in cell viability and an increase in LDH release, caspase­3 activity and apoptosis rate. Furthermore, salidroside mitigated ox­LDL­induced reactive oxygen species production, upregulated malondialdehyde content and NADPH oxidase 2 expression and decreased superoxide dismutase and glutathione peroxidase activities, while these effects were also reversed by siSIRT1 transfection. In addition, it was demonstrated that salidroside suppressed ox­LDL­induced mitochondrial dysfunction as demonstrated by the increase in mitochondrial membrane potential and decreases in cytochrome c expression, and Bax/Bcl­2 reductions. However, these effects were eliminated by SIRT1 knockdown. Finally, it was demonstrated that salidroside significantly upregulated the phosphorylated­AMPK expression in ox­LDL­treated HUVECs and AMPK knockdown induced by transfection with AMPK siRNA (siAMPK) leads to elimination of the salidroside­induced increase in cell viability and the decrease in LDH release. Notably, siAMPK transfection further decreased the expression of SIRT1. In conclusion, these results suggested that salidroside protects HUVECs against ox­LDL injury through inhibiting oxidative stress and improving mitochondrial dysfunction, which were dependent on activating the AMPK/SIRT1 pathway.

Diversity-generating Retroelements in Phage and Bacterial Genomes.

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.

Structure of the essential diversity-generating retroelement protein bAvd and its functionally important interaction with reverse transcriptase.

Diversity-generating retroelements (DGRs) are the only known source of massive protein sequence variation in prokaryotes. These elements transfer coding information from a template region (TR) through an RNA intermediate to a protein-encoding variable region. This retrohoming process is accompanied by unique adenine-specific mutagenesis and, in the prototypical BPP-1 DGR, requires a reverse transcriptase (bRT) and an accessory variability determinant (bAvd) protein. To understand the role of bAvd, we determined its 2.69 Å resolution structure, which revealed a highly positively charged pentameric barrel. In accordance with its charge, bAvd bound both DNA and RNA, albeit without a discernable sequence preference. We found that the coding sequence of bAvd functioned as part of TR but identified means to mutate bAvd without affecting TR. This mutational analysis revealed a strict correspondence between retrohoming and interaction of bAvd with bRT, suggesting that the bRT-bAvd complex is important for DGR retrohoming.

Dual PI3K/mTOR inhibitor NVP-BEZ235-induced apoptosis of hepatocellular carcinoma cell lines is enhanced by inhibitors of autophagy.

Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling has been found in several types of human cancer, including hepatocellular carcinoma (HCC). NVP-BEZ235 is a novel, orally bioavailable dual PI3K/mTOR inhibitor that has exhibited promising activity against HCC in preclinical models. Autophagy is a cellular lysosomal degradation pathway essential for the regulation of cell survival and death to maintain homeostasis. This process is negatively regulated by mTOR signaling and often counteracts the efficacy of certain cancer therapeutic agents. In this study, we explored the role of autophagy in apoptosis induced by NVP-BEZ235 in two HCC cell lines, Hep3B and PLC/PRF/5, and identified the mechanism of combinatorial treatment. NVP-BEZ235 was effective in inhibiting the growth of the two HCC cell lines possibly though induction of apoptosis. NVP-BEZ235 also potently increased the expression of LC3-II and decreased the expression of p62, indicating induction of autophagy. When NVP-BEZ235 was used in combination with Atg5 siRNA or the autophagy inhibitor 3-methyladenine (3-MA), enhancement of the inhibitory effects on the growth of HCC cells was detected. In addition, enhanced induction of apoptosis was observed in cells exposed to the combination of NVP-BEZ235 and Atg5 siRNA or 3-MA. Thus, induction of autophagy by NVP-BEZ235 may be a survival mechanism that counteracts its anticancer effects. Based on these data, we suggest a strategy to enhance the anticancer efficacy of BEZ235 by blockade of autophagy. Thus, our study provides a rationale for the clinical development of combinations of NVP-BEZ235 and autophagy inhibitors for the treatment of HCC and other malignancies.

A new topology of the HK97-like fold revealed in Bordetella bacteriophage by cryoEM at 3.5 A resolution.

Bacteriophage BPP-1 infects and kills Bordetella species that cause whooping cough. Its diversity-generating retroelement (DGR) provides a naturally occurring phage-display system, but engineering efforts are hampered without atomic structures. Here, we report a cryo electron microscopy structure of the BPP-1 head at 3.5 Å resolution. Our atomic model shows two of the three protein folds representing major viral lineages: jellyroll for its cement protein (CP) and HK97-like ('Johnson') for its major capsid protein (MCP). Strikingly, the fold topology of MCP is permuted non-circularly from the Johnson fold topology previously seen in viral and cellular proteins. We illustrate that the new topology is likely the only feasible alternative of the old topology. ß-sheet augmentation and electrostatic interactions contribute to the formation of non-covalent chainmail in BPP-1, unlike covalent inter-protein linkages of the HK97 chainmail. Despite these complex interactions, the termini of both CP and MCP are ideally positioned for DGR-based phage-display engineering. DOI: http://dx.doi.org/10.7554/eLife.01299.001.