Huatao Guo

Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria

Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1–22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.

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 1026 unique nucleotide sequences in its target gene, legionella determinent target A (ldtA), creating a repertoire of 1019 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.

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

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.

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.

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

Abstract 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

Significance Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacteria, archaea, and their viruses. DGRs use a reverse transcriptase (RT)-mediated mechanism to diversify protein-encoding genes to facilitate adaptation of their hosts to changing environments. Here, we demonstrate that the Bordetella phage DGR-encoded RT uses the 3′-OH of a nicked template RNA to initiate reverse transcription, during which random nucleotides are incorporated when adenine residues in the template are copied into complementary DNA (cDNA). We further show that this mutated, covalently linked RNA-cDNA molecule is required for DGR-mediated sequence diversification, revealing a mechanism of accelerated evolution with broad practical applications. 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.

Atomic Structure of Bordetella Bacteriophage Reveals a Jellyroll Fold in CementProtein and a Topologically Distinct HK97-like Fold in Major Capsid Protein.

Bordetella Bacteriophage (BPP-1) infects both Bordetella bronchiseptica, and B. pertussis, the bacterium that causes whooping cough. BPP-1 is a short-tailed dsDNA virus and a member in the Podoviridae family. It has a T=7 icosahedral head with a diameter of ~700 A and a tail on one of the twelve icosahedral vertices [1]. The capsid head is composed of two proteins: a major capsid protein (MCP) and a cement protein, and neither has recognizable sequence similarity with proteins in other well studied dsDNA bacteriophages like HK97 and PRD1 [2, 3]. We have determined the structure of the icosahedral head of BPP-1 to 3.4A resolution using single particle cryo-electron microscopy (cryoEM) (Fig. 1a) and derived an atomic model of BPP-1. Briefly, cryoEM images were recorded on Kodak SO-163 micrographs at 59K magnification in an FEI Titan Krios microscope at 300kV with a dosage of ~25 e−2/A on the specimen. ~43,000 particles from 340 micrographs were selected for image processing and three dimensional (3D) reconstruction (Fig. 1b, c). Local averaging of the 7 copies of the cement or MCP protein in the asymmetric unit was performed to further improve resolution, model building was performed using Coot, and atomic models were refined using CCP4 (Fig. 1c–e) [4]. Fig. 1 Structure of BPP-1 at 3.4A resolution determined by cryoEM The BPP-1 head reconstruction has an angular shape with 420 MCP subunits making up the capsid shell and 210 dimers of the cement protein joining neighboring MCPs, together forming chainmail (Fig. 1c). Side chains of amino acids are clearly resolved in both proteins and were used for atomic model building. All but the last amino acid of the 140 amino acid residues of the cement protein were visible in the cryoEM density. The atomic model of the cement protein shows a jellyroll fold and a ~40A C-terminal arm (Fig. 1d). The interactions between the two monomers in the cement protein dimer are limited to the F-strand of the jellyroll (Fig. 1d), primarily through a pair of Pro residues, one on each monomer. In contrast, each cement protein monomer interacts with five underlying MCP molecules, mediated through loops of the jellyroll and through a β augmentation of the C-terminal arm (Fig. 1c). This use of the jellyroll fold for an auxiliary stabilizing role is quite remarkable because such fold has only been observed as a building block for viral capsids. Of the 331 residues of the BPP-1 MCP, we traced Thr6 to Val330 in our density map. The N and C termini are both located on the surface, but the first 5 and the last one amino acids are not visible, suggesting their flexibility (Fig. 1e). The BPP-1 MCP has the fold/architecture of the capsid protein of HK97 bacteriophage. We hereby divide it into three building blocks from N to C termini: chainmail-forming (cyan), β sheet-forming (purple) and capsomer-forming (green) (Fig. 1e) [2]. Surprisingly, the topologies of folding these three building blocks into this architecture are different between BPP-1 and HK97 (Fig. 1e, f): The last two of the three building blocks in gp5 of HK97 are swapped as compared to the sequence in BPP-1, in a rare, non-circular permutation, as found in DNA methyltransferases [5]. The difference in the topologies in folding suggests convergent evolution in selecting the characteristic HK97-like fold to build chainmail in the two viruses. It has been found that the internal pressure inside bacteriophages is very high due to the high packing density of the viral genome inside the viral capsid [6], and that different bacteriophages adopt distinctive strategies to stabilize their capsids to withstand the high pressure. HK97 uses covalent isopeptide bonds to link adjacent gp5 proteins to stabilize its capsid [2]. Having the same building blocks but lacking isopeptide bonds to join the chainmail-forming building blocks, BPP-1 uses an extra cement protein with the jellyroll fold to complete the chainmail (Fig. 1c). Our structure highlights the diverse strategies to build a highly stable dsDNA virus and suggests different maturation processes between the two bacteriophages.

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

BackgroundTransposon 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.ResultsWe 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.ConclusionWe 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.