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PubMed1992

Sequence diversity of the 1.3 kb retron (retron-Ec107) among three distinct phylogenetic groups of Escherichia coli.

In the preceding paper, we showed that a new 1.3 kb retron (retron-Ec107) in Escherichia coli is responsible for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec107). Here, we show that this retron occurs in strains from different branches, A, B1, and D of a well-defined phylogenetic tree of a collection of wild E. coli. Sequence comparisons of the retrons from these three branches were carried out. Sequence homology was well conserved among the strains within the same branch and the retron sequence from branch A was exactly the same with that from branch D, while there were 18 base substitutions between the retrons from branch B1 and A or D, resulting in seven amino acid substitutions in reverse transcriptase. No substitutions were found in the msDNA- and msdRNA-coding regions, and there was no difference in the ability of msDNA production between them. These results suggest that the retron has probably been integrated into at least one of the three branches at an early stage of evolution and subsequently transferred to the other two branches, and also that the msDNA-producing system has been conserved during evolution with some mutations in the retron.

Base Sequence↗

PubMed1995

The role of ribonuclease H in multicopy single-stranded DNA synthesis in retron-Ec73 and retron-Ec107 of Escherichia coli.

Bacterial reverse transcriptase is responsible for the synthesis of multicopy single-stranded DNA (msDNA). Reverse transcriptases from retron-Ec73 and retron-Ec107 do not contain an RNase H domain. Cellular RNase H is therefore considered to be required to make the mature form of msDNA. We found that RNase HI, but not RNase HII, is required for the production of the mature form of both msDNAs.

Base Sequence↗

PubMed2025

New retron systems from environmental bacteria identify triggers of anti-phage defense and expand tools for genome editing.

Retrons are bacterial immune systems that protect a bacterial population against phages by killing infected hosts. Retrons typically comprise a reverse transcriptase (RT), a template noncoding RNA that is partially reverse transcribed into RT-DNA, and a toxic effector. The reverse transcriptase (RT), noncoding RNA, and RT-DNA complex sequester the toxic effector until triggered by phage infection, at which point the toxin is released to induce cell death. Due to their ability to produce single-stranded DNA in vivo, retrons have also been engineered to produce donor templates for genome editing in both prokaryotes and eukaryotes. However, the current repertoire of experimentally characterized retrons is limited, with most retrons sourced from clinical and laboratory strains of bacteria. To better understand retron biology and natural diversity, and to expand the current toolbox of retron-based genome editors, we developed a pipeline to isolate retrons and their bacterial hosts from a variety of environmental samples. Here, we identify seven new retron systems, each isolated from a different host bacterium. We characterize DNA production by these retrons and test their ability to defend against a panel of Escherichia coli phages. We find that two of these retrons are disrupted by other elements, in one case a group II intron and in another a separate defense system, yet both retrons still produce RT-DNA. For two other retrons, we further unravel their mechanism of defense by identifying the phage genes responsible for triggering abortive infection. Finally, we engineer these retrons for genome editing in E. coli, demonstrating their potential use in a biotechnological application.

Gene Editing↗

PubMed1992

Retron-Ec107 is inserted into the Escherichia coli genome by replacing a palindromic 34bp intergenic sequence.

Some natural isolates of Escherichia coli have been shown to produce a unique branched RNA-linked single-stranded DNA called msDNA. These bacteria contain a retro-element called retron consisting of the msr-msd region and the gene for reverse transcriptase (RT). All three E. coli retrons characterized to date have been shown to be integrated into a prophage or to be associated with phage-related genes. In this report, we identified a new msDNA from an E. coli wild strain. Using the msDNA as a probe, the retron for the msDNA was cloned and its DNA sequence was determined. The retron was found to consist of a 1.3kb DNA fragment, making it the smallest retron isolated to date. The msDNA produced from the retron consists of a 107 base single-stranded DNA, which is considered to be branched out from the 18th G residue of a 75-base RNA molecule by a 2',5'-phosphodiester linkage. Thus, the msDNA and the retron were designated msDNA-Ec107 and retron-Ec107, respectively. Most significantly, retron-Ec107 was inserted into the E. coli genome by replacing a 34bp intergenic sequence between the pyrE and ttk genes located at 82 min on the E. coli chromosome. Interestingly, the retron contains palindromic structures at both ends and the E. coli 34bp intergenic sequence also contains a 10bp inverted repeat structure. These palindromic structures might have played a role in the integration of retron-Ec107 into the E. coli genome.

Amino Acid Sequence↗

PubMed2026

Landscape of retron diversity across the SPIRE microbial metagenome resource reveals candidate novel type XI-like lineages.

Retrons are bacterial genetic elements encoding a specialized reverse transcriptase (RT) that synthesizes multicopy single-stranded DNA and are increasingly recognized as components of bacterial anti-phage defense systems. However, their diversity and ecological distribution across large-scale genomic resources remain poorly characterized. Here, we surveyed retron RTs across the SPIRE representative metagenome collection, a non-redundant, species-level data set spanning diverse microbial habitats. Using a curated panel of type-specific hidden Markov models, we identified retrons representing all canonical types together with additional divergent lineages. Retron distribution showed strong taxonomic and ecological structuring, with some groups restricted to specific bacterial phyla, whereas others were broadly distributed across environmental categories. Systematic novelty assessment identified two candidate type XI-like lineages, TXI_C2like and TXI_noncan_h, characterized by protease-independent architectures and distinct accessory modules associated with WYL- and DnaB_C-containing proteins, respectively. De novo covariance-based analyses further identified candidate msr/msd-like non-coding RNA structures in both lineages, supporting conservation of the canonical RT-ncRNA organizational framework despite extensive sequence divergence. Together, these findings expand the known diversity of retron systems and identify type XI-like retrons as a dynamic and previously underexplored evolutionary group.IMPORTANCERetrons are bacterial genetic elements that are increasingly exploited as programmable tools for genome editing, molecular recording, and biosensing in addition to their natural role in anti-phage defense. Despite this growing biotechnological interest, the true diversity of retrons across the bacterial world has remained largely unmapped. By mining a resource of over 100,000 processed microbial metagenomes, we uncovered thousands of retron sequences spanning known types as well as previously unrecognized lineages and found that their distribution is strongly shaped by both bacterial taxonomy and ecological niche. Among these, we identified two candidate new lineages related to type XI retrons that lack the protease domain typical of this group but instead carry distinct accessory proteins, expanding the known architectural diversity of these systems. These findings broaden the catalog of retron diversity available for functional characterization and biotechnological engineering and provide a framework for prioritizing candidate lineages for future experimental validation.

effectors↗

PubMed1996

The Escherichia coli retrons Ec67 and Ec86 replace DNA between the cos site and a transcription terminator of a 186-related prophage.

Retrons are unusual, reverse transcriptase-encoding elements found in bacteria. Although there are a number of indications that retrons are mobile elements, their transposition has not been observed. The Escherichia coli retrons Ec67 and Ec86 are different retrons inserted at the same site and we have further characterized this site in search of clues to the mechanism of retron transposition. We confirm, by extending previous sequence analysis, that Ec67 and Ec86 are inserted into prophages related to coliphage 186. Comparison with the recently published sequence of the 186 96-2% region indicates that the retrons have replaced approximately 180 bp of DNA between the phage cohesive end site (cos) and the transcription terminator of a phage DNA-packaging gene. These features--DNA replacement at the insertion site and the location of retron junctions near transcription terminators or DNA cleavage sites--are shared with other retrons and suggest ways in which retron transposition might have occurred.

Amino Acid Sequence↗

PubMed1991

Structure of two retrons of Escherichia coli and their common chromosomal insertion site.

It has been shown that certain strains of myxobacteria and of Escherichia coli have a genetic element encoding a reverse transcriptase (RT). This element, called a 'retron', produces a covalently linked RNA-DNA compound (msDNA-RNA). Here, I report the complete nucleotide sequence of retron EC-86, the retron in E. coli B, together with its flanking regions. Retron EC-86 contains genes for msDNA-RNA (msd, and msr), a gene for RT (ret) and a gene for an open reading frame whose function is unknown. The upstream junction is composed of the sequence GCGCGCGC, but there are no direct or inverted repeats at the retron-host junctions. It is also shown that another retron of E. coli, EC-67, which was isolated originally from the clinical strain CL1 and was later found to be present also in a clinical E. coli isolate from Brazil, is inserted at the same chromosomal site as retron EC-86. Retron EC-67 contains only msd, msr, and ret. I suggest that these two retrons were independently inserted into the same site of their host strains via a novel mechanism of integration.

Amino Acid Sequence↗

PubMed1995

Phylogenetic comparison of retron elements among the myxobacteria: evidence for vertical inheritance.

Twenty-eight myxobacterial strains, representing members from all three subgroups, were screened for the presence of retron elements, which are novel prokaryotic retroelements encoding reverse transcriptase. The presence of retrons was determined by assaying strains for a small satellite DNA produced by reverse transcription called multicopy, single-stranded DNA (msDNA). An msDNA-producing retron appeared to be absent from only one of the strains surveyed. DNA hybridization experiments revealed that retron elements similar to retron Mx162, first identified in Myxococcus xanthus, were found only among members of the Myxococcus subgroup; that is, each of the seven different genera which constitute this subgroup contained a Mx162 homolog. Another retron element also appeared to have a clustered distribution, being found exclusively within the Nannocystis subgroup of the myxobacteria. A retron element of the Mx162 type was cloned from Melittangium lichenicola, and its DNA sequence was compared with those of similar elements in M. xanthus and Stigmatella aurantiaca. Together, the degree of sequence diversity, the codon bias of the reverse transcriptase genes, and the clustered distribution of these retrons suggest a possible evolutionary scenario in which a common ancestor of the Myxococcus subgroup may have acquired this retroelement.

Amino Acid Sequence↗

PubMed1995

Analysis of a retron EC86 and EC67 insertion site in Escherichia coli.

The Escherichia coli clinical strain 49 contains the integration sites for the retrons EC86 and EC67 but not the retrons themselves. To compare the chromosomal structures before and after retron integration, the DNA sequence of the integration site in strain 49 was determined and compared to the corresponding sequences in strains containing EC86 and EC67. The results suggested that when these retrons inserted into the E. coli chromosome they replaced a 3.5-kb fragment of chromosomal DNA. It is proposed that the replacement of preexisting DNA by a retron may be a general mechanism for retron integration, since in the three examples in which the integration sites are known, the retrons appear to have integrated into the chromosome by replacing a preexisting DNA segment.

Amino Acid Sequence↗

PubMed2005

Retrons, msDNA, and the bacterial genome.

Retrons are distinct DNA sequences that code for a reverse transcriptase (RT) similar to the RTs produced by retroviruses and other types of retroelements. Retron DNAs are commonly associated with prophage DNA and are found in the genomes of a wide variety of different bacteria. The retron RT is used to synthesize a strange satellite DNA known as msDNA. msDNA is actually a complex of DNA, RNA, and probably protein. It is composed of a small, single-stranded DNA, linked to a small, single-stranded RNA molecule. The 5' end of the DNA molecule is joined to an internal guanosine residue of the RNA molecule by a unique 2'-5' phosphodiester bond. msDNA is produced in many hundreds of copies per cell, but its function remains unknown. Although retrons are absent from the genome of most members of a population of related bacteria, retrons may not be entirely benign DNAs. Evidence is beginning to suggest that retron elements may produce small but potentially significant effects on the host cell. This includes the generation of repeated copies of the msDNA sequence in the genome, and increasing the frequency of spontaneous mutations. Because these events involve the retron RT, this may represent a source of reverse transcription in the bacterial cell. Thus, the process of reverse transcription, a force that has profoundly affected the content and structure of most eukaryotic genomes, may likewise be responsible for changes in some prokaryotic genomes.

DNA Transposable Elements↗

PubMed1993

Diversity of retron elements in a population of rhizobia and other gram-negative bacteria.

Genetic elements called retrons reside on the chromosome of Escherichia coli and the myxobacteria and represent the first reverse transcriptase-encoding element to be found in a prokaryotic cell. All known retrons produce a functionally obscure RNA-DNA satellite molecule called multicopy single-stranded DNA (msDNA). We report here the presence of msDNA-producing retron elements in a number of new bacterial groups, including strains of the genera Proteus, Klebsiella, Salmonella, Nannocystis, Rhizobium, and Bradyrhizobium. Among a population of 63 rhizobia strains, only 16% contain a retron element. The rhizobia retrons appear to be heterogeneous in nucleotide sequence and show little similarity to previously studied retrons of E. coli and the myxobacteria.

DNA, Single-Stranded↗

PubMed1993

Reverse transcriptases from bacterial retrons require specific secondary structures at the 5'-end of the template for the cDNA priming reaction.

Multicopy single-stranded DNA (msDNA) is a peculiar molecule consisting of a single-stranded DNA that is branched out from an internal G residue of an RNA molecule (msdRNA) via a 2',5'-phosphodiester linkage. The genetic unit required for msDNA synthesis is designated "retron" and consists of msr (a gene for msdRNA), msd (a gene for msDNA), and a gene for reverse transcriptase (RT) in a single operon. To date, four different msDNAs have been isolated from Escherichia coli. They do not share any primary sequences in either RNA or DNA. To elucidate the specificity of bacterial RT for msDNA synthesis, the msr-msd region from retron-Ec67 was introduced into E. coli cells producing RT-Ec73, or the msr-msd region from retron-Ec73 into E. coli cells producing RT-Ec67. In both cases, msDNA was not synthesized. However, when the msdRNA coding regions (msr) for retron-Ec67 and -Ec73 were mutually exchanged and the chimeric genes were introduced into E. coli cells producing either RT-Ec67 or RT-Ec73, it was thus found that msDNA was produced only when msr and RT were from the same retron. Requirement of the msr region for msDNA synthesis by RT was further investigated by mutations in the msr region for retron-Ec67. These analyses revealed that there is a strict requirement for specific primary sequences as well as the secondary structure in msdRNA. This finding is discussed in relationship to the mechanism of the priming reaction of cDNA synthesis by eukaryotic retroviral RTs using tRNAs.

Bacterial Proteins↗

PubMed2005

Retron reverse transcriptase (rrtT) can be lost in multidrug resistant Salmonella enterica serovar Typhimurium DT 104 strains and influences virulence for mice.

In Salmonella enterica serovar Typhimurium, retron reverse transcriptase (rrtT), which is part of St 85 retron, is quite ubiquitous and is located in the thdF -yidY intergenic region. In this study, we showed that rrtT is relatively unstable in multidrug resistant, Salmonella genomic island 1 (SGI 1) positive strains. Out of 365 field strains, 55 were free of retron. In 54 of the rrtT negative strains, the excision must have occurred by the same mechanism in which the rrtT together with five other genes was excised. Altogether 8164 bp was missing in the chromosome of the rrtT negative strains. Since the deletion happened exactly between the right inverted repeat of IS 6100 and inside the yieE gene, we propose that intramolecular transposition of IS 6100 followed by homologous recombination was responsible for the excision. Excision of retron together with the right end of SGI 1 may also result in its stabilisation in the Salmonella typhimurium genome. Experimental deletion of rrtT resulted in an accelerated course of infection in orally infected mice. Since the retron excision occurred exclusively in multidrug resistant S. typhimurium, it cannot be excluded that such strains may increase their virulence in the future.

Animals↗

PubMed2003

Avoidance of false PCR results with the integron-retron junction in multiple antibiotic resistant Salmonella enterica serotype Typhimurium.

Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Another concern with this pathogen is the ability to acquire integrons that confer resistance to multiple antibiotics. For multiresistant Salmonella enterica serotype Typhimurium, the most common multiresistant Salmonella serotype, an integron structure can be found between thdF and a retron. Our objective was to investigate the utility of a 450 bp thdF-retron amplicon as an indicator of an insertless thdF-retron junction thus indicating an integron-free strain. Surprisingly, we found that the 450 bp thdF-retron amplicon was present, and thus incorrectly suggesting an integron-free status, in some multiresistant S. enterica serotype Typhimurium isolates. However, this phenomenon was not observed if the isolate was enriched in the presence of two antibiotics. This demonstrates that, within some individual clinical isolates of multiresistant S. enterica serotype Typhimurium, there exists a small subpopulation of integron-free bacteria. Consequently, it appears that the thdF-retron amplicon is an inaccurate predictor of integron status in S. enterica serotype Typhimurium unless multiresistance is used as a selection tool during enrichment.

Animals↗

PubMed1990

Two independent retrons with highly diverse reverse transcriptases in Myxococcus xanthus.

A reverse transcriptase (RT) was recently found in Myxococcus xanthus, a Gram-negative soil bacterium. This RT has been shown to be associated with a chromosomal region designated a retron responsible for the synthesis of a peculiar extrachromosomal DNA called msDNA (multicopy single-stranded DNA). We demonstrate that M. xanthus contains two independent, unlinked retrons, one for the synthesis of msDNA-Mx162 and the other for msDNA-Mx65. The structural analysis of the retron for msDNA-Mx65 revealed that the coding regions for msdRNA (msr) and msDNA (msd), and an open reading frame (ORF) downstream of msr are arranged in the same manner as found for the Mx162 retron. The ORF encodes a polypeptide of 427 amino acid residues. The amino-terminal domain (residues 1-138) shows no striking similarity to these proteins presently available in the data bases including the msDNA-Mx162 ORF, while the sequence from residues 139-394 can be aligned with various known RT sequences and has 47% identity with the RT domain of the msDNA-Mx162 ORF. On the basis of these findings, possible origins of two highly diverse retrons on the M. xanthus chromosome are discussed.

Amino Acid Sequence↗

PubMed2024

Reducing competition between msd and genomic DNA improves retron editing efficiency.

Retrons, found in bacteria and used for defense against phages, generate a unique molecule known as multicopy single-stranded DNA (msDNA). This msDNA mimics Okazaki fragments during DNA replication, making it a promising tool for targeted gene editing in prokaryotes. However, existing retron systems often exhibit suboptimal editing efficiency. Here, we identify the msd gene in Escherichia coli, which encodes the noncoding RNA template for msDNA synthesis and carries the homologous sequence of the target gene to be edited, as a critical bottleneck. Sequence homology causes the msDNA to bind to the msd gene, thereby reducing its efficiency in editing the target gene. To address this issue, we engineer a retron system that tailors msDNA to the leading strand of the plasmid containing the msd gene. This strategy minimizes msd gene editing and reduces competition with target genes, significantly increasing msDNA availability. Our optimized system achieves very high retron editing efficiency, enhancing performance and expanding the potential for in vivo techniques that rely on homologous DNA synthesis.

Gene Editing↗

PubMed2003

Retron reverse transcriptase rrtT is ubiquitous in strains of Salmonella enterica serovar Typhimurium.

Bacterial retron reverse transcriptases are unusual enzymes which utilise the same RNA molecule as a template and also as a primer for initiation of the reverse transcription. Except for their relatively frequent presence in Myxococcus spp., they are considered as quite rare proteins. However, in this study we proved that retron reverse transcriptase is frequently found in certain serovars of Salmonella enterica. Using polymerase chain reaction (PCR), in strains of serovar Typhimurium, the rrtT (retron reverse transcriptase Typhimurium) gene was detected in 158 out of 175 tested field strains. On the other hand, in none of the 18 tested serovar Enteritidis strains the rrtT was detected in their genome. Detailed computer analysis allowed us to predict the sequence of msDNA and to propose that the final msDNA is free of any RNA. Furthermore, we predict that there are at least three different classes of retron reverse transcriptases.

Base Sequence↗

PubMed2003

msDNA-St85, a multicopy single-stranded DNA isolated from Salmonella enterica serovar Typhimurium LT2 with the genomic analysis of its retron.

Bacterial reverse transcriptase is responsible for the production of a small satellite DNA-RNA complex called multicopy single-stranded DNA (msDNA) that has been found in a wide variety of Gram-negative bacteria. Here we describe the isolation and characterization of a novel msDNA, msDNA-St85, from Salmonella enterica serovar Typhimurium LT2. We determined the nucleotide sequence of msDNA-St85 and the location of retron-St85 on the chromosome that is responsible for msDNA-St85 production by analyzing the complete genomic sequence of S. typhimurium LT2. It was found that the G+C content and the codon usage of retron-St85 were significantly different from those of the S. typhimurium genome, indicating that retron-St85 was probably acquired recently in this bacterium. This is the first report for identification of an msDNA in the genus Salmonella with the complete description and analysis of its retron.

Amino Acid Sequence↗