TheEscherichia coliRetrons Ec67 and Ec86 Replace DNA between thecosSite and a Transcription Terminator of a 186-Related Prophage (original) (raw)
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Bacterial retrons function in anti-phage defense
2020
Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA. The RT uses the non-coding RNA as a template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. In this study we report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the non-coding RNA, and an effector protein. Retron-containing systems are abundant in genomic “defense islands”, suggesting a role for most retrons in phage resistance. By cloning multiple retron systems into a retron-lessEscherichia colistrain, we show that these systems confer defense against a broad range of phages, with different retrons defending against different phages. Focusing on a single retron, Ec48, we show evidence that it is a “guardian” of RecBCD, a complex with central anti-phage functions in the bacterial...
Nucleic Acids Research
Bacterial retrons consist of a reverse transcriptase (RT) and a contiguous non-coding RNA (ncRNA) gene. One third of annotated retrons carry additional open reading frames (ORFs), the contribution and significance of which in retron biology remains to be determined. In this study we developed a computational pipeline for the systematic prediction of genes specifically associated with retron RTs based on a previously reported large dataset representative of the diversity of prokaryotic RTs. We found that retrons generally comprise a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions. These retron systems are highly modular, and their components have coevolved to different extents. Based on the additional module, we classified retrons into 13 types, some of which include additional variants. Our findings provide a basis for future studies on the biological function of retrons and for expanding their biotechnolo...
Diversity of retron elements in a population of rhizobia and other gram-negative bacteria
Journal of bacteriology, 1993
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.
A novel mechanism of self-primed reverse transcription defines a new family of retroelements
Molecular and Cellular Biology, 1995
Retroviruses and long terminal repeat (LTR)-containing retrotransposons initiate reverse transcription by using a specific tRNA primer that anneals to the primer-binding site of the retroelement transcript. Sequences from a large number of retroviruses and LTR-containing retrotransposons had indicated that the role of tRNAs in priming reverse transcription is universal among these LTR-containing retroelements. Data presented here strongly support the surprising conclusion that Tf1, a highly active LTR-containing retrotransposon isolated from Schizosaccharomyces pombe, undergoes a novel self-priming process that requires hybridization between the primer-binding site and the first 11 bases of the Tf1 transcript. Single-base mutations in these regions block transposition and reverse transcription, while compensatory mutations that reestablish complementarity rescue both defects. In addition, the sequence of the minus-strand RNA primer of reverse transcription was consistent with its being derived from the 5 end of the Tf1 transcript. Evidence that this mechanism defines a new family of retroelements is presented.
Journal of Bacteriology, 2001
Retron reverse transcriptases are unusual procaryotic enzymes capable of synthesis of low-molecular-weight DNA by reverse transcription. All of the so-far-described DNA species synthesized by retron reverse transcriptases have been identified as multicopy single-stranded DNA. We have shown that Salmonella enterica serovar Enteritidis is also capable of synthesis of the low-molecular-weight DNA by retron reverse transcriptase. Surprisingly, Salmonella serovar Enteritidis-produced low-molecular-weight DNA was shown to be a doublestranded DNA with single-stranded overhangs (sdsDNA). The sdsDNA was 72 nucleotides (nt) long, of which a 38-nt sequence was formed by double-stranded DNA with 19-and 15-nt single-stranded overhangs, respectively. Three open reading frames (ORFs), encoded by the 4,053-bp plasmid, were essential for the production of sdsDNA. These included an ORF with an unknown function, the retron reverse transcriptase, and an ORF encoding the cold shock protein homologue. This plasmid was also able to confer phage resistance onto the host cell by a mechanism which was independent of sdsDNA synthesis.
Short-patch reverse transcription in Escherichia coli
Genetics, 1995
Chimeras of RNA and DNA have distinctive physical and biological properties. Chimeric oligonucleotides that contained one, two or three ribonucleotides whose phosphodiester backbone was covalently continuous with DNA were synthesized. Site-directed mutagenesis was used to assess genetic information transfer from the ribonucleotide positions. Transfer was scored by the formation or reversion of an ochre site that also corresponded to a restriction cleavage site. This allowed physical as well as genetic assay of mutational events. Bases attached to the ribonucleotides were able to accurately direct the synthesis of progeny DNA. The results suggest that in vivo DNA polymerases utilize a "running start" on a DNA backbone to continue across a covalent backbone junction into a region of ribonucleotides and then back again onto a normal DNA backbone. The phenomenon is designated short-patch reverse transcription (SPRT) by analogy to short-patch mismatch correction and reverse tra...
Journal of Molecular Biology, 1989
Two plasmid systems, containing the easily assayable galK and 1acZ functions, were employed to study the regulation of the bacteriophage Pl tail-fibre and dar operons. Various PI DNA fragments carrying either the 5' end of 2ydA (the 1st gene in the dar operon) or the tail-fibre gene 19 precede the promoterless coding region of galK or were fused, in-frame, to the 1acZ gene. In the presence of an induced Pl prophage, GalK and LacZ activities were both detected after a 20 to 30 minute lag period, indicating that the dar and tail-fibre operons are expressed from positively regulated, late promoters. The corresponding DNA region of the closely related p15B plasmid exhibits comparable promoter properties. Deletion analysis mapped the promoter of a gene 19-&Z fusion to a DNA region upstream from gene R, an open reading frame that precedes the coding frame of gene 19. The tail-fibre gene thus forms the second gene in a three gene operon (genes R, 19 (S) and U). Sequence comparison between this promoter region, upstream sequences of the 1ydA gene and the corresponding portions of the p15B genome allowed the identification of a highly conserved 38 base-pair sequence, which most likely represents a Pl-specific late promoter. This was confirmed by 5' mapping of Pl mRNA. Transcription of both the tail-fibre and dar operons is initiated at sites five and six base-pairs, respectively, downstream from the first conserved nucleotide of this sequence. The conserved motif consists of a standard Escherichia coli -10 region followed by a nine base-pair palindromic sequence located centrally about position -22.
Expression of enzymatically active reverse transcriptase in Escherichia coli
Proceedings of the National Academy of Sciences, 1985
Reverse transcriptase of murine retroviruses is a monomeric protein of-80,000 daltons, which is encoded by the central portion of the viral pol gene. To prepare large quantities of the enzyme, we have constructed gene fusions between the trpE gene and portions of the poi gene of Moloney murine leukemia virus. The insertedpol gene sequences include the entire coding region for the mature enzyme and various amounts of additional coding sequences. Many of these constructs express high levels of reverse transcriptase activity even though the NH2 and COOH termini of the protein product only approximate the correct termini of the authentic protein.