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The multi-copy single-stranded DNA (msDNA) is yielded by the action of reverse transcriptase of retro-element in a wide range of pathogenic bacteria. Upon this phenomenon, it has been shown that msDNA is only produced by Eubacteria because many Eubacteria species contained reverse transcriptase in their special retro-element. We have screened around 111 Archaea at KEGG (Kyoto Encyclopedia of Genes and Genomes) database available at genome net server and observed three Methanosarcina species (M.acetivorans, M.barkeri and M.mazei), which also contained reverse transcriptase in their genome sequences. This observation of reverse transcriptase in Archaea raises questions regarding the origin of this enzyme. The evolutionary relationship between these two domains of life (Eubacteria and Archaea) hinges upon the phenomenon of retrons. Interestingly, the evolutionary trees based on the reverse transcriptases (RTs) and 16S ribosomal RNAs point out that all the Eubacteria RTs were descended from Archaea RTs during their evolutionary times. In addition, we also have shown some significant structural features among the newly identified msDNA-Yf79 in Yersinia frederiksenii with other of its related msDNAs (msDNA-St85, msDNA-Vc95, msDNA-Vp96, msDNA-Ec78 and msDNA-Ec83) from pathogenic bacteria. Together the degree of sequence conservation among these msDNAs, the evolutionary trees and the distribution of these ret (reverse transcriptase) genes suggest a possible evolutionary scenario. The single common ancestor of the organisms of Eubacteria and Archaea subgroups probably achieved this ret gene during their evolution through the vertical descent rather than the horizontal transformations followed by integration into this organism genome by a mechanism related to phage recognition and/or transposition.

Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules., Author Summary Multicopy single-stranded DNA (msDNA) is a unique molecule consisting of both an RNA and DNA portion. This molecule is produced by a reverse transcriptase and has no known natural function despite more than 30 years of study. We report that msDNA is important for both Salmonella Typhimurium and an enteropathogenic E. coli, two pathogens that cause diarrhea in susceptible hosts, to survive in the intestine. Using mutant strains incapable of producing msDNA, we show that msDNA is needed for Salmonella to grow in the absence of oxygen. Mutants grown in oxygen-deficient conditions have substantial changes in overall protein composition, including numerous proteins known to be important for anaerobic metabolism and growth in the intestine. Our findings link msDNA to the ability of Salmonella to thrive in an oxygen-deficient environment similar to the conditions inside the gut. We report that msDNA regulates the quantity of proteins, the first natural function attributed to this molecule. msDNA may represent a new class of regulatory molecules.

Multicopy single-stranded DNA is found as a small single-stranded RNA-DNA complex in certain wild-type strains of Escherichia coli as well as in other gram-negative bacteria. Using the promoter region of the previously characterized retron-Ec107 from E. coli ECOR70, I constructed a chromosomally located lacZ operon fusion. Examination of expression from the PEc107 promoter showed that activity increased sharply when cells entered stationary phase in rich medium or when they were starved for phosphate. The nucleotide guanosine-3',5'-bispyrophosphate was found to be a positive regulator of retron-Ec107 expression. Its presence is required for starvation-induced transcription of retron-Ec107 and multicopy single-stranded DNA production. It was also found that expression from the retron promoter is independent of the sigma factor sigmaS.

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.

A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.

Retron is a retroelement that encodes msDNA (multicopy single-stranded DNA) which was significantly found mainly in Gram-negative pathogenic bacteria. We screenedYersinia frederikseniiATCC 33641 contig01029 for the presence of retroelement by using bioinformatics tools and characterized a novel retron-Yf79 on the chromosome that encodes msDNA-Yf79. In this study, we perceived that, the codon usage of retron-Yf79 were noteworthy different from those of theY. frederikseniigenome. It demonstrates that, the retron-Yf79 was a foreign DNA element and integrated into this organism genome during their evolution. In addition to this, we have observed a transposase gene which is located just downstream of retron-Yf79. So, the enzyme might be responsible for the transposition of this novel retron element.

Summary It has been shown that retrons, retro-elements in bacteria, produce a reverse transcriptase (RT) and multicopy single-stranded DNA(msDNA) whose 5′ end is covalently linked to RNA (msdRNA) by a 2′-5′ phosphodiester bond. Here, I show that a retron in clinical Escherichia coli strain 161 produces an msDNA unlinked to RNA. The msDNA produced by this retron is a 79-nucleotlde-long single-stranded DNA with monophosphate on its 5′ terminus. When the retron in strain 161 is cloned into E. coli K-12, the majority of msDNA produced in the clone is the same as the msDNA in the clinical strain. However, in the K-12 clone, about 10% of the msDNA produced is present as a DNA covalently linked to RNA. The DNA part of this RNA-DNA compound is an 83 nucleotides long with the same sequence as the unbranched msDNA, except for the presence of four additional nucleotides at the 5′ side. From the analysis of the RNA-DNA compound and the results of in vitro synthesis, I show that the primary product of reverse transcription in this retron is an 83-nucleotide-tong DNA covalently linked to RNA. This RNA-DNA compound is further processed to the final product, the 79-nucleotide-long msDNA with a terminal 5′ monophosphate, by an endonucleolytic cleavage between the fourth and fifth positions of the DNA component of the RNA-DNA compound. The minimum region required for the production of such msDNA free of RNA contains only genes known to be required for the synthesis of branched msDNA-RNA compound in other retrons (msd, msr and ret). This suggests that either the RT has an endonuclease activity or that the msDNA-RNA compound is autoca-talytically processed.

To determine the evolutional relationship of bacterial retroelements of Myxococcus xanthus and Stigmatella aurantiaca, the nucleotide sequence of 3,060 bases encompassing msr, msd, and the upstream region of msd (downstream of msr) of S. aurantiaca DW4 was determined and compared with the same region from M. xanthus. An open reading frame was found 92 bases upstream of msd which encoded a polypeptide of 480 amino acid residues having 73% identity with the reverse transcriptase of M. xanthus. Together with high homologies in msr (86%) and msd (81%) regions, the present data indicate that the reverse transcriptase genes as well as the retrons of M. xanthus (retron-Mx162) and S. aurantiaca (retron-Sa163) were derived from a common progenitor retron which possibly before the two myxobacterial species diverged.

Myxobacteria and a variety of strains of Escherichia coli contain an unusual extrachromosomal element, a small single-stranded branched copolymer of DNA and RNA (msDNA). Interest in msDNA stems from the presence of a 2'-5' linkage between its DNA and RNA moieties and the possible involvement of reverse transcriptase in its synthesis. Two groups have proposed a model for the synthesis of msDNA that involves the following sequence of events: 1) synthesis of an RNA precursor; 2) addition of a dNTP in a 2'-5' linkage to the RNA precursor (branch priming); 3) synthesis by reverse transcriptase of complementary DNA on the primed RNA precursor; 4) concomitant processing of the RNA precursor by RNase H; and 5) further processing of the RNA precursor by RNase H; and 5) further processing of the branched polymer. The branch priming hypothesis (step 2) was originally based on pulse-chase experiments using a lengthy pulse of 30-min duration. Our experiments with shorter pulse durations demonstrate a variety of intermediate forms not predicted by this model. Specifically, we find that early in synthesis, intermediate msDNA forms appear that are apparently not branch-linked to corresponding RNA moieties. The concomitant RNase H hypothesis (step 4) was based in part on intermediate trapping experiments using dideoxy NTPs to interrupt msDNA synthesis. These experiments showed an apparent complementary relationship between DNA length and RNA length in the trapped forms. In contrast, our experiments show that DNA elongation and RNA processing follow different kinetics. These results suggest an alternate model for synthesis of msDNA in which a conventionally primed DNA moiety is joined with the RNA moiety in a branch ligation event taking place several minutes after the synthesis of both moieties.

The region (retron-Ec67) required for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec67) from a clinical isolate of Escherichia coli was mapped at a position equivalent to 19 min on the K-12 chromosome. The element containing the retron consisted of a unique 34-kilobase sequence that was flanked by direct repeats of a 26-base-pair sequence found in the K-12 chromosomal DNA. This suggests that the 34-kilobase element was probably integrated into the E. coli genome by a mechanism related to transposition or phage integration. In the 34-kilobase sequence an open reading frame of 285 residues was found, which displays 44% sequence identity with the E. coli Dam methylase. Interestingly, there are three GATC sequences, the site of Dam methylation, in the promoter region of the gene for reverse transcriptase.

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.

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.

A minor population of wild strains of Escherichia coli contains a retron, a retroelement responsible for the synthesis of multicopy single-stranded DNA (msDNA). The retron is a genetic element consisting of the gene for reverse transcriptase (RT) and the msr-msd region under a single promoter. A single RNA transcript from the msr-msd region serves not only as a template but also as a primer for msDNA synthesis. Here, using a cell-free system with purified RT from retron Ec73, we examined whether the reaction can occur in a bimolecular reaction with use of separately expressed msr and msd transcripts. DNA sequencing of the cell-free product revealed that the sequence of the 5′-end region was identical to that of msDNA-Ec73, indicating that the cDNA synthesis was primed from the 2′-OH group of the specific internal G residue of the primer RNA, identical to the branching G residue in the RNA molecule of msDNA-Ec73. The present results raise an intriguing possibility for a role of bacterial retrons in vivo, the possibility that cellular mRNAs can be converted into cDNAs in retron-harboring cells if the mRNAs contain a sequence complementary to the sequence directly upstream of the branching G residue of the msr RNA transcript.

Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. Such molecules are encoded by genetic elements called retrons. Unlike other retrons, retron EC83 isolated from Escherichia coli 161 produces RNA-free msDNA by site-specific cleavage of msDNA at 5'-TTGA/A-3', where the slash indicates the cleavage site. In order to investigate factors responsible for the msDNA cleavage, retron EC83 was treated with hydroxylamine and colonies were screened for cleavage-negative mutants. We isolated three mutants which were defective in msDNA cleavage and produced RNA-linked msDNA. They were all affected in msd, a gene for msDNA, with a base substitution at the bottom part of the msDNA stem. In contrast, base substitution at and around the cleavage site has no marked effect on msDNA synthesis or its cleavage. From these results, we concluded that the nucleotides at the bottom of the msDNA stem, but not the nucleotides at the cleavage site, play a major role in the recognition and cleavage of msDNA EC83.

A minor population of wild Escherichia coli strains contain retroelements called retrons, which produce a peculiar satellite DNA, multicopy single-stranded DNA (msDNA). It has been reported that mismatched base pairs in the secondary structure formed in msDNA are mutagenic in E. coli [Maas et al. (1994) Mol. Microbiol. 14,437–441; Maas et al. (1996) Mol. Microbiol. 19, 505–509]. We reexamined this proposal by converting mismatched base pairs to matched base pairs using a single msDNA species, msDNA-Ec86, or by deleting mismatched regions using msDNA-Ec73. We also examined the effect of reverse transcriptases (RT) without msDNA production on mutagenesis. All the constructs are under the lppllac promoter-operator control so that their mutagenic effects can be tested in the absence and the presence of a lac inducer. It was found that when the production of msDNA-Ec86 or Ec73 was induced, reversion frequencies from Lac− to Lac+ significantly increased in the case of a Lac− mutation caused by a frame-shift mutation, but much less by a substitution mutation. The removal of mismatched base pairs eliminated the high mutation frequencies, and the inducible expression of RT alone was not mutagenic. These results are consistent with the hypothesis of Maas and his associates that mismatched base pairs in msDNA sequester a cellular mismatch repair system, resulting in the increase of frame-shift mutations.

Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. In Escherichia coli, such molecules are encoded by genetic elements called retrons. The DNA moieties of msDNAs have characteristic stem-loop structures, and most of these structures contain mismatched base pairs. Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids. In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E. coli. To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs. We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination. We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.

Multicopy single-stranded DNA (msDNA) is produced by bacterial retroelements called retrons. It consists of single-stranded DNA that is linked to an internal G residue of an RNA molecule by a 2‘,5‘-phosphodiester linkage. It has been demonstrated that specific primary sequences, as well as the secondary structures immediately downstream of the G residue, are essential for the cDNA priming reaction (Shimamoto, T., Hsu, M.-Y., Inouye, S., and Inouye, M. (1993) J. Biol. Chem. 268, 2684-2692). We have now examined the requirement of the structures in the region corresponding to DNA for msDNA synthesis. The upper stem region consisting of 71 bases of msDNA-Ec107 was found not to be essential, and this region could be deleted to efficiently produce a truncated msDNA containing only a 36-base single-stranded DNA. Various mutations including base replacements, deletions, and insertions were constructed in the lower stem region. It was found that any mutations resulting in more stable secondary structures caused reduction in msDNA synthesis. The results indicated that reverse transcriptase requires a loose secondary structure in the template RNA near the cDNA priming site for cDNA elongation.

Multicopy single-stranded DNA (msDNA), a branched DNA-RNA molecule, has been shown in Escherichia coli B and clinical strain Cl-1 to be synthesized by reverse transcriptase. We report that 13% of the strains of the ECOR collection, a sample of 72 E. coli isolates representing the breadth of genetic variation of the species, produce msDNA. Three of the four major subspecific groups include msDNA-producing strains. Screening of 25 isolates that are genetically related to msDNA-producing clinical strains uncovered 22 additional msDNA-producing strains. A phylogenetic tree based on allelic variation detected electrophoretically at 20 enzyme-encoding loci revealed two major clusters and several deep branches composed of strains that synthesize msDNA. Although E. coli K-12 does not harbor msDNA, other closely related strains of the K-12 family do. The results support the hypothesis that msDNA-synthesizing systems, including reverse transcriptase genes, were acquired recently and independently in different lineages of E. coli.

A gram-negative bacterium, Myxococcus xanthus, was found to contain 500 to 700 copies per chromosome of a short single-stranded linear DNA fragment. When this DNA (multicopy single-stranded DNA; msDNA) labeled at the 5′ end with kinase was used as a probe against total chromosomal blots, it hybridized to unique high molecular weight bands, which were cloned and sequenced. Labeling of msDNA was also possible using the Klenow fragment of DNA polymerase I as well as terminal deoxynucleotidyl transferase, permitting direct sequencing. The 5′ end of msDNA was found to be primed by a short RNA segment. The DNA portion of msDNA consisted of 163 bases. Exact correspondence was seen between the msDNA sequence and the sequence of a chromosomal clone. An elaborate secondary structure is postulated for the msDNA sequence. A similar satellite DNA was also found in another myxobacterium, Stigmatella aurantiaca.

We have found a branched DNA-RNA compound in E. coli B, that is similar in its secondary structure, but not its nucleotide sequence, to the previously described branched DNA-RNA compounds in myxobacteria. This compound is not produced in E. coli K12. We have cloned a 3.5 kb chromosomal segment of E. coli B, which, when transferred into E. coli K12, leads to the production of the DNA-RNA compound. We describe the isolation of the DNA-RNA compound, the determination of its nucleotide sequence, and the nucleotide sequence of the genes required for its formation. The sequence contains the coding regions for the DNA component, the RNA component, and an open reading frame encoding a reverse transcriptase. This reverse transcriptase is shown to be required for the formation of the DNA-RNA compound in vivo and in vitro.

Multicopy single-stranded DNA (msDNA) is a short single-stranded linear DNA originally discovered in Myxococcus xanthus and subsequently found in Stigmatella aurantiaca. It exists at an estimated 500 to 700 copies per chromosome (T. Yee, T. Furuichi, S. Inouye, and M. Inouye, Cell 38:203-209, 1984). We found msDNA in other myxobacteria, including Myxococcus coralloides, Cystobacter violaceus, Cystobacter ferrugineus (Cbfe17), Nannocystis exedens, and nine independently isolated strains of M. xanthus. The presence of msDNA in N. exedens would extend its phylogenetic distribution into another family of myxobacteria. Flexibacter elegans, a Cytophaga-like gliding bacteria which may be even more distantly related, also contained an msDNA but at a much lower copy number. msDNA was not detected in closely related strains of the myxobacteria Cystobacter fuscus and C. ferrugineus (Cbfe16 and Cbfe18) and the more distantly related eubacteria Herpetosiphon giganteus, Taxeobacter ocellatus, Lysobacter antibioticus, Lysobacter enzymogenes, Cytophaga johnsonae, Rhodopseudomonas sphaeroides, and Rhodospirillum rubrum. Thus far, msDNA has been found in certain gliding bacteria but not in others.

Branched RNA-linked multicopy single-stranded DNA (msDNA) originally detected in myxobacteria has now been found in a clinical isolate of Escherichia coli. Although lacking homology in the primary structure, the E. coli msDNA is similar in secondary structure to the myxobacterial msDNA's, including the 2',5'-phosphodiester linkage between RNA and DNA. A chromosomal DNA fragment responsible for the production of msDNA was cloned in an E. coli K12 strain; its DNA sequence revealed an open reading frame (ORF) of 586 amino acid residues. The ORF shows sequence similarity with retroviral reverse transcriptases and ribonuclease H. Disruption of the ORF blocked msDNA production, indicating that this gene is essential for msDNA synthesis.

Myxobacteria have been shown to contain a large number of branched RNA-linked single-stranded DNA (multicopy single-stranded DNA (msDNA] molecules. In addition, we found that Myxococcus xanthus contains another smaller msDNA-like molecule, designated mrDNA, consisting of a 65-base single-stranded DNA covalently linked by a 2',5'-phosphodiester linkage to a 49-base branched RNA. In spite of their different primary sequences, the RNA-linked mrDNA is remarkably similar in secondary structure to msDNA, sharing similar stem-loop folding as well as the unique 2',5'-phosphodiester linkage. These results indicate that these novel molecules are synthesized by common molecular mechanisms.

A precursor RNA molecule (pre-msdRNA) of approximately 375 bases is considered to form a stable secondary structure which serves as a primer as well as a template to synthesize the branched RNA-linked multicopy single-stranded DNA (msDNA) of Myxococcus xanthus. When 3-base mismatches were introduced into the stem structure immediately upstream of the branched rG residue to which msDNA is linked by a 2',5'-phosphodiester linkage, the production of msDNA was almost completely blocked. However, if additional 3-base substitutions were made on the other strand to resume the complementary base pairing, msDNA production was restored, being consistent with the proposed model of msDNA synthesis. We also found that the branched rG residue of pre-msdRNA could not be replaced with either rC or rA, while the 5' end (dC) of msDNA which is linked to the branched rG could be substituted with a dG residue. Together with several other mutations, the structural requirements of pre-msdRNA are discussed with respect to the mechanism of msDNA biosynthesis.

Stigmatella aurantiaca, a gram-negative bacterium, contains approximately 500 copies per cell of a short single-stranded linear DNA (multicopy single-stranded DNA: msDNA). This DNA is attached to a branched RNA (msdRNA) by its 5′ end. The entire sequence of msdRNA was determined and found to consist of 76 bases. The msDNA is linked at the 19th G residue of msdRNA by a 2′, 5′ phosphodiester linkage. The coding region for msdRNA ( msr ) is located downstream of the coding region for msDNA ( msd ). These coding regions exist in opposite orientation with respect to each other and overlap by 8 bases at their 3′ ends. Biosynthesis of RNA-linked msDNA was characterized and mechanisms of synthesis are proposed.