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Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7208-7212, September 1989 Microbiology Extensive diversity of branched-RNA-linked multicopy single-stranded in clinical strains of (reverse transcriptase/polymorphisms/2'-5' phosphodiester/evolution) JING SUN*, PETER J. HERZER*, MELVIN P. WEINSTEINt, BERT C. LAMPSON*, MASAYORI INOUYE*, AND SUMIKO INOUYE* *Department of , Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854; and tDepartment of Clinical Microbiology, Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903 Communicated by Dale Kaiser, May 31, 1989

ABSTRACT Recently it was shown that a clinical strain of ofE. coli were examined for the existence of msDNA, and 7 Escherichia colicontains a reverse transcriptase that is essential were found to contain msDNA. for the synthesis of a branched-RNA-linked multicopy single- stranded DNA (msDNA). We now have examined 113 inde- MATERIALS AND METHODS pendent clinical isolates of E. coli for the existence of msDNA and found that 7 strains contained msDNA. Four ofthem were Bacterial Strain. The clinical E. coli strains were isolated further analyzed by hybridization analysis, which indicated from patients with urinary tract infection or with blood that three ofthe msDNAs were different, having little sequence infection in the Microbiology Laboratory, Robert Wood . When the reverse transcriptase associated with Johnson University Hospital (New Brunswick, NJ). All one of these msDNAs was used as a probe, it did not hybridize strains were identified with the use of the API-20E identifi- with chromosomal DNA from the other strains containing cation system (API Laboratory Products, Sherwood Medi- msDNA. These results indicate that some clinical E. col strains cal, Plainview, NY) and gave a typical E. coli profile. carry their own unique msDNA-synthesizing systems; msDNAs Materials. Restriction enzymes were purchased from New produced by these systems have little, if any, sequence homol- England Biolabs and Boehringer Mannheim. Avian myelo- ogy in their RNA and DNA molecules and the reverse tran- blastosis virus (AMV) RT was from Boehringer Mannheim. scriptases required for the production ofmsDNA also have little All enzymes were used as recommended by suppliers. RNase sequence similarity. Such extensive diversity of the msDNA- A was from Worthington. [a-32P]dCTP (5000 Ci/mmol; 1 Ci the that were = 37 GBq) was purchased from Amersham. synthesizing systems supports notion they ac- Preparation ofChromosomal DNA and RNA. Chromosomal quired by theE. colU genome late during the evolution ofE. col. DNA was prepared according to Yee et al. (2). Total RNA was prepared according to the methods described by Mani- Reverse transcriptase (RT; RNA-directed DNA polymerase, atis et al. (10), but without proteinase K treatment. EC 2.7.7.49) was recently found in a clinical Escherichia coli Preparation ofDNA Probes. To obtain 32P-labeled msDNAs strain isolated from a patient with a urinary tract infection (1). as probes, total RNA prepared from a 5-ml culture was This RT was shown to be associated with a chromosomal incubated in a 50-pul reaction mixture containing 50 mM DNA region responsible for the synthesis of a peculiar Tris/HCl buffer (pH 8.3), 6 mM MgC12, 40 mM KC1, 5 mM satellite DNA called msDNA (multicopy single-stranded dithiothreitol, 1 jLM each dATP, dTTP, and dGTP, 0.2 uM DNA) and to be essential for its production. This msDNA [a-32P]dCTP, and 10 units of AMV RT. The reaction was (msDNA-Ec67) consisted of a 67-base single-stranded DNA, carried out as described (1). The reaction mixture was then and its 5' end was linked to a branched RNA of 58 bases by electrophoresed in a 4% acrylamide/8 M urea gel and the a 2'-5' phosphodiester linkage at the 2' position of the 15th 32P-labeled msDNA band was eluted from the gel. residue, a guanosine. msDNAs were originally found in DNA fragments containing the gene for Cl-1 RT, o- factor, myxobacteria, Gram-negative bacteria living in soil as mul- or Era were nick-translated (11) for use as probes. ticellular organisms (2-6). From work on myxobacterial Southern Blot Hybridization. Chromosomal DNA prepara- msDNA, it was predicted that msDNA is synthesized by a tions were digested with BamHI or Pst I and -3 Ag of each novel mechanism using an RNA precursor as a primer for digest was applied to a 0.7% agarose gel. After electropho- initiating msDNA synthesis as well as a template to form the resis the DNA was blotted to a nitrocellulose filter by branched-RNA-linked msDNA. It was proposed that RT is capillary transfer (12), and hybridization was carried out in required for this reaction (5). Indeed a gene for RT was 50%o (vol/vol) formamide/5x SSPE/5x Denhardt's solu- recently demonstrated to be closely associated with the tion/0.3% SDS at 420C. (SSPE is 180 mM NaCl/10 mM msDNA coding region on the of Myxococcus sodium phosphate, pH 7.4/10 mM EDTA; Denhardt's solu- xanthus (7) and E. coli clinical strain Cl-1 (1). It was also tion is 0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bo- demonstrated that RT activity is required for msDNA syn- vine serum albumin.) thesis in both M. xanthus (8) and E. coli strain Cl-1 (1). Another msDNA (msDNA-Ec86) was reported to exist in E. coli B (9). In spite of a lack of any primary sequence RESULTS homology with msDNA-Ec67, msDNA-Ec86 not only shares Screening of msDNA-Containing Strains. For screening all the key structural features ofmsDNA but also has its own msDNA-containing strains we used the RT labeling method RT gene closely associated with the msDNA coding region. (1, 9), which is much more sensitive than staining msDNA by In the work described here, 113 independent clinical isolates ethidium bromide (2). Since msDNA contains a DNA-RNA duplex structure, the 3' end of the DNA molecule serves as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: AMV, avian myeloblastosis virus; RT, reverse tran- in accordance with 18 U.S.C. §1734 solely to indicate this fact. scriptase; msDNA, multicopy single-stranded DNA. 7208 Downloaded by guest on September 28, 2021 Microbiology: Sun et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7209 an intramolecular primer and the RNA molecule as a tem- observed for msDNAs from myxobacteria (3, 5). In the case plate for RT. RNA was extracted from 113 independent of strain Cl-23, two bands appeared at =152 and 139 bases clinical isolates. Fifty were from patients with urinary tract before RNase treatment (lane 7), and one band appeared at infection, and 63 from patients with blood infection. As =122 bases afterthe treatment (lane 8). As shown by Lim and previously shown (1), the total RNA preparation from strain Maas (9), E. coli B produces msDNA (msDNA-Ec86) result- Cl-1 labeled with AMV RT gives two distinct, low molecular ing in a band at =160 bases before RNase A treatment and a weight bands of -130 bases (Fig. 1A, lane 3). It was reported band at =136 bases after the treatment (lanes 11 and 12, previously that msDNA from strain Cl-1 migrated at 160 respectively). After RNase A treatment, bands from strains bases and 105 bases before and after RNase treatment, Cl-13, Cl-23, and Cl-30 migrated between the band for strain respectively. In the present experiment msDNA from Cl-i Cl-1 (lane 4) and the band for E. coli B (lane 12), indicating migrated at 130 and 100 bases. This abnormal migration was that the sizes of their msDNAs are between 67 and 86 bases. due to the electrophoretic properties of the single-stranded From patients with blood infection, 3 strains were found to DNA. When the labeled sample was digested with RNase A contain msDNA, and 1 of them is shown in Fig. 1B. prior to gel electrophoresis, a single band was detected at the in msDNAs. Next, to examine the position corresponding to 100-base single-stranded DNA sequence homology between the msDNAs from different (lane 4). The labeled bands before and after the RNase A strains, Southern blot hybridization analysis (12) with labeled treatment can be explained by the structure of the msDNA msDNAs as probes was carried out for the four strains from molecule from strain Cl-i (msDNA-Ec67) determined previ- urinary tract infections. Chromosomal DNA was isolated ously (1). As reported (1), no labeled bands were detected in from each strain and digested with BamHI or Pst I. Fig. 2A E. coli K-12 strain JA221 (Fig. 1A, lanes 1 and 2). shows that msDNA-Ec67 (msDNA from strain Cl-1) - Among 50 strains from patients with urinary tract infection, ized only with its own chromosomal DNA; furthermore, only 3 strains in addition to Cl-i were found to contain msDNA; a single band appeared for each digestion: a >23-kilobase strains Cl-13, Cl-23, and Cl-30. Both Cl-13 (Fig. LA, lane 5) (kb) band for the BamHI digestion (lane 3) and a 2.8-kb band and Cl-30 (lane 9) produced a band of =190 bases before for the Pst I digestion (lane 10). Under the condition for RNase A treatment. In the case of strain Cl-30 an extra band Southern blot hybridization used for Fig. 2A, any sequences appeared at -172 bases. However, after RNase A treatment with less than =80%o homology with msDNA-Ec67 cannot be both Cl-13 and Cl-30 gave rise to a single band at 125 bases detected. A similar result was obtained with msDNA from (lanes 6 and 10, respectively). Therefore, the lower band of strain Cl-23 (Fig. 2C); it did not hybridize with chromosomal strain Cl-30 was most likely due to processing of the RNA DNA from any other strain (Cl-1, Cl-13, Cl-30, and Cl-48; molecule associated with the higher band, as previously strain Cl-48 was used as a control for strains that do not A B 5 6 7 8 9 10 11 12 S 1 2 *, *.10 4

62 1 2

527- 404- 622- 309- X 527-,vli.

247- 407- A 180- ,I 309- 160-

122- 247- * 180- 160- 4b 90-

122-

90-

FIG. 1. Detection of msDNA in clinical strains ofE. coli. Total RNA prepared from a 5-ml culture was added to 50 A.l of a reaction mixture containing 50 mM Tris/HCl buffer (pH 8.3), 6 mM MgCl2, 40 mM KCl, 5 mM dithiothreitol, 1 ,uM dATP, dTTP, and dGTP, 0.2 j1M [a-32P]dCTP, and 10 units of AMV RT (Boehringer Mannheim). The reaction was carried out as described (1). The samples were electrophoresed in a 4% acrylamide/8 M urea gel. Lanes S, molecular size markers (lengths in bases at left), Msp I digest of pBR322 end-labeled with [a-32P]dCTP and the Klenow fragment of DNA polymerase I. (A) Lanes 1 and 2, E. coli K-12 strain JA221 (13); lanes 3 and 4, strain Cl-1; lanes 5 and 6, strain Cl-13; lanes 7 and 8, strain Cl-23; lanes 9 and 10, strain Cl-30; lanes 11 and 12, E. coli B. Odd-numbered lanes contained samples prior to RNase A treatment; even-numbered lanes contained RNase A-treated samples. Positions of single-stranded DNA are shown by dots. Relative sample volumes applied to the gel were 1, 1/20, 1, 1/10, 1, and 1/80 for K-12, Cl-1, Cl-13, Cl-23, CI-30, and B, respectively. (B) Sample from strain BC43 before (lanes 1) and after (lane 2) RNase A treatment. Downloaded by guest on September 28, 2021 7210 Microbiology: Sun et al. Proc. Natl. Acad. Sci. USA 86 (1989)

A BamH I Pst I B BamH I Pst I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 >23- w >23- to

7.0-

2.8- :.. .a:

D C I Pst I I - BamH BamH I Pst 1 2 3 4 5 6 7 8 9 10 U 12 13 14 1 2 3 4 5 7 8 9 10 11 12 13 14 >23- >23- _ 15-

7.0-

FIG. 2. Southern blot analysis ofthe chromosomal DNA from various E. coli strains containing msDNAs. Chromosomal DNA preparations were digested with BamHI (lanes 1-7) or Pst I (lanes 8-14). For each lane, -3 jLg of the DNA digest was applied to a 0.7% agarose gel. After electrophoresis and blot transfer to a nitrocellulose filter, hybridization analysis was carried out using various msDNA probes. The msDNA probes were prepared by incubation of total RNA with AMV RT and [a-32P]dCTP, followed by polyacrylamide gel electrophoresis (see Fig. 1). The labeled msDNAs from strains Cl-1, Cl-13, CI-23, and Cl-30 were used as probes for A, B, C, and D, respectively. Lanes 1 and 8, DNA from E. coli K-12 strain JA221; lanes 2 and 9, from E. coli B; lanes 3 and 10, from strain Cl-1; lanes 4 and 11, from strain Cl-13; lanes 5 and 12, from strain Cl-23; lanes 6 and 13, from strain Cl-30; lanes 7 and 14, from strain Cl-48, which does not contain msDNA, as a control. Numbers at left indicate molecular size in kilobases.

contain msDNA). Only a single band was obtained for each entire msd-msr region as well as a part ofthe RT coding region digestion; a >23-kb band for BamHI digestion (lane 5) and a ( residues 1-126; ref. 1), hybridized not only with 15.0-kb band for Pst I digestion (lane 12). Strains Cl-13 and the Cl-1 DNA (Fig. 3A, lanes 3 and 10) but also with the E. coli Cl-30 produced exactly the same pattern with both msDNAs, B DNA (lanes 2 and 9). However, the band intensity with the giving a single 7.0-kb band when hybridized with the BamHI E. coli B DNA was less than that with the Cl-1 DNA. The chromosomal digest from either strain Cl-13 or strain Cl-30 probe did not hybridize with DNA from any other strain. (lanes 4 and 6, respectively, in both B and D of Fig. 2). When the 0.9-kb HindIII-Pst I fragment that covers the region Similarly, they gave rise to a single >23-kb band when coding for RT residues 127-430 (1) was used as a probe, it hybridized with the Pst I digest from either strain Cl-13 or hybridized only with the Cl-1 DNA (Fig. 3B, lanes 3 and 10). strain Cl-30 (lanes 11 and 13, respectively, in both B and D). It should be noted that this region contains the Tyr-Xaa- These results indicate that the msDNAs from strains Cl-13 Asp-Asp (YXDD) box, the most in all and Cl-30 are identical or very closely related. This is RTs so far identified. Thus the results shown in Fig. 3 A and supported by the fact that the sizes of msDNA from strains B indicate that the RT that are assumed to be associated Cl-13 and Cl-30 were identical (Fig. 1A, lanes 6 and 10, with the synthesis of msDNAs from strains Cl-13, Cl-23, and respectively). These results demonstrate that there are at Cl-30 have little homology with the Cl-i RT gene. Since the least three unique msDNAs, among 50 strains from urinary region encompassing the 5' end of the msd-msr region and tract infection, that differ in DNA size and in RNA size and encoding the amino-terminal 126 residues of RT from strain share little sequence homology. Indeed, the primary se- Cl-i has little homology with the corresponding region of E. quence of msDNA from strain Cl-23 did not show any coli B (1, 9), the result in Fig. 3A indicates that there is a unique homology with that of msDNA-Ec67 (unpublished data). sequence upstream of the msd-msr region of strain Cl-1 that Sequence Homology in the RT Genes. Next, we examined can hybridize only with the E. coli B DNA. whether DNA from the RT gene, whose product is associated Polymorphisms Between the Strains. When two endoge- with the synthesis ofmsDNA-Ec67 from strain Cl-1 (1), would nous, essential genes-rpoD, encoding the cr factor (v.70; ref. hybridize with chromosomal DNA from strains other than 14), and era, encoding the Ras-like protein (15, 16)-were Cl-i. The 1.9-kb Pst 1-HindIII fragment, which includes the used as probes, they hybridized with every chromosomal Downloaded by guest on September 28, 2021 Microbiology: Sun et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7211

A BamH I PstI B BamH I Pst I 1 2 3 4 5 6 7 8 9 10 II 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 >23- _ >23- i

2.9-

3.0- 2.4- V

C BamH I Pst I D Bami I Pst I 12 3 4 5 6 7 8 9 10 12 13 14 1 2 3 4 5 6 7 8 9 10 U 12 13 14

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4.6- t --4.6 4.1-_ _, 4-4 _w _8 to

1-6-_ No 40 elmft -~~~~~~~~~~~~~~~~~~~1.

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FIG. 3. Southern blot analysis ofthe chromosomal DNA from various E. coli strains with DNA probes ofdifferent E. coli genes. The filters were prepared as described for Fig. 2. Chromosomal DNA was digested with BamHI (lanes 1-7) or Pst I (lanes 8-14). Nick-translated DNA fragments used as probes were as follows. (A) The 1.9-kb Pst I-HindIII fragment encompassing the entire msd-msr region and a part of the RT gene from strain Cl-1 (1). The RT region in this probe covers an amino-terminal part of RT from the initiation methionine to residue 126. (B) The 0.9-kb HindIII-Pst I fragment encompassing RT codons 127-430 from strain Cl-1. (C) The 3.0-kb Pvu II fragment from the gene for the major E. coli af factor (o.70; ref. 14). (D) The 1.6-kb BamHI fragment from the era gene (15). Lanes 1 and 8, DNA from E. coli K-12 strain JA221; lanes 2 and 9, from E. coli B; lanes 3 and 10, from strain Cl-i; lanes 4 and 11, from strain Cl-13; lanes 5 and 12, from strain Cl-23; lanes 6 and 13, from strain Cl-30; lanes 7 and 14, from strain Cl-48. Molecular sizes are indicated in kilobases.

DNA (Fig. 3 C and D, respectively). However, there are synthesizing systems and that these systems are markedly polymorphisms for the restriction sites; the 0.-factor probe different, having their own RTs which share little sequence hybridized to a 9.4-kb BamHI fragment from all E. coli strains similarity and producing msDNAs which also share little, if tested (Fig. 3C, lanes 1-7), while it hybridized with a 4.1-kb any, sequence homology in the RNA and DNA BamHI fragment from E. coli K-12 (lane 1), B (lane 2), strain molecules. Recently, the structures of msDNA from E. coli Cl-23 (lane 5), and strain Cl-48 (lane 7) but not from strain Cl-1 B (msDNA-Ec86; ref. 9) and strain Cl-1 (msDNA-Ec67; ref. (lane 3), strain Cl-13 (lane 4), and strain Cl-30 (lane 6). For 1) were determined and were found to share no primary strain Cl-1 a 4.6-kb fragment (lane 3) was observed instead of sequence homology in either RNA or DNA. Further, the the 4.1-kb fragment, and for both strains Cl-13 (lane 4) and domain structures were found to be remarkably different Cl-30 (lane 6) a 5.4-kb fragment was detected. Hybridization between E. coli B RT and strain Cl-1 RT, even though both to the Pst I digests showed polymorphism of the Pst I sites are required for their own msDNA synthesis (1, 9). In in strains Cl-1, Cl-13, and Cl-30 (Fig. 3C, lanes 10, 11, and 13, addition, the amino acid sequences were quite different, with respectively). This result again indicates that strains Cl-13 35.3% sequence identity between the E. coli B RT (9) and the and Cl-30 are probably identical, having not only the same amino-terminal region of the Cl-1 RT from residue 19 to 328 msDNA but also the same chromosomal DNA restriction-site (1). The structure of msDNA from strain Cl-23 has also been patterns. When the era gene was used as a probe, the probe determined, and it showed no primary sequence homology hybridized to a 1.6-kb BamHI fragment in all strains tested with msDNA-Ec86 and msDNA-Ec67 (J.S., M.I., and S.I., (Fig. 3D, lanes 1-7), whereas different patterns were ob- unpublished data). served for E. coli K-12 and E. coli B for the Pst I digestion The marked diversity ofthe msDNA-synthesizing systems (lanes 8 and 9, respectively). ofE. coli suggests that these systems, including the RT genes, were acquired by the E. coli genome from some other source DISCUSSION late during the evolution of E. coli. This notion is supported by the fact that the codon usage of the RT gene of strain Cl-1 The present data demonstrate that only a minor population of is remarkably different from that of other E. coli genes (1, 7). E. coli clinical strains (7 out of 113, or =6%) carry msDNA- In sharp contrast with the E. coli msDNA-synthesizing Downloaded by guest on September 28, 2021 7212 Microbiology: Sun et al. Proc. Natl. Acad. Sci. USA 86 (1989) systems, msDNA-Mxl62 from M. xanthus is highly con- 1. Lampson, B. C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J. & served among nine independent M. xanthus strains isolated Inouye, S. (1989) Science 243, 1033-1038. from various sites (17). In addition, the codon usage ofthe M. 2. Yee, T., Furuichi, T., Inouye, S. & Inouye, M. (1984) Cell 38, 203-209. xanthus RT is very similar to that of other M. xanthus genes 3. Furuichi, T., Dhundale, A., Inouye, M. & Inouye, S. (1987) (7). Therefore, the M. xanthus msDNA system including the Cell 48, 47-53. RT gene is likely to be as old as other essential genomic 4. Furuichi, T., Inouye, M. & Inouye, S. (1987) Cell 48, 55-62. genes. Furthermore, Stigmatella aurantiaca, another myxo- 5. Dhundale, A., Lampson, B., Furuichi, T., Inouye, M. & bacterium, contains an msDNA that is highly homologous Inouye, S. (1987) Cell 51, 1105-1112. with msDNA-Mxl62 (3, 4). Thus, myxobacterial RT most 6. Dhundale, A., Inouye, M. & Inouye, S. (1988) J. Biol. Chem. likely existed in an ancestral myxobacterium before M. 263, 9055-9058. 7. Inouye, S., Hsu, M.-Y., Eagle, S. & Inouye, M. (1989) Cell 56, xanthus and S. aurantiaca diverged. It is now ofgreat interest 709-717. to examine whether the msDNA-synthesizing systems in E. 8. Lampson, B. C., Inouye, M. & Inouye, S. (1989) Cell 56, coli have any association with the source of infection or 701-707. specific diseases. However, msDNA may not be associated 9. Lim, D. & Maas, W. (1989) Cell 56, 891-904. with any pathological causes, since we have recently found 10. Maniatis, T., Fritsch, E. R. & Sambrook, J. (1982) Molecular E. coli strains containing msDNAs in stool samples from Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold people without any obvious diseases. There appears to be Spring Harbor, NY). only a single site for the msDNA-synthesizing system on the 11. Rigby, P. W., Dieckmann, M., Rhodes, C. & Berg, P. (1977) J. the basis of the Southern blot analysis Mol. Biol. 133, 237-251. chromosome, on 12. Southern, E. (1975) J. Mol. Biol. 98, 503-517. shown in Fig. 2. Further work is needed to determine the 13. Clarke, L. & Carbon, J. (1987) J. Mol. Biol. 120, 517-532. chromosomal location of each msDNA-synthesizing system 14. Burton, Z., Burgess, R. R., Lim, J., Moore, D., Holder, S. & and to examine whether the system can be transferred to Gross, C. A. (1981) Nucleic Acids Res. 9, 2889-2903. other E. coli strains. 15. Ahnn, J., March, P. E., Takiff, H. E. & Inouye, M. (1986) Proc. Natl. Acad. Sci. USA 83, 8849-8853. We are grateful to Dr. J. Peterson for obtaining clinical strains used 16. March, P. E., Ahnn, J. & Inouye, M. (1985) Nucleic Acids Res. in this study. This work was partially supported by a grant from the 13, 4677-4685. U.S. Public Health Service (GM26843) and a grant from Takara 17. Dhundale, A., Furuichi, T., Inouye, S. & Inouye, M. (1985) J. Shuzo Co., Ltd. Bacteriol. 164, 914-917. Downloaded by guest on September 28, 2021