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Theoriof Filamentous Phage φLf Is Located within the Gene Encoding the Replication Initiation Protein
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
228, 246–251 (1996)
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The ori of Filamentous Phage fLf Is Located within the Gene Encoding the Replication Initiation Protein Nien-Tsung Lin and Yi-Hsiung Tseng1 Institute of Molecular Biology and Department of Botany, National Chung Hsing University, Taichung 402, Taiwan, Republic of China Received September 23, 1996
fLf is a filamentous phage of Xanthomonas campestris pv. campestris. In this study, the origin for fLf replication was located in a 121-bp TaqI fragment within gII, the gene encoding the replication initiation protein. This fragment, ligating with a GmR cartridge, was able to be maintained as a plasmid (pT2) in strain Xc17 with the gII being provided in trans. ssDNA of pT2 was detected in the cells, indicating that pT2 may replicate by a rolling circle replication mechanism. Upon superinfection of Xc17 containing pT2 with fLf, transducing particles containing ssDNA of pT2 were released, suggesting the presence of packaging signal in the 121-bp TaqI fragment. This fragment contains a sequence homologous to the nicking sites for superfamily I Rep proteins of the rolling circle-replicating replicons, in concert with the presence of conserved amino acid sequence motifs of the superfamily I Rep proteins in the fLf gIIP. q 1996 Academic Press, Inc.
fLf is a filamentous phage of Xanthomonas campestris pv. campestris, the causative agent of black rot in crucifers (1) and the microorganism of choice for xanthan gum production in industry (2). Similar to other filamentous phages, fLf contains a single-stranded (ss) circular DNA genome (6.0 kb), uses RF as replication intermediate, and manifests a non-lytic life cycle, propagating without lysis of the host cell. However, it differs from other filamentous phages in possessing a mechanism to integrate its genome into the host chromosome (3, 4). We have previously determined the nucleotide sequences for the intergenic region (IG), and the genes encoding replication initiation protein(s) (gII, gX), the ssDNA binding protein (gV), two of the minor coat proteins (gVII, gIX), the major coat protein (gVIII), and the adsorption protein (gIII) (Fig. 1, ref. 5-7). These genes are organized in the order IG-gII-gX-gV-gVIIgIX-gVIII-gIII, similar to that of the Ff phages f1, fd and M13 (8). Within this sequenced region, the 2,028-bp fragment containing IG and gII was found to be maintained as an autonomous mini-replicon (5). In addition, ssDNA of this mini-replicon can be packaged and released as transducing particles upon superinfection of the cells containing the mini-replicon with the wild-type fLf (5). Therefore, it was thought that fLf IG contains origins for replication of the viral and the complementary strands as well as the signal for phage packaging, as it is in other filamentous phages (8). In this study, efforts were made to seek for the ori for fLf replication. Surprisingly, the ori was found to be contained within the gII coding region. Sequence comparison of the fLf ori-containing fragment revealed a region similar to the nicking sites for the superfamily I Rep proteins of rolling circle replicons, RCR (9). 1
To whom correspondence should be addressed. Abbreviations: dsDNA, double-stranded DNA; GmR, getamycin-resistance; IHF, integration host factor; IG, intergenic region; ori, origin of replication; PFU, plaque forming unit; RCR, rolling circle replicon; ssDNA, singlestranded DNA; TcR, tetracycline-resistance. 246 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Phage and bacterial strains. Bacteriophage fLf was isolated in our laboratory (10). Xc17, a wild-type strain, has been described previously (11). Escherichia coli DH5a (12) was used for gene cloning using pUC18 (13), pOK12 (14) or the broad-host-range vector pRK415 (15) as the vectors. Luria broth and L agar (16) were the general-purpose media for growing E. coli at 377C or X. c. pv. campestris strains at 287C. Antibiotics used were ampicillin (50 mg/ ml), kanamycin (50 mg/ml), tetracycline (15 mg/ml) and gentamycin (15 mg/ml). Phage titer assay. Determination of phage titer was carried out as described previously (10), using the non-mucoid mutant XcP20H (17) as the indicator host. Titer of transducing particle was assayed by infecting Xc17, following the procedure described previously (5) except that gentamycin was used for selection. DNA techniques. The procedures described in Sambrook et al. (18) were used for plasmid extraction, preparation of phage ssDNA, DNA cloning, preparation of a-[32P]-labeled probes, Southern hybridization, and transformation of E. coli. RF DNA and intracellular ssDNA of the recombinant phages were extracted by the alkaline lysis method (19). The rapid screening method described by Weng et al. (20) was used for detection of plasmid. Transformation of X. c. pv. campestris was performed by electroporation (21). Incompatible fragments were blunt-ended with Klenow enzyme before ligation, following the procedures described in Sambrook et al. (18). Restriction enzymes, T4 DNA ligase, Bal31 and other enzymes were purchased from New England Biolabs (Beverly, MA) and used following the instructions provided by the supplier. a-[32P]-dCTP was purchased from Amersham Life Sciences (England, UK). Determination of DNA sequence. DNA fragments cloned in pUC18 were sequenced using the dideoxy chain termination method of Sanger et al. (22) on double-stranded templates using the kit purchased from Amersham Life Sciences. Appropriate synthetic oligomers were used as primers for the reactions.
RESULTS AND DISCUSSION
Cloning of the fLf ori Ff phages (M13, f1 and fd) have an intergenic region (IG, 508 bp) that does not encode any protein product (8). This region contains two subregions, a core and an enhancer. The core includes the origins for replication of the viral and the complementary strands, and the phage packaging signal, whereas the enhancer is a 150-bp AT-rich sequence containing two integration host factor (IHF)-binding sites (23). It has been demonstrated in fd that DNA fragments within IG containing ori are able to maintain as plasmids with the replication initiation protein gene (gII) being provided in trans (24). In fLf, we have previously identified a region capable of autonomous replication, a 2,028-bp Sau3A1 partial fragment which contains IG and gII (5). Therefore, a strategy similar to that for cloning fd ori was employed to identify the fLf ori in this study. Firstly, to provide the fLf gII in trans, the 1,537-bp HincII-XhoI fragment containing gII (Fig. 1, ref. 5) was cloned into the broad-host-range vector pRK415 (TcR, ref. 15) forming pXH15RK. This plasmid was introduced into Xc17 by electroporation, resulting in the construction of strain Xc17(pXH15RK). Then, the 1,280-bp PstI-SphI fragment containing IG (Fig. 1) was tested for maintenance. For this test, the fragment was ligated with the gene encoding gentamycin resistance (GmR cartridge) from pUCGM (25) followed by electroporating the ligation mixture into Xc17(pXH15RK), screening for GmR and TcR. Some transformants were obtained; however, only pXH15RK but no other plasmid was detected in the transformants. Because the PstI-SphI fragment can mediate integration (4), the recombinant molecule was integrated into the chromosome (data not shown). These results suggest that fLf ori is not located within IG. Therefore, the 1,273-bp NruI-XhoI fragment containing gII was ligated with the GmR cartridge, forming plasmid pN1 (Fig. 1), and tested for maintenance. Interestingly, pN1 was found to be maintained in Xc17 without pXH15RK, indicating that both gII and ori are contained in the NruI-XhoI fragment and most likely overlapping. fLf ori Is Located in the 121-bp TaqI Fragment within gII
From the pN1 insert (Fig. 1), two kinds of subfragments, the restriction enzyme-digested and the Bal31-deleted, were tested for maintenance in Xc17(pXH15RK). The restriction enzymedigested ones were ligated with the GmR cartridge after being blunt-ended, whereas the Bal31deleted were cloned into pUC18, verified by nucleotide sequencing and then recovered, blunt247
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FIG. 1. (A) Physical map of fLf RF DNA linearized at the unique PstI site. Numbers in the parentheses represent nucleotide positions counting from the PstI site. The arrows with Roman numerals are positions of the genes already assigned. IG denotes intergenic region. I and C between the ClaI and EcoRI sites represent the predicted integration host factor (IHF)-binding site and the core sequence for fLf integration, respectively. (B) The deletion clones tested for maintenance, with fragment designations on the left and results of the tests on the right. ‘‘/’’ represents being maintained, whereas ‘‘0’’ not. ‘‘ND’’ means maintenance not tested. Nucleotide position numbers at the ends of the fragments represent the deletion end points obtained by Bal31 treatment, whereas the ends without number are the restriction enzyme cleavage sites.
ended and ligated with the GmR cartridge. Twelve clones containing serial deletion of the pN1 insert were obtained. Only pKFP1 which carried the fragment from bp 1,386 to the XhoI site was able to maintain in Xc17 without helper plasmid (Fig. 1), indicating that this fragment still contains functional gII. Nucleotide position 1,386 is within codon 13 predicted for gII (5). Since truncation made here did not abolish the gII function, it seems that the previous prediction may not be correct. Thus, ATG at codon 34 (bp 1,446) may be the true translation start site, using 5*-GAAGAA-3* 13 bp upstream for ribosome binding (5). Detailed analysis is needed for determination of the true initiation codon. Seven out of the 12 deletion clones derived from pN1 were able to maintain in Xc17(pXH15RK). The smallest one was pN19 which contained the 289-bp fragment starting from the NsiI site within the gII coding region (Fig. 1B). This fragment was further reduced to 121 bp by cutting with TaqI, resulting in plasmid pT2, without affecting the ability to maintain. The 121-bp TaqI fragment occupies nucleotide positions 1,591 to 1,711 counting from the unique PstI site of the fLf RF, which is corresponding to aa 82 to 122 of gIIP (Fig. 1B). These results indicate that the location of fLf ori is different from those of other filamen248
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FIG. 2. Detection of plasmid pT2, ssDNA intermediate of pT2 replication, and ssDNA from the pT2 transducing particles by Southern hybridization using probes indicated. DNAs in lane 1 of panels A, B, C, and D were extracted from Xc17(pT2, pXH15RK), whereas that in lane 2 is the GmR cartridge (0.85 kb) from pUCGM as the control. DNAs in lane 1 of panels E and F were prepared from the culture supernatant of Xc17(pT2, pXH15RK) superinfected with fLf, whereas that in lane 2 is the ssDNA from fLf particles as the control. DNAs were denatured (B, D) or non-denatured (A, C, E and F) prior to Southern transfer.
tous phages which are located in IG (8). In addition, fLf ori is short, comparing to those of other filamentous phages, such as phages Ff and Ike (8). Only a few phages have been found to contain their ori within the Rep protein gene, which include fX174 (26), P2 (27) and 186 (28). The Rep proteins of these three phages are classified into the superfamily I of RCR (9). Thus, in terms of the location of ori, fLf is similar to these phages. ssDNA of pT2 Can Be Packaged to Form Transducing Particles pT2 and the other fLf ori-carrying clones were maintained as double-stranded plasmids. They could also form ssDNA molecules detectable by hybridization of the DNAs which were Southern-transferred without prior denaturation. Without denaturation, ssDNA could be hybridized efficiently but dsDNA could not. Fig. 2 shows the results observed on pT2, as an example. These results suggest that the fLf ori-containing DNAs may use rolling circle mechanism for replication in which ssDNA molecules are produced as the intermediate. Upon superinfection of Xc17(pT2, pXH15RK) with the wild-type fLf, transducing recombinant phage particles containing pT2 ssDNA were released (Fig. 2). The transducing particles could also be detected by transforming Xc17 into GmR using the culture supernatant of superinfected Xc17(pT2, pXH15RK). The transducing particles thus detected in overnight cultures titered 0.8 to 2.3 1 106 particles per ml versus 1.7 to 3.3 1 1011 PFU/ml of the wild-type fLf in the same culture supernatants. These results suggest that the 121-bp fragment contains the ori sequences for replication of both viral and complementary strands as well as the phage packaging signal. It was interesting to note that no ssDNA intermediate of pXH15RK was detected in Xc17(pT2, pXH15RK) and no transducing particle of pXH15RK was produced upon superinfection with fLf (Fig. 2). Since pXH15RK contains two oris, it is possible that RK2 ori in stead of fLf ori had been used for replication in Xc17. Should this be the case, ssDNA would not be produced as the replication intermediate. To test whether this prediction is true, further experiments are needed to be carried out. 249
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FIG. 3. Nucleotide sequence of the 121-bp TaqI fragment spanning bp 1,591 to 1,711, counting from the unique PstI site of the fLf RF. The position of this fragment corresponds to bp 1,221 to 1,341 in the 2,028-bp Sau3A1 fragment described by Lin et al. (1996). The inverted repeats are shown by dashed arrows. The possible recognition sequence for the fLf gIIP is bold-faced.
Comparison of the fLf ori Sequence The 121-bp TaqI fragment had a G/C content of 63%, similar to that of the X. c. pv. campestris chromosome (29) and the other regions of the fLf genome already sequenced (57). Inverted repeats of 17 bp having potential to form hairpin structure were noticed, lying between bp 1,619 and 1,652 (Fig. 3, ref. 5). The function of this structure remains to be studied. No extensive homology was found between the sequence of the whole fragment and the other sequences in database. The Rep proteins involved in initiation of rolling circle replication are classified into two groups: the replication and the mobilization group (9). The replication group is further divided into two superfamilies and several smaller families. Superfamily I includes phage P2 and the related phage 186, the isometric ssDNA phages G4 and fX174, the small phagemid phasyl, several cyanobacterial and archaebacterial plasmids, and others (9) (see Fig. 4). Filamentous phages are RCRs; however, the Rep proteins (gIIP) of the known filamentous phages do not belong to any of the superfamilies, since these proteins do not show similarity to those within the known families (9; Koonin, personal communication). In a previous report, we have described the presence of conserved amino acid sequence motifs in the fLf gIIP which are similar to those occurring in the superfamily I Rep proteins (5). The nicking sites for the superfamily I Rep proteins have a conserved sequence of 5*-CTtG3*, with the nicking occurs on the 3* side of the G residue (9). Within the 121-bp TaqI fragment, a 5*-CTTG-3* sequence was found to occur at bp 57 (Fig. 3), corresponding to bp 1,647 counting from the unique PstI site of the fLf RF DNA (Fig. 1). Therefore, with similarities in the putative nicking site and the conserved amino acid sequence motifs (9), fLf
FIG. 4. Alignment of the predicted fLf ori sequence with those of the rolling circle DNA replicons. Only 6 of the conserved ori sequences compiled by Koonin and Ilyina (1993) are quoted here. Asterisk denotes that the origin and nick site are putative. Common sequence/bases found in the binding regions of some ori sequences are indicated by boxes. 250
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gIIP appears to fit in with superfamily I Rep proteins of the RCRs. In constrast to fLf ori, the oris of other filamentous phages, such as Ff, are located in loops D and E (8), where no conserved sequence in the nicking sites has been proposed. CONCLUSION
In this study, we have identified the ori region of fLf, based on its ability to maintain as a plasmid and produce ssDNA intermediate. The results of this study indicate that in addition to being able to integrate, fLf has additional properties different from other filamentous phages including 1) the gIIP being similar to the superfamily I Rep proteins, 2) the ori region being located within gII instead of IG, and 3) presence of a sequence in the ori which is similar to the nicking sites for superfamily I Rep proteins. All these properties are unique among the filamentous phages studied thus far. ACKNOWLEDGMENTS This research was supported by National Science Council of the Republic of China Grant NSC84-2311-B-005-029. We thank Dr. E. Koonin for providing information about classification of the RCR Rep proteins, and Dr. C. N. Sun for reading the manuscript.
REFERENCES 1. Williams, P. H. (1980) Plant Dis. 64, 736–742. 2. Sandford, P. A., and Baird, J. (1983) in The Polysaccharides (Aspinall, G. O., Ed.), Vol. 2, pp. 411–490, Academic Press, New York. 3. Lee, S.-J. (1991) M.S. thesis, National Chung Hsing University, Taiwan. 4. Fu, J.-F., Chang, R.-Y., and Tseng, Y.-H.(1992) Appl. Microbiol. Biotechnol. 37, 225–229. 5. Lin, N.-T., Wen, F.-S., and Tseng, Y.-H. (1996) Biochem. Biophys. Res. Commun. 218, 12–16. 6. Wen, F.-S., and Tseng, Y.-H. (1994) J. Gen. Virol. 75, 15–22. 7. Wen, F.-S., and Tseng, Y.-H. (1996) Gene 172, 161–162. 8. Model, P., and Russel, M. (1988) in The Bacteriophages (Calendar, R., Ed.), Vol. 2, pp. 375–455, Plenum, New York. 9. Koonin, E. V., and Ilyina, T. V. (1993) Biosystems 30, 241–268. 10. Tseng, Y.-H., Lo, M.-C., Lin, K.-C., Pan, C.-C., and Chang, R.-Y. (1990) J. Gen. Virol. 71, 1881–1884. 11. Yang, B.-Y., and Tseng, Y.-H. (1988) Bot. Bull. Acad. Sinica 29, 93–99. 12. Hanahan, D. (1983) J. Mol. Biol. 166, 557. 13. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene 33, 103–119. 14. Vieira, J., and Messing, J. (1991) Gene 100, 189–194. 15. Keen, N. T., Tamaki, S., Kobayashi, D., and Trollinger, D. (1988) Gene 70, 191–197. 16. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 17. Yang, B.-Y., Tsai, H.-F., and Tseng, Y.-H. (1988) Chin. J. Microbiol. Immunol. 21, 231–240. 18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 19. Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513–1523. 20. Weng, S.-F., Lin, N.-T., Fan, Y.-F., Lin, J.-W., and Tseng, Y.-H. (1996) Bot. Bull. Acad. Sinica 37, 93–98. 21. Wang, T.-W., and Tseng, Y.-H. (1992) Lett. Appl. Microbiol. 14, 65–68. 22. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–5467. 23. Greenstein, D., Zinder, N. D., and Horiuchi, K. (1988) Proc. Natl. Acad. Sci. USA 85, 6262–6266. 24. Meyer, T. F., and Geider, K. (1981) Proc. Natl. Acad. Sci. USA 78, 5416–5420. 25. Schweizer, H. P. (1993) BioTechnology 15, 831–832. 26. Langeveld, S. A., van Mansfeld, A. D. M., Baas, P. D., Jansz, H. S., Van Arkel, G. A., and Weisbeek, P. J. (1978) Nature 271, 417. 27. Liu, Y., Saha, S., and Haggard-Ljungquist, E. (1993) J. Mol. Biol. 231, 361–374. 28. Sivaprasad, A. V., Jarvinen, R., Puspurs, A., and Egan, J. B. (1990) J. Mol. Biol. 213, 449–463. 29. Bradbury, J. F. (1984) in Bergey‘s Manual of Systematic Bacteriology (Krieg, N. R. Ed.), Vol. 1, pp. 199–210, Williams & Wilkins, Baltimore.
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The ori of Filamentous Phage fLf Is Located within the Gene Encoding the Replication Initiation Protein Nien-Tsung Lin and Yi-Hsiung Tseng1 Institute of Molecular Biology and Department of Botany, National Chung Hsing University, Taichung 402, Taiwan, Republic of China Received September 23, 1996
fLf is a filamentous phage of Xanthomonas campestris pv. campestris. In this study, the origin for fLf replication was located in a 121-bp TaqI fragment within gII, the gene encoding the replication initiation protein. This fragment, ligating with a GmR cartridge, was able to be maintained as a plasmid (pT2) in strain Xc17 with the gII being provided in trans. ssDNA of pT2 was detected in the cells, indicating that pT2 may replicate by a rolling circle replication mechanism. Upon superinfection of Xc17 containing pT2 with fLf, transducing particles containing ssDNA of pT2 were released, suggesting the presence of packaging signal in the 121-bp TaqI fragment. This fragment contains a sequence homologous to the nicking sites for superfamily I Rep proteins of the rolling circle-replicating replicons, in concert with the presence of conserved amino acid sequence motifs of the superfamily I Rep proteins in the fLf gIIP. q 1996 Academic Press, Inc.
fLf is a filamentous phage of Xanthomonas campestris pv. campestris, the causative agent of black rot in crucifers (1) and the microorganism of choice for xanthan gum production in industry (2). Similar to other filamentous phages, fLf contains a single-stranded (ss) circular DNA genome (6.0 kb), uses RF as replication intermediate, and manifests a non-lytic life cycle, propagating without lysis of the host cell. However, it differs from other filamentous phages in possessing a mechanism to integrate its genome into the host chromosome (3, 4). We have previously determined the nucleotide sequences for the intergenic region (IG), and the genes encoding replication initiation protein(s) (gII, gX), the ssDNA binding protein (gV), two of the minor coat proteins (gVII, gIX), the major coat protein (gVIII), and the adsorption protein (gIII) (Fig. 1, ref. 5-7). These genes are organized in the order IG-gII-gX-gV-gVIIgIX-gVIII-gIII, similar to that of the Ff phages f1, fd and M13 (8). Within this sequenced region, the 2,028-bp fragment containing IG and gII was found to be maintained as an autonomous mini-replicon (5). In addition, ssDNA of this mini-replicon can be packaged and released as transducing particles upon superinfection of the cells containing the mini-replicon with the wild-type fLf (5). Therefore, it was thought that fLf IG contains origins for replication of the viral and the complementary strands as well as the signal for phage packaging, as it is in other filamentous phages (8). In this study, efforts were made to seek for the ori for fLf replication. Surprisingly, the ori was found to be contained within the gII coding region. Sequence comparison of the fLf ori-containing fragment revealed a region similar to the nicking sites for the superfamily I Rep proteins of rolling circle replicons, RCR (9). 1
To whom correspondence should be addressed. Abbreviations: dsDNA, double-stranded DNA; GmR, getamycin-resistance; IHF, integration host factor; IG, intergenic region; ori, origin of replication; PFU, plaque forming unit; RCR, rolling circle replicon; ssDNA, singlestranded DNA; TcR, tetracycline-resistance. 246 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Phage and bacterial strains. Bacteriophage fLf was isolated in our laboratory (10). Xc17, a wild-type strain, has been described previously (11). Escherichia coli DH5a (12) was used for gene cloning using pUC18 (13), pOK12 (14) or the broad-host-range vector pRK415 (15) as the vectors. Luria broth and L agar (16) were the general-purpose media for growing E. coli at 377C or X. c. pv. campestris strains at 287C. Antibiotics used were ampicillin (50 mg/ ml), kanamycin (50 mg/ml), tetracycline (15 mg/ml) and gentamycin (15 mg/ml). Phage titer assay. Determination of phage titer was carried out as described previously (10), using the non-mucoid mutant XcP20H (17) as the indicator host. Titer of transducing particle was assayed by infecting Xc17, following the procedure described previously (5) except that gentamycin was used for selection. DNA techniques. The procedures described in Sambrook et al. (18) were used for plasmid extraction, preparation of phage ssDNA, DNA cloning, preparation of a-[32P]-labeled probes, Southern hybridization, and transformation of E. coli. RF DNA and intracellular ssDNA of the recombinant phages were extracted by the alkaline lysis method (19). The rapid screening method described by Weng et al. (20) was used for detection of plasmid. Transformation of X. c. pv. campestris was performed by electroporation (21). Incompatible fragments were blunt-ended with Klenow enzyme before ligation, following the procedures described in Sambrook et al. (18). Restriction enzymes, T4 DNA ligase, Bal31 and other enzymes were purchased from New England Biolabs (Beverly, MA) and used following the instructions provided by the supplier. a-[32P]-dCTP was purchased from Amersham Life Sciences (England, UK). Determination of DNA sequence. DNA fragments cloned in pUC18 were sequenced using the dideoxy chain termination method of Sanger et al. (22) on double-stranded templates using the kit purchased from Amersham Life Sciences. Appropriate synthetic oligomers were used as primers for the reactions.
RESULTS AND DISCUSSION
Cloning of the fLf ori Ff phages (M13, f1 and fd) have an intergenic region (IG, 508 bp) that does not encode any protein product (8). This region contains two subregions, a core and an enhancer. The core includes the origins for replication of the viral and the complementary strands, and the phage packaging signal, whereas the enhancer is a 150-bp AT-rich sequence containing two integration host factor (IHF)-binding sites (23). It has been demonstrated in fd that DNA fragments within IG containing ori are able to maintain as plasmids with the replication initiation protein gene (gII) being provided in trans (24). In fLf, we have previously identified a region capable of autonomous replication, a 2,028-bp Sau3A1 partial fragment which contains IG and gII (5). Therefore, a strategy similar to that for cloning fd ori was employed to identify the fLf ori in this study. Firstly, to provide the fLf gII in trans, the 1,537-bp HincII-XhoI fragment containing gII (Fig. 1, ref. 5) was cloned into the broad-host-range vector pRK415 (TcR, ref. 15) forming pXH15RK. This plasmid was introduced into Xc17 by electroporation, resulting in the construction of strain Xc17(pXH15RK). Then, the 1,280-bp PstI-SphI fragment containing IG (Fig. 1) was tested for maintenance. For this test, the fragment was ligated with the gene encoding gentamycin resistance (GmR cartridge) from pUCGM (25) followed by electroporating the ligation mixture into Xc17(pXH15RK), screening for GmR and TcR. Some transformants were obtained; however, only pXH15RK but no other plasmid was detected in the transformants. Because the PstI-SphI fragment can mediate integration (4), the recombinant molecule was integrated into the chromosome (data not shown). These results suggest that fLf ori is not located within IG. Therefore, the 1,273-bp NruI-XhoI fragment containing gII was ligated with the GmR cartridge, forming plasmid pN1 (Fig. 1), and tested for maintenance. Interestingly, pN1 was found to be maintained in Xc17 without pXH15RK, indicating that both gII and ori are contained in the NruI-XhoI fragment and most likely overlapping. fLf ori Is Located in the 121-bp TaqI Fragment within gII
From the pN1 insert (Fig. 1), two kinds of subfragments, the restriction enzyme-digested and the Bal31-deleted, were tested for maintenance in Xc17(pXH15RK). The restriction enzymedigested ones were ligated with the GmR cartridge after being blunt-ended, whereas the Bal31deleted were cloned into pUC18, verified by nucleotide sequencing and then recovered, blunt247
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FIG. 1. (A) Physical map of fLf RF DNA linearized at the unique PstI site. Numbers in the parentheses represent nucleotide positions counting from the PstI site. The arrows with Roman numerals are positions of the genes already assigned. IG denotes intergenic region. I and C between the ClaI and EcoRI sites represent the predicted integration host factor (IHF)-binding site and the core sequence for fLf integration, respectively. (B) The deletion clones tested for maintenance, with fragment designations on the left and results of the tests on the right. ‘‘/’’ represents being maintained, whereas ‘‘0’’ not. ‘‘ND’’ means maintenance not tested. Nucleotide position numbers at the ends of the fragments represent the deletion end points obtained by Bal31 treatment, whereas the ends without number are the restriction enzyme cleavage sites.
ended and ligated with the GmR cartridge. Twelve clones containing serial deletion of the pN1 insert were obtained. Only pKFP1 which carried the fragment from bp 1,386 to the XhoI site was able to maintain in Xc17 without helper plasmid (Fig. 1), indicating that this fragment still contains functional gII. Nucleotide position 1,386 is within codon 13 predicted for gII (5). Since truncation made here did not abolish the gII function, it seems that the previous prediction may not be correct. Thus, ATG at codon 34 (bp 1,446) may be the true translation start site, using 5*-GAAGAA-3* 13 bp upstream for ribosome binding (5). Detailed analysis is needed for determination of the true initiation codon. Seven out of the 12 deletion clones derived from pN1 were able to maintain in Xc17(pXH15RK). The smallest one was pN19 which contained the 289-bp fragment starting from the NsiI site within the gII coding region (Fig. 1B). This fragment was further reduced to 121 bp by cutting with TaqI, resulting in plasmid pT2, without affecting the ability to maintain. The 121-bp TaqI fragment occupies nucleotide positions 1,591 to 1,711 counting from the unique PstI site of the fLf RF, which is corresponding to aa 82 to 122 of gIIP (Fig. 1B). These results indicate that the location of fLf ori is different from those of other filamen248
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FIG. 2. Detection of plasmid pT2, ssDNA intermediate of pT2 replication, and ssDNA from the pT2 transducing particles by Southern hybridization using probes indicated. DNAs in lane 1 of panels A, B, C, and D were extracted from Xc17(pT2, pXH15RK), whereas that in lane 2 is the GmR cartridge (0.85 kb) from pUCGM as the control. DNAs in lane 1 of panels E and F were prepared from the culture supernatant of Xc17(pT2, pXH15RK) superinfected with fLf, whereas that in lane 2 is the ssDNA from fLf particles as the control. DNAs were denatured (B, D) or non-denatured (A, C, E and F) prior to Southern transfer.
tous phages which are located in IG (8). In addition, fLf ori is short, comparing to those of other filamentous phages, such as phages Ff and Ike (8). Only a few phages have been found to contain their ori within the Rep protein gene, which include fX174 (26), P2 (27) and 186 (28). The Rep proteins of these three phages are classified into the superfamily I of RCR (9). Thus, in terms of the location of ori, fLf is similar to these phages. ssDNA of pT2 Can Be Packaged to Form Transducing Particles pT2 and the other fLf ori-carrying clones were maintained as double-stranded plasmids. They could also form ssDNA molecules detectable by hybridization of the DNAs which were Southern-transferred without prior denaturation. Without denaturation, ssDNA could be hybridized efficiently but dsDNA could not. Fig. 2 shows the results observed on pT2, as an example. These results suggest that the fLf ori-containing DNAs may use rolling circle mechanism for replication in which ssDNA molecules are produced as the intermediate. Upon superinfection of Xc17(pT2, pXH15RK) with the wild-type fLf, transducing recombinant phage particles containing pT2 ssDNA were released (Fig. 2). The transducing particles could also be detected by transforming Xc17 into GmR using the culture supernatant of superinfected Xc17(pT2, pXH15RK). The transducing particles thus detected in overnight cultures titered 0.8 to 2.3 1 106 particles per ml versus 1.7 to 3.3 1 1011 PFU/ml of the wild-type fLf in the same culture supernatants. These results suggest that the 121-bp fragment contains the ori sequences for replication of both viral and complementary strands as well as the phage packaging signal. It was interesting to note that no ssDNA intermediate of pXH15RK was detected in Xc17(pT2, pXH15RK) and no transducing particle of pXH15RK was produced upon superinfection with fLf (Fig. 2). Since pXH15RK contains two oris, it is possible that RK2 ori in stead of fLf ori had been used for replication in Xc17. Should this be the case, ssDNA would not be produced as the replication intermediate. To test whether this prediction is true, further experiments are needed to be carried out. 249
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FIG. 3. Nucleotide sequence of the 121-bp TaqI fragment spanning bp 1,591 to 1,711, counting from the unique PstI site of the fLf RF. The position of this fragment corresponds to bp 1,221 to 1,341 in the 2,028-bp Sau3A1 fragment described by Lin et al. (1996). The inverted repeats are shown by dashed arrows. The possible recognition sequence for the fLf gIIP is bold-faced.
Comparison of the fLf ori Sequence The 121-bp TaqI fragment had a G/C content of 63%, similar to that of the X. c. pv. campestris chromosome (29) and the other regions of the fLf genome already sequenced (57). Inverted repeats of 17 bp having potential to form hairpin structure were noticed, lying between bp 1,619 and 1,652 (Fig. 3, ref. 5). The function of this structure remains to be studied. No extensive homology was found between the sequence of the whole fragment and the other sequences in database. The Rep proteins involved in initiation of rolling circle replication are classified into two groups: the replication and the mobilization group (9). The replication group is further divided into two superfamilies and several smaller families. Superfamily I includes phage P2 and the related phage 186, the isometric ssDNA phages G4 and fX174, the small phagemid phasyl, several cyanobacterial and archaebacterial plasmids, and others (9) (see Fig. 4). Filamentous phages are RCRs; however, the Rep proteins (gIIP) of the known filamentous phages do not belong to any of the superfamilies, since these proteins do not show similarity to those within the known families (9; Koonin, personal communication). In a previous report, we have described the presence of conserved amino acid sequence motifs in the fLf gIIP which are similar to those occurring in the superfamily I Rep proteins (5). The nicking sites for the superfamily I Rep proteins have a conserved sequence of 5*-CTtG3*, with the nicking occurs on the 3* side of the G residue (9). Within the 121-bp TaqI fragment, a 5*-CTTG-3* sequence was found to occur at bp 57 (Fig. 3), corresponding to bp 1,647 counting from the unique PstI site of the fLf RF DNA (Fig. 1). Therefore, with similarities in the putative nicking site and the conserved amino acid sequence motifs (9), fLf
FIG. 4. Alignment of the predicted fLf ori sequence with those of the rolling circle DNA replicons. Only 6 of the conserved ori sequences compiled by Koonin and Ilyina (1993) are quoted here. Asterisk denotes that the origin and nick site are putative. Common sequence/bases found in the binding regions of some ori sequences are indicated by boxes. 250
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gIIP appears to fit in with superfamily I Rep proteins of the RCRs. In constrast to fLf ori, the oris of other filamentous phages, such as Ff, are located in loops D and E (8), where no conserved sequence in the nicking sites has been proposed. CONCLUSION
In this study, we have identified the ori region of fLf, based on its ability to maintain as a plasmid and produce ssDNA intermediate. The results of this study indicate that in addition to being able to integrate, fLf has additional properties different from other filamentous phages including 1) the gIIP being similar to the superfamily I Rep proteins, 2) the ori region being located within gII instead of IG, and 3) presence of a sequence in the ori which is similar to the nicking sites for superfamily I Rep proteins. All these properties are unique among the filamentous phages studied thus far. ACKNOWLEDGMENTS This research was supported by National Science Council of the Republic of China Grant NSC84-2311-B-005-029. We thank Dr. E. Koonin for providing information about classification of the RCR Rep proteins, and Dr. C. N. Sun for reading the manuscript.
REFERENCES 1. Williams, P. H. (1980) Plant Dis. 64, 736–742. 2. Sandford, P. A., and Baird, J. (1983) in The Polysaccharides (Aspinall, G. O., Ed.), Vol. 2, pp. 411–490, Academic Press, New York. 3. Lee, S.-J. (1991) M.S. thesis, National Chung Hsing University, Taiwan. 4. Fu, J.-F., Chang, R.-Y., and Tseng, Y.-H.(1992) Appl. Microbiol. Biotechnol. 37, 225–229. 5. Lin, N.-T., Wen, F.-S., and Tseng, Y.-H. (1996) Biochem. Biophys. Res. Commun. 218, 12–16. 6. Wen, F.-S., and Tseng, Y.-H. (1994) J. Gen. Virol. 75, 15–22. 7. Wen, F.-S., and Tseng, Y.-H. (1996) Gene 172, 161–162. 8. Model, P., and Russel, M. (1988) in The Bacteriophages (Calendar, R., Ed.), Vol. 2, pp. 375–455, Plenum, New York. 9. Koonin, E. V., and Ilyina, T. V. (1993) Biosystems 30, 241–268. 10. Tseng, Y.-H., Lo, M.-C., Lin, K.-C., Pan, C.-C., and Chang, R.-Y. (1990) J. Gen. Virol. 71, 1881–1884. 11. Yang, B.-Y., and Tseng, Y.-H. (1988) Bot. Bull. Acad. Sinica 29, 93–99. 12. Hanahan, D. (1983) J. Mol. Biol. 166, 557. 13. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene 33, 103–119. 14. Vieira, J., and Messing, J. (1991) Gene 100, 189–194. 15. Keen, N. T., Tamaki, S., Kobayashi, D., and Trollinger, D. (1988) Gene 70, 191–197. 16. Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 17. Yang, B.-Y., Tsai, H.-F., and Tseng, Y.-H. (1988) Chin. J. Microbiol. Immunol. 21, 231–240. 18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 19. Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513–1523. 20. Weng, S.-F., Lin, N.-T., Fan, Y.-F., Lin, J.-W., and Tseng, Y.-H. (1996) Bot. Bull. Acad. Sinica 37, 93–98. 21. Wang, T.-W., and Tseng, Y.-H. (1992) Lett. Appl. Microbiol. 14, 65–68. 22. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–5467. 23. Greenstein, D., Zinder, N. D., and Horiuchi, K. (1988) Proc. Natl. Acad. Sci. USA 85, 6262–6266. 24. Meyer, T. F., and Geider, K. (1981) Proc. Natl. Acad. Sci. USA 78, 5416–5420. 25. Schweizer, H. P. (1993) BioTechnology 15, 831–832. 26. Langeveld, S. A., van Mansfeld, A. D. M., Baas, P. D., Jansz, H. S., Van Arkel, G. A., and Weisbeek, P. J. (1978) Nature 271, 417. 27. Liu, Y., Saha, S., and Haggard-Ljungquist, E. (1993) J. Mol. Biol. 231, 361–374. 28. Sivaprasad, A. V., Jarvinen, R., Puspurs, A., and Egan, J. B. (1990) J. Mol. Biol. 213, 449–463. 29. Bradbury, J. F. (1984) in Bergey‘s Manual of Systematic Bacteriology (Krieg, N. R. Ed.), Vol. 1, pp. 199–210, Williams & Wilkins, Baltimore.
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