Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, Taipei, 112, Taiwan1
Department of Microbiology and Immunology, Chang-Gung University, Kwei-Shan, 333, Taiwan2
Author for correspondence: Shih-Tung Liu. Tel: +886 3 328 0292. Fax: +886 3 328 0292. e-mail: cgliu{at}mail.cgu.edu.tw
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: plasmid replication, replication control
The GenBank accession number for the sequence of the minimal replicon of pSW800 is AF310258.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As is generally known, the expression of replication initiation proteins (Rep) is often closely regulated to maintain plasmid copy number and stability (Asano & Mizobuchi, 1998a ; Burian et al., 1999
; del Solar et al., 1998
). Plasmids in the IncB, IncI
and IncL/M groups, which are regulated post-transcriptionally by antisense RNA, are typical examples (Asano et al., 1999
; Athanasopoulos et al., 1995
; Siemering et al., 1993
). In these plasmids the gene encoding the Rep protein overlaps with an upstream gene that is less than 90 bp in length (Asano & Mizobuchi, 1998b
) and contains a ribosome-binding sequence (RBS) hidden in a stemloop structure preventing the translation of Rep (Asano et al., 1999
; Athanasopoulos et al., 1995
; Siemering et al., 1993
). Although the peptide translated from the upstream gene is unimportant for plasmid replication, the translation process is believed to destablize the stemloop structure (Praszkier et al., 1992
). However, translation of this small peptide is still insufficient for the translation of Rep; formation of an RNA pseudoknot structure between the stemloop region and a sequence upstream is also required to further destabilize the structure to allow the translation of Rep to occur (Athanasopoulos et al., 1999
). Plasmids regulated by this mechanism also transcribe an antisense RNA molecule, forming a duplex RNA structure to prevent the formation of an RNA pseudoknot (Asano & Mizobuchi, 2000
; Asano et al., 1998
) and thus inhibiting the translation of Rep. This work demonstrates that a mechanism similar to that regulating plasmid replication in the IncB, IncI
and IncL/M groups also regulates pSW800 replication.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Transfer of pSW800 and selection of E. coli HB101(pSW800).
P. stewartii SW800(pBR322) and E. coli HB100 were cultured in LB broth overnight. Approximately 1x108 c.f.u. were taken from each strain and mixed. Cells were incubated for 1 h at 30 °C and transconjugants were selected on LB agar containing 50 µg ampicillin ml-1, 15 µg tetracycline ml-1 and 25 µg streptomycin ml-1.
Southern blot analysis.
The 2 kb PvuII fragment from pSW830 was labelled with 50 µCi [-32P]dCTP (3000 Ci mmol-1; Amersham), using a random-prime labelling kit (Amersham). DNA in an agarose gel was then transferred to Zeta-Probe membrane (Bio-Rad) according to an alkaline-transfer method described by the manufacturer. Finally, hybridization was performed according to a method described by Sambrook & Russell (2000)
.
Determination of plasmid copy number.
Plasmids in P. stewartii SW2 were isolated by using the method of Kado & Liu (1981) and separated by agarose gel electrophoresis. Plasmid bands were then photographed over a UV transilluminator by a digital camera after staining the gel with ethidium bromide. The intensity of the plasmid bands was measured using Gel-Pro Software (Media Cybernetics) on an Apple Macintosh computer. Plasmids pSW500 and pSW1200, with copy numbers of two per cell and one per cell, respectively (Fu et al., 1996
, 1997
), were used as references to determine the copy number of pSW800 in P. stewartii SW2. The same method was used to measure the copy number of pSW800 in E. coli HB101 with pMAK705, which has a copy number of five per cell, serving as a reference.
DNA sequencing.
DNA sequencing was performed according to the chain-termination method (Sanger et al., 1977 ), using double-stranded plasmid DNA as template.
Incompatibility analysis.
Plasmid pSW891 and its deletion derivatives (Apr) were transformed into E. coli HB101(pSW820) and transformants were selected on LB agar containing ampicillin and kanamycin. The cells were then subcultured in LB-ampicillin broth and the presence of pSW820 in the transformants was determined by replica-plating the colonies on LB agar containing ampicillin and kanamycin.
Northern blot analysis.
Cells were cultured in LB broth at 37 °C for 3 h to exponential phase. Total RNA was prepared from the cells according to the acid/phenol extraction method of Aiba et al. (1981) . RNA (50 µg) was then separated on a 1·5% formaldehyde/1% agarose gel at 27 mA for 2 h, with 23S rRNA (2904 nt), 16S rRNA (1473 nt) and 5S rRNA (128 nt) as size markers. The RNA was transferred and cross-linked to Zeta-Probe membrane and probes specific to RNAI and RNAII were transcribed in vitro with 50 µCi [
-32P]CTP (3000 Ci mmol-1; Amersham) and SP6 and T7 RNA polymerase, respectively, using SmaI-digested pSW891 as template. Hybridization was performed according to a method described by Chang et al. (1998)
.
Primer extension.
Primers P-RNA-II (5'-GTTTTTGCATTCAGGGGTTAG-3'), complementary to the region between map locations 112 and 132, and P-RNAI (5'-CTCGAACTTGCCGGGACGAA-3'), which comprises a sequence between map locations 156 and 175 (see Fig. 2), were end-labelled with 3 µCi [-32P]ATP (6000 Ci mmol-1; Amersham) and 5 U T4 polynucleotide kinase (Promega). Primer extension was then performed with 50 µg RNA and 1 U AMV reverse transcriptase (Promega) according to the method described by Lin et al. (1999)
. Finally, labelled cDNA was analysed on an 8% urea/polyacrylamide gel (Sambrook & Russell, 2000
).
Prediction of RNA structure.
The structures of the untranslated region of RNAII were predicted using SeqWeb version 1.1 MFold software [Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, WI, USA].
Construction of fusion plasmids.
A repB-lacZ translation fusion (pSW851) was constructed by inserting the PvuIIBsrFI fragment from pSW830 (see Fig. 2) into the HindIII site of pKM005. A repA-lacZ translational fusion (pSW852) was then generated by inserting the PvuIIXmnI fragment (see Fig. 2) into the HindIII site of pKM005. Meanwhile, transcriptional fusions in repB (pSW855) and repA (pSW856) were constructed by inserting the PvuIIBsrFI and PvuIIXmnI fragments into the XbaI site in pKM005. The -35 sequence of the RNAI promoter in pSW851 and pSW852 was changed to 5'-CTCAGA-3' by a PCR mutagenesis method of Ho et al. (1989) to generate pSW851M and pSW852M, respectively. The initiation codon of repB in pSW851M and pSW852M was changed to CCG by the same method to generate pSW853 and pSW854, respectively. The initiation codon of repA in pSW852M was also mutated to CCG to generate pSW857. Regions Z12Z17 in pSW852M were substituted with a 5'-CTGCAG-3' sequence to generate pSW861pSW866, respectively. Sequences in regions a, b, c and d in stemloop VI in pSW852M were mutated to GCA (pSW871), CCC (pSW872), TGC (pSW873) and GGG (pSW874), respectively. The region e sequence in pSW873 was mutated to GCA to generate pSW875. The region f sequence in pSW874 was mutated to CCC to generate pSW876. Plasmid pSW877 was generated by mutating the region a sequence in pSW875 to GCA; the region b sequence in pSW876 was mutated to CCC to generate pSW878. These fusion plasmids were transformed into E. coli CSH50 and ß-galactosidase activity was assayed as described by Miller (1972)
. The assay was performed in duplicate and each experiment was repeated at least three times.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
Analysis of the sequence in stemloop III
The target region in RNAII for RNAI is probably located between +58 and +130 of RNAII (see Discussion; Fig. 6a). This region potentially contains two stemloop structures, III and IV (Fig. 6a
). To elucidate the importance of stemloop III, we changed the sequence of every 6 nt from +67 (Fig. 6b
) (map location 136, Fig. 2
) to 5'-CTGCAG-3' (a PstI site) in pSW852M by site-directed mutagenesis, subsequently generating six consecutive mutants, Z12Z17 (Fig. 6b
). The Z12 mutation, which contains a sequence change between +67 and +72 (pSW861), did not influence the expression of ß-galactosidase. The enzyme activity exhibited by the plasmid (2084 Miller units) was roughly equal to that displayed by the parent plasmid, pSW852M (Table 3
). However, the Z13 mutation (pSW862), which contains a sequence change from +73 (Fig. 6b
), reduced ß-galactosidase expression by 58% to 872 Miller units (Table 3
). Furthermore, the Z14 mutation (pSW863), which contains a sequence change from +79 (Fig. 6b
), reduced ß-galactosidase expression by 65% to 711 Miller units (Table 3
). Moreover, the Z15 mutation (pSW864), which contains a sequence change from +85 (Fig. 6b
), reduced the expression even further, decreasing activity by 83% to 364 Miller units (Table 3
). Finally, the Z16 mutation reduced the ß-galactosidase expression by 58%, while the Z17 mutation decreased it by 76% to 496 Miller units (Table 3
). These results suggest that the structure of stemloop III and the nucleotide sequence in loop III (Fig. 6a
) are essential for repA expression. Similar mutations were also generated in stemloop IV (Fig. 6a
), but reduced the expression of ß-galactosidase less than the mutations in stemloop III (data not shown), indicating that stemloop IV is less important than stemloop III in regulating the translation of repA.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Translation of repA and repB is coupled
Translation of repA and repB is likely to be coupled in pSW800 since mutation of the initiation codon of repB (pSW853 and pSW854) prevents the translation of repA (Table 2). Translation coupling is also important for expressing a replication initiation protein from the plasmids in the IncFII (Blomberg et al., 1992
), IncI
, IncL/M and IncB groups (Asano & Mizobuchi, 1998
; Athanasopoulos et al., 1995
; Praszkier et al., 1992
). In these plasmids, and in pSW800, repB (repY) typically overlaps with repA (repZ). This unusual organization seems crucial for the translation of Rep, since mutation of the termination codon or generation of a nonsense mutation in repB in pUM604, pUM720 and repY in ColIb-P9 significantly reduces the expression of the Rep protein (Asano et al., 1999
; Athanasopoulos et al., 1995
; Siemering et al., 1993
). These studies suggest that, in translation coupling, ribosome translation of the upstream gene has to terminate at an exact location so that the ribosome can translate the downstream gene directly without leaving the mRNA (Athanasopoulos et al., 1995
). Furthermore, the RBS of repA or repZ of pMU604, pMU720, ColIb-P9 and pSW800 is typically hidden in a stemloop structure (Fig. 6a
; Asano & Mizobuchi, 2000
; Athanasopoulos et al., 1999
; Praszkier et al., 1992
). The RBS becomes available for ribosome binding, allowing the translation of Rep to occur, if the stemloop structure is destroyed by mutagenesis (Table 2
; pSW873 and pSW874). However, translation coupling between repB and repA of pMU604 and pMU720 also depends on the formation of an RNA pseudoknot structure between the sequence in the stemloop structure and a sequence located upstream (Athanasopoulos et al., 1995
; Praszkier et al., 1992
). In the case of pUM604, a lack of repA translation when an RNA pseudoknot structure is absent is partly attributed to the poor RBS sequence of the gene (Athanasopoulos et al., 1995
). Because repA of pSW800 also contains a short ShineDalgarno sequence (Fig. 2
; 5'-CGGA-3'), this may explain why pSW871 and pSW872 express ß-galactosidase at a background level (Table 3
) and formation of an RNA pseudoknot structure is required for the translation of repA (Table 3
; pSW877 and pSW878).
Expression of repA and repB is regulated post-transcriptionally
Translational fusions (pSW851 and pSW852) revealed that repA and repB are translated at a low level (Table 2), which can probably be attributed to the transcription of RNAI, since a -35 mutation in the RNAI promoter significantly raises the expression, and presence of pSW897, which transcribes RNAI, can again repress the expression from pSW8521M and pSW852M (Table 2
). Furthermore, transcriptional fusions generated in repA and repB (pSW855 and pSW856) also revealed that repA and repB are regulated post-transcriptionally at the translational level. According to our analysis, transcription of RNAI begins from map location 199 (Fig. 5a
), which is only 4 nt upstream from the RBS of repB (Fig. 2
). Although RNAI does not directly form a duplex with the region containing the RBS of repB, the duplex region in RNAII is probably close enough to impede the binding of ribosome to the repB RBS, thus inhibiting the translation of repB, and ultimately repA. This may indeed be the case, since the RBS of repB and the transcription start site of RNAI in pMU604 are also separated by 4 nt (Athanasopoulos et al., 1995
).
RNase III is not required for control of repA expression
Since interaction between antisense and target RNAs will create double-stranded RNA structures, it was conceivable that cleavage by the double-stranded RNA-specific RNase III could affect control of repA expression. To test whether RNase III influences the replication, we transformed pSW830 into E. coli HT115 (Franch et al., 1999 ), an RNase III-deficient strain, revealing that the plasmid replicates normally and is stably maintained in the strain, and thus suggesting that RNase III does not influence pSW800 replication.
The sequence in loop III is essential for repA translation
Transcription of RNAI from pSW800 is likely to terminate at the 3' region of stemloop III, since the stemloop structure resembles a -independent transcription stop signal (Fig. 6a
). If transcription of RNAI terminates here, RNAI should be 73 nt long, consistent with the estimated size from Northern blot analysis (Fig. 4a
). The target area for RNAI in RNAII of pSW800 thus contains stemloops III and IV (Fig. 6b
). Linker scanning analysis of stemloop III reveals that the Z12 mutation in pSW852M, which does not significantly influence the stemloop structure, does not influence expression (Fig. 6b
and Table 3
). However, the mutations that disrupt the stemloop structure in pSW852M, including mutations Z13, Z16 and Z17, significantly reduced ß-galactosidase expression (Fig. 6b
and Table 3
). Varying the sequence in loop III reduced the expression even further, to just 17% of the level expressed by pSW852M (Table 3
). These experimental results clearly demonstrate not only the critical role the stemloop structure and the loop sequence play, but also that the interaction between loop III and a downstream region affecting the translation of repA, probably stemloop VI, are crucial.
Interaction between stemloops III and VI is important for the expression of repA
In the plasmids of IncI, IncL/M and IncB groups, formation of an RNA pseudoknot structure is required before Rep proteins can be expressed (Asano & Mizobuchi, 1998b
; Athanasopoulos et al., 1995
; Siemering et al., 1993
). These RNA pseudoknot structures are frequently formed between an upstream loop and the stem of the stemloop structure containing the RBS of repA (Asano & Mizobuchi, 1998b
), and these two regions are normally separated by approximately 100 nt (Asano & Mizobuchi, 1998
; Athanasopoulos et al., 1995
; Praszkier et al., 1992
). However, the length of the sequence involving pseudoknot formation in these plasmids generally varies. For example, in ColIb-P9, only five of the seven complementary bases are important for pseudoknot formation (Asano & Mizobuchi, 1998b
). Meanwhile, pseudoknot formation in pUM604 requires six of the eight complementary bases (Athanasopoulos et al., 1999
). If pseudoknot formation is also required for repA translation in pSW800, a sequence that complements loop III and stem VI may appear. Our results reveal that mutation in region c (Fig. 6a
, Table 3
), which partially disrupts the structure of stemloop VI and destroys the pairing between regions a and c, does not influence the expression of ß-galactosidase from pSW852M (Table 3
). Meanwhile, mutation of the sequence of region e (pSW875), to restore the structure of stemloop VI in pSW873, reduced the expression of ß-galactosidase by 89%, indicating that the structure of stem VI prevents the translation of repA. Changing the region a sequence (pSW875) to restore the pairing between regions a and c in pSW875 again increases the expression of ß-galactosidase (Table 3
). This behaviour demonstrates that the two regions must interact, probably forming a pseudoknot structure, for repA to be expressed. Since a mutation in region c (pSW873) causes repA translation, this mutation may destabilize the structure of stemloop VI to a degree that a pseudoknot structure is no longer necessary for repA translation. Similar to the mutation in region c, a mutation in region d does not significantly influence expression. However, in contrast to the sequence changes in regions c and e (pSW875), mutations in regions of d and f (pSW876) reduced the expression of ß-galactosidase only by 58% (Table 3
). It is unclear why a mutation in regions d and f does not decrease the expression as much as a mutation in regions c and e. It is likely that the structure in regions d and f may also be influenced by the ribosome that occupies the region where repB terminates, explaining why changing the sequence alone does not completely abolish the translation. We also found that restoring the base pairing between regions b and d (pSW878) in pSW876 still increases ß-galactosidase expression to 86% of that exhibited by pSW874 (Table 3
), showing that interaction between regions b and d can enhance the translation of repA.
Results in this study have demonstrated that a mechanism similar to that regulating plasmid replication in the IncB, IncI and IncL/M groups also regulates pSW800 replication. Translational coupling between repA and repB and interactions between loop III and stem VI are required for repA of pSW800 to be expressed.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asano, K. & Mizobuchi, K. (1998a). Copy number control of Inc plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site. EMBO J 17, 5201-5213.
Asano, K. & Mizobuchi, K. (1998b). An RNA pseudoknot as the molecular switch for translation of the repZ gene encoding the replication initiator of IncI plasmid ColIb-P9. J Biol Chem 273, 11815-11825.
Asano, K. & Mizobuchi, K. (2000). Structural analysis of late intermediate complex formed between plasmid ColIb-P9 Inc RNA and its target RNA. How does a single antisense RNA repress translation of two genes at different rates? J Biol Chem 275, 1269-1274.
Asano, K., Niimi, T., Yokoyama, S. & Mizobuchi, K. (1998). Structural basis for binding of the plasmid ColIb-P9 antisense Inc RNA to its target RNA with the 5'-rUUGGCG-3' motif in the loop sequence. J Biol Chem 273, 11826-11838.
Asano, K., Hama, C., Inoue, S., Moriwaki, H. & Mizobuchi, K. (1999). The plasmid ColIb-P9 antisense Inc RNA controls expression of the RepZ replication protein and its positive regulator repY with different mechanisms. J Biol Chem 274, 17924-17933.
Athanasopoulos, V., Praszkier, J. & Pittard, A. J. (1995). The replication of an IncL/M plasmid is subject to antisense control. J Bacteriol 177, 4730-4741.[Abstract]
Athanasopoulos, V., Praszkier, J. & Pittard, A. J. (1999). Analysis of elements involved in pseudoknot-dependent expression and regulation of the repA gene of an IncL/M plasmid. J Bacteriol 181, 1811-1819.
Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1523.[Abstract]
Blomberg, P., Nördstrom, K. & Wagner, E. G. H. (1992). Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation. EMBO J 11, 2675-2683.[Abstract]
Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L. & Boyer, H. W. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95-113.[Medline]
Boyer, H. W. & Roulland-Dessoix, D. (1969). A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41, 459-472.[Medline]
Burian, J., Stuchlik, S. & Kay, W. W. (1999). Replication control of a small cryptic plasmid of Escherichia coli. J Mol Biol 294, 49-65.[Medline]
Chang, P. J., Chang, Y. S. & Liu, S. T. (1998). Role of Rta in the translation of bicistronic BZLF1 of EpsteinBarr virus. J Virol 72, 5128-5136.
Coplin, D. L., Rowan, R. G., Chisholm, D. A. & Whitmoyer, R. E. (1981). Characterization of plasmids in Erwinia stewartii. Appl Environ Microbiol 42, 599-604.[Medline]
Coplin, D. L., Frederick, R. D. & McCammon, S. L. (1985). Characterization of a conjugative plasmid from Erwinia stewartii. J Gen Microbiol 131, 2985-2991.
Franch, T., Thisted, T. & Gerdes, K. (1999). Ribonuclease III processing of coaxially stacked RNA helices. J Biol Chem 274, 26572-26578.
Frederick, R. D. & Coplin, D. L. (1986). Transformation of Escherichia coli by plasmid DNA from Erwinia stewartii. Mol Plant Pathol 76, 1353-1356.
Fu, J. F., Chang, H. C., Chen, Y. M., Chang, Y. S. & Liu, S. T. (1995). Sequence analysis of an Erwinia stewartii plasmid, pSW100. Plasmid 34, 75-84.[Medline]
Fu, J. F., Chang, H. C., Chen, Y. M., Chang, Y. S. & Liu, S. T. (1996). Characterization of the replicon of plasmid pSW500 of Erwinia stewartii. Mol Gen Genet 250, 699-704.[Medline]
Fu, J. F., Ying, S. W. & Liu, S. T. (1997). Cloning and characterization of the ori region of pSW1200 of Erwinia stewartii: similarity with plasmid P1. Plasmid 38, 141-147.[Medline]
Fu, J. F., Hu, J. M., Chang, Y. S. & Liu, S. T. (1998). Isolation and characterization of plasmid pSW200 from Erwinia stewartii. Plasmid 40, 100-112.[Medline]
Hama, C., Takizawa, T., Moriwaki, H., Urasaki, Y. & Mizobuchi, K. (1990). Organization of the replication control region of plasmid ColIb-P9. J Bacteriol 172, 1983-1991.[Medline]
Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P. & Kushner, S. R. (1989). New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol 171, 4617-4622.[Medline]
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59.[Medline]
Kado, C. I. & Liu, S. T. (1981). Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145, 1365-1373.[Medline]
Lin, T. P., Chen, C. L., Chang, L. K., Tschen, J. S. & Liu, S. T. (1999). Functional and transcriptional analyses of a fengycin synthetase gene, fenC, from Bacillus subtilis. J Bacteriol 181, 5060-5067.
Masui, Y., Coleman, J. & Inouye, M. (1983). Multipurpose Expression Cloning Vehicles in Escherichia coli. New York: Academic Press.
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Oka, A., Sugisaki, H. & Takanami, M. (1981). Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147, 217-226.[Medline]
Praszkier, J., Wilson, I. W. & Pittard, A. J. (1992). Mutations affecting translational coupling between the rep genes of an IncB miniplasmid. J Bacteriol 174, 2376-2383.[Abstract]
Saadi, S., Maas, W. K., Hill, D. F. & Bergquist, P. L. (1987). Nucleotide sequence analysis of RepFIC, a basic replicon present in IncFI plasmids P307 and F, and its relation to the RepA replicon of IncFII plasmids. J Bacteriol 169, 1836-1846.[Medline]
Sambrook, J. & Russell, D. (2000). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]
Siemering, K. R., Praszkier, J. & Pittard, A. J. (1993). Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication. J Bacteriol 175, 2895-2906.[Abstract]
del Solar, G., Giraldo, R., Ruiz-Echevarria, M. J., Espinosa, M. & Diaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev 62, 434-464.
Wu, R. P., Womble, D. D. & Rownd, R. H. (1985). Incompatibility mutants of IncFII plasmid NR1 and their effect on replication control. J Bacteriol 163, 973-982.[Medline]
Received 6 March 2001;
revised 1 June 2001;
accepted 12 June 2001.