Summary
Derivatives of the conjugative streptococcal plasmid pIP501 replicate stably in Bacillus subtilis. The region essential for replication of plP501 has been narrowed down to a 2.2 kb DNA segment, the sequence of which has been determined. This region comprises two genes, copR and repR, proposed to be involved in copy control and replication. By in vitro and in vivo transcriptional analysis we characterized three active promoters, pII pII and pIII within this region. A putative fourth promoter (PTV) was neither active in vitro nor in vivo. We showed that copR is transcribed from promoter pI while the repR, gene is transcribed from promoter pII located just downstream of copR The pII transcript encompasses a 329 nucleotide (nt) long leader sequence. A counter transcript that was complementary to a major part of this leader was found to originate from a third promoter pIII The secondary structure of the counter transcript revealed several stem-loop regions. A regulatory function for this antisense RNA in the control of repR, expression is proposed. Comparative analysis of the replication regions of pAMβ 1 and pSM19035 suggested a similar organization of transcriptional units, suggesting that an antisense RNA is produced by these plasmids also.
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References
Alonso JC (1989) DNA replication of plasmids from Gram-positive bacteria in Bacillus subtilis. Plasmid pUB 110 as a model system. Microbiologia (SEM) 5:5–12
Barthelemy I, Salas M, Mellado RP (1987) In vivo transcription of bacteriophage φ29 DNA-transcription initiation sites. J Virol 60:874–879
Behnke D, Ferretti JJ (1980) Physical mapping of plasmid pDB101: a potential vector plasmid for molecular cloning in streptococci. Plasmid 4:130–138
Behnke D, Gilmore MS (1981) Location of antibiotic resistance determinants, copy control and replication functions on the double-selective streptococcal cloning vector pGB301. Mol Gen Genet 184:115–120
Behnke D, Klaus S (1983) Double or triple sets of replication functions as inverted and direct repeats on in vitro reconstructed streptococcal MLS resistance plasmids. Z Allg Mikrobiol 23:539–547
Behnke D, Malke H, Hartmann M, Walter F (1979) Post-transcriptional rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid. Plasmid 2:605–616
Behnke D, Tomich PK, Clewell DB (1980) Electron microscopic mapping of deletions on a streptococcal plasmid carrying extraordinarily long inverted repeats. Plasmid 4:139–142
Behnke D, Gilmore M, Ferretti JJ (1981) Plasmid pGB301, a new multiple resistance streptococcal vehicle and its use in cloning of a gentamycin/kanamycin resistance determinant. Mol Gen Genet 182:414–421
Brand S, Nowak A, Behnke D, Alonso JC (1989) Revision of the nucleotide sequence of the Streptococcus pyogenes plasmid pSM19035 reps gene. Nucleic Acids Res 17:10110
Brand S, Behnke D, Alonso JC (1990) Molecular analysis of the replication region of the conjugative plasmid pIP501 of Streptococcus agalactiae in Bacillus subtilis. Comparative analysis to plasmids pAMβ1 and pSM19035. Nucleic Acids Res 18:4783–4790
Brantl S, Behnke D (1992) Copy number control of the streptococcal plasmid plP501 occurs at three levels. Nucleic Acids Res 20:395–400
Bron S (1990) Plasmids. In: Harwood CR, Cutting SM (eds) Molecular biology methods for Bacillus. John Wiley & Sons, pp 75–138.
Bron S, Luxen E (1985) Segregational instability of pUB110 derived recombinant plasmids in Bacillus subtilis. Plasmid 14:235–244
Bruand C, Ehrlich SD, Janniŕe L (1991) Unidirectional theta replication of the structurally stable Enterococcus faecalis plasmid pAMβ1. EMBO J 10:2171–2177
Clewell DB, Yagi Y, Dunny GM, Schultz SK (1974) Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: Identification of a plasmid determining erythromycin resistance. J Bacteriol 117:283–289
Ehrlich SD, Noirot P, Petit MA, Jannière L, Michel B, te Riele H (1986) Structural instability of Bacillus subtilis plasmids. In: Setlow JK, Hollaender A (eds) Genetic engineering, vol. 8. Plenum Publishing, New York, pp 71–83
Gruss A, Ehrlich SD (1989) The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev 53:231–241
Horodniceanu T, Bouanchaud DH, Bieth G, Chabbert YA (1976) R-plasmids in Streptococcus agalactiae (group B). Antimicrob Agents Chemother 10:795–801
Jannière L, Bruand C, Ehrlich SD (1990) Structurally stable Bacillus subtilis cloning vectors. Gene 87: 53–61
Kawamura F, Doi RH (1984) Construction of Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol 160:442–444
Masai H, Arai K-I (1988) RI plasmid replication in vitro. repA- and dnaA-dependent initiation at oriR. In: Robb EM, Summers WC (eds) DNA replication and mutagenesis. American Society for Microbiology, Washington DC, pp 113–121
Maxam AM, Gilbert V (1977) A new method for sequencing DNA. Proc Natl Acad Sci USA 74:560–564
Mellado RP, Barthelemy I, Salas M (1986) In vitro transcription of bacteriophage φ29 DNA. Correlation between in vitro and in vivo promoters. Nucleic Acids Res 14:4731–4741
Novick RP (1987) Plasmid incompatibility. Microbiol Rev 51:381–395
Novick RP, Adler GK, Projan SJ, Carleton S, Highlander S, Gruss A, Khan SA, Iordanescu S (1984) Control of pT181 replication. I. The pT181 copy control function acts by inhibiting the synthesis of replication protein. EMBO J 3:2399–2405
Persson C, Wagner EGH, Nordström K (1990a) Control of replication of plasmid Rl: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT. EMBO J 9:3767–3775
Persson C, Wagner EGH, Nordström K (1990b) Control of replication of plasmid Rl: formation of an initial transient complex is rate-limiting for antisense RNA-target RNA pairing. EMBO J 9:3777–3785
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edition. Cold Spring Harbor Laboratory Press, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sogo JM, Inciarte MR, Corral J, Vinuela E, Salas M (1979) RNA polymerase binding sites and transcription map of the DNA of Bacillus subtilis phage φ29. J Mol Biol 127:411–436
Sorokin A, Khazak V (1989) Structure of pSM19035 replication region and MLS resistance gene. In: Butler LO, Harwood C, Moseley BEB (eds) Genetic transformation and expression. Intercept, Andover, UK, pp 269–281
Stougaard P, Molin S, Nordström (1981) RNAs involved in copy number control and incompatibility of plasmid R1. Proc Natl Acad Sci USA 78:6008–6012
Swinfield TJ, Oultram JD, Thompson DE, Brehm JK, Minton NP (1990) Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAMβ1. Gene 87:79–90
Zuker M, Stiegler P (1981) Description of the method used in RNAFOLD. Nucleic Acids Res 9:133–148
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Brantl, S., Nuez, B. & Behnke, D. In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501. Molec. Gen. Genet. 234, 105–112 (1992). https://doi.org/10.1007/BF00272351
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DOI: https://doi.org/10.1007/BF00272351