Abstract
The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 by region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR 1) showed some homology with other previously described SSOs of the SSOA type, as well as with the conversion signal of the Escherichia coli phage ΦX174. This IR was shown to have a partial, RNA polymerise-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerise-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arai K, Kornberg A (1981) Unique primed start of ϕX174 DNA replication and mobility of the primosome in a direction opposite chain synthesis. Proc Natl Acad Sci USA 78:69–73
Baas PD, Jansz HS (1988) Single-stranded DNA phage origins. Curr Topics Microbiol Immunol 136:31–70
Birch P, Khan SA (1992) Replication of single-stranded plasmid pT181 DNA in vitro. Proc Natl Acad Sci USA 89:290–294
Boe L, Gros M-F, te Riele H, Ehrlich SD, Gruss A (1989) Replication origins of single-stranded-DNA plasmid pUB110. J Bacteriol 171:3366–3372
Bron S (1990) Plasmids. In: Harwood CR, Cutting SM (eds) Molecular biological methods for Bacillus. John Wiley and Sons, Chichester, UK, pp 75–175
Bron S, Luxen E, Swart P (1988) Instability of recombinant pUB110 plasmids in Bacillus subtilis: plasmid-encoded stability function and effects of DNA inserts. Plasmid 19:231–241
Bron S, Meijer W, Holsappel S, Haima P (1991) Plasmid instability and molecular cloning in Bacillus subtilis. Res Microbiol 142:875–883
Burdett V (1980) Identification of tetracyclin resistant R-plasmids in Streptococcus agalactiae (group B). Antimicrob Agents Chemother 18:753–760
De Vos WM (1987) Gene cloning and expression in lactic streptococci. FEMS Microbiol Rev 46:281–295
Del Solar GH, Moscoso M, Espinosa M (1993) Rolling circlereplicating plasmids from Gram-positive and Gram-negative bacteria: a wall falls. Mol Microbiol 8:789–796
Del Solar GH, Puyet A, Espinosa M (1987) Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1. Nucleic Acids Res 15:5561–5580
Gruss AD, Ehrlich SD (1989) The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev 53:231–241
Gruss AD, Ross HF, Novick RP (1987) Functional analysis of a palindromic sequence required for normal replication of several staphylococcal plasmids. Proc Natl Acad Sci USA 84:2165–2169
Holo H, Nes IF (1989) High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55:3119–3123
Jannière L, Niaudet B, Pierre E, Ehrlich SD (1985) Stable gene amplification in the chromosome of Bacillus subtilis. Gene 40:47–55
Kiewiet R, Kok J, Seegers JFML, Venema B, Bron S (1993) The mode of replication is a major factor in segregational plasmid instability in Lactococcus lactis. Appl Environ Microbiol 59:358–364
Kok J, van der Vossen JMBM, Venema G (1984) Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Streptococcus lactis. Appl Environ Microbiol 48:726–731
Kreiswirth B, Lofdahl S, Betley MJ, O'Reilly M, Schlievert PM, Bergdoll MS, Novick RP (1983) The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305:709–712
Leenhouts KJ, Tolner B, Bron S, Kok J, Venema G, Seegers JFML (1991) Nucleotide sequence and characterization of the broadhost-range lactococcal plasmid pWVO1. Plasmid 26:55–66
Leer RJ, van Luijk N, Posno M, Pouwels PH (1992) Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014. Mol Gen Genet 234:265–274
Meijer WJJ, van der Lelie D, Venema G, Bron S (1995a) Effects of the generation of single-stranded DNA on the maintenance of plasmid pMV158 and derivatives in Lactococcus lactis. Plasmid 33:91–99
Meijer WJJ, van der Lelie D, Venema G, Bron S (1995b) Effects of the generation of single-stranded DNA on the maintenance of plasmid pMV158 and derivatives in different Bacillus subtilis strains. Plasmid 33:79–89
Murray RW, Koepsel RR, Khan SA (1989) Synthesis of singlestranded plasmid pT181 DNA in vitro. J Biol Chem 264:1051–1057
Nordström K, Austrin SJ (1989) Mechanisms that contribute to the stable segregation of plasmids. Annu Rev Genet 23:37–69
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Te Riele H, Michel B, Ehrlich SD (1986a) Are single-stranded circles intermediates in plasmid DNA replication? EMBO J 5:631–637
Te Riele H, Michel B, Ehrlich SD (1986b) Single-stranded plasmid DNA in Bacillus subtilis and Staphylococcus aureus. Proc Natl Acad Sci USA 83:2541–2545
Terzaghi BE, Sandine WE (1975) Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29:807–813
Xu F-F, Pearce LE, Yu P-L (1991) Genetic analysis of a lactococcal plasmid replicon. Mol Gen Genet 227:33–39
Author information
Authors and Affiliations
Additional information
Communicated by H. Böhme
Rights and permissions
About this article
Cite this article
Seegers, J.F.M.L., Meijer, W.J.J., Venema, G. et al. Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01. Molec. Gen. Genet. 249, 43–50 (1995). https://doi.org/10.1007/BF00290234
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00290234