Summary
At least ten polypeptides larger than 6 kilodaltons (K) are produced in minicells from the miniplasmid pSM1 in vivo. pSM1 (5804 bp) is a small derivative of the drug resistance plasmid R100 (ca. 90 kb) and carries the R100 essential replication region as well as some non-essential functions. Cloned restriction fragments of pSM1 and plasmids with deletions within pSM1 sequences were used to assign eight of the ten oberserved polypeptides to specific coding regions of pSM1. Two of these polypeptides were identified as RepA1 and RepA2, proteins encoded by the essential replication region of pSM1/R100. The nucleotide sequence consisting of 885 bp outside the essential replication region is presented here. This sequence contains an open reading frame,orf4, for a protein 22.9 K in size, and one of the pSM1-encoded polypeptides was identified as theorf4 gene product. Five additional polypeptides were shown to be the products of other open reading frames mapping outside the essential replication region. Specific functions have been assigned to four of these polypeptides and tentatively to the fifth.
Similar content being viewed by others
Abbreviations
- K :
-
kilodaltons
- kb :
-
kilobasepairs
- bp :
-
basepairs
- a.a. :
-
amino acid
References
Andres I, Slocombe PM, Cabello F, Timmis JK, Lurz R, Burkhardt JH, Timmis KN (1979) Plasmid replication functions. II Cloning analysis of therepA replication region of antibiotic resistance plasmid R6-5. Mol Gen Genet 168: 1–25
Armstrong, K, Bauer W (1980) Preferential site-dependent cleavage by restriction endonucleasePstl. Nuc Acids Res 10:993–1007
Armstrong KA, Acosta R, Ledner E, Machida Y, Pancotto M, McCormick M, Ohtsubo H, Ohtsubo E (1984) A 37×103 molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1. J Mol Biol 175:331–347
Armstrong K, Rosen J, Ryder T, Ohtsubo E, Ohtsubo H (1981) Structure and function of the replication origin region of the resistance factors R100 and R1. In: Molecular biology, pathogenicity, and ecology of bacterial plasmids, Levy, SB, Clowes RC, Koenig EL (eds) Plenum Publishing Co. LTD, New York, pp 279–289
Bonner WM, Laskey RA, (1974) A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83–88
Brawner ME, Jaskunas SR (1982) Identification of polypeptides encoded by the replication region of resistance factor R100. J Mol Biol 159: 35–55
Burger KJ, Steinbauer J, Rollich G, Kollek R, Goebel W (1981) Copy number control and incompatibility of plasmid R1: Identification of a protein that seems to be involved in both processes. Mol Gen Genet 182: 44–52
Cheah K-C, Ray A, Skurray R (1984) Cloning and molecular analysis of thefinO region from the antibiotic-resistance plasmid R6-5. Plasmid 12: 222–226
Cleveland DW, Fisher SG, Kirschner MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252: 1102–1106
Danbara H, Brady G, Timmis JK, Timmis KN (1981) Regulation of DNA replication: “Target” determinant of the replication control elements of plasmid R6-5 lies within a control element gene. Proc Natl Acad Sci USA 78: 4699–4703
Dempsey WB, McIntire SA (1979) Lambda transducing phages derived from afinO − R100-lambda cointegrate plasmid. Proteins encoded by the R100 replication/incompatibility region and the antibiotic resistance determinant. Mol Gen Genet 176: 319–334
Dempsey WB, McIntire SA (1983) ThefinO gene of antibiotic resistance plasmid R100. Mol Gen Genet 190: 444–451
Dougan G, Sherratt D (1977) The transposon Tn1 as a probe for studying ColE1 structure and function. Mol Gen Genet 151: 151–160
Dougan G, Saul M, Twigg A, Gill R, Sherratt D (1979) Polypeptides expressed inEscherichia coli K-12 minicells by transposition elements Tn1 and Tn3. J Bacteriol 138: 48–54
Duffaud GD, Lehnhardt FK, March PE, Inouye M (1985) Structure and function of the signal polypeptide. In: Current topics in membranes and transport, Coo JS (ed) Academic Press, New York
Kahn M, Kolter R, Thomas C, Figurski D, Meyer R, Remaut E, Helinski DR (1979) Plasmid cloning vehicles derived from plasmids ColE1, F, R6K and RK2. Meth Enzymol 68: 268–280
Katz L, Kingsbury DT, Helinski DR (1973) Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol 114: 577–591
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685
Light J, Molin S (1983) Post transcriptional control of expression of therepA gene of plasmid R1 mediated by a small RNA molecule. EMBO J 12: 93–98
Liu C-PL, Churchward G, Caro L (1983) Therep A2 gene of the plasmid R100 encodes a repressor of plasmid replication. Plasmid 10: 148–155
Lovett MA, Helinski DR (1976) Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'Km plasmid. J Bacteriol 127: 982–987
Machida Y, Machida C, Ohtsubo E (1984) Insertion element IS1 encodes two structural genes required for its transposition. J Mol Biol 177: 229–246
Madel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53: 159–162
Maxam A, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci USA 74: 560–564
Messing J (1983) New M13 vector for cloning. Meth Enzymol 101: 20–78
Mickel S, Bauer WR (1976) Isolation, by tetracycline selection, of small plasmids derived from R-factor R12 inEscherichia coli K12. J Bacteriol 127: 644–655
Mickel S, Ohtsubo E, Bauer W (1977) Heteroduplex mapping of small plasmids derived from R-factor R12: In vivo recombination occurs at IS1 insertion sequences. Gene 2: 193–210
Miki T, Easton AM, Rownd RH (1980) Cloning of replication, incompatibility, and stability functions of R plasmid NR1. J Bacteriol 141: 87–99
Molin, S., Stougaard P, Light J, Nordstrom M, Nordstrom K (1981) Isolation and characterization of new copy mutants of plasmid R1, and identification of a polypeptide involved in copy number control. Mol Gen Genet 181: 123–130
Ohtsubo H, Ohtsubo E (1978) Nucleotide sequence of an insertion element IS1. Proc Natl Acad Sci USA 75: 615–619
Ohtsubo H, Ohmori H, Ohtsubo E (1978) Nucleotide sequence analysis of the ampicillin resistance transposon Tn3: Implication for insertion and deletion. Cold Spring Harbor Symp Quant Biol 43: 1269–1277
Ohtsubo H, Nyman K, Doroszkiewicz W, Ohtsubo E (1981) Multiple copies of iso-insertion sequences of IS1 inShigella dysenteriae chromosome Nature 292: 640–643
Ohtsubo H, Vassino B, Ryder T, Ohtsubo E (1982) A simple method for shortening a plasmid genome using a system of plasmid cointegration mediated by a Tn3 mutant. Gene 20: 245–254
Ohtsubo E, Feingold J, Ohtsubo H, Mickel S, Bauer W (1977) Unidirectional replication of three small plasmids derived from R factor R12 inEscherichia coli. Plasmid 1: 8–18
Ohtsubo E, Rosenbloom M, Schrempf H, Goebel W, Rosen J (1978) Site-specific recombination involved in the generation of small plasmids. Mol Gen Genet 159: 131–141
Ohtsubo H, Ryder TB, Maeda Y, Armstrong KA, Ohtsubo E (1986) DNA replication of the resistance plasmid R100 and its control. In: Genetic systems. Advances in biophysics, vol. 21 (Special vol. 1986) Ozeki H, Osawa S, Uchida H, Shimura Y, Ishihama A (eds) Japan Scientific Societies, Tokyo/Springer, Berlin Heidelberg New York Tokyo, pp 115–133
Ohtsuka A (1981) Recovery of DNA fragments inserted by the “tailing” method: regeneration ofPstI restriction sites. Gene 13: 339–346
Oka A, Sugisaki H, Takanami M (1981) Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147: 217–226
Riise E, Stougaad P, Binzdslev B, Nordstrom K, Molin S (1982) Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1. J Bacteriol 151: 1136–1145
Rosen J, Ohtsubo H, Ohtsubo E (1979) The nucleotide sequence of the region surounding the replication origin of an R100 resistance factor derivative. Mol Gen Genet 171: 287–293
Rosen J, Ryder R, Inokuchi H, Ohtsubo H, Ohtsubo E (1980) Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis. Mol Gen Genet 179: 527–537
Rosen J, Ryder T, Ohtsubo H, Ohtsubo E (1981) Role of RNA transcripts in replication, incompatibility and copy number control in antibiotic resistance plasmid derivatives Nature 290: 794–797
Ryder T, Davison DB, Rosen JI, Ohtsubo E, Ohtsubo H (1982) Analysis of plasmid genome evolution based on nucleotidesequence comparison of two related plasmids ofEscherichia coli. Gene 17: 299–310
Ryder T, Rosen J, Armstrong K, Davison D, Ohtsubo E, Ohtsubo H (1981) Dissection of the replication region controlling incompatibility, copy number and initiation of DNA synthesis in the resistance plasmids R100 and R1. In: Ray DS (ed) Initiation of DNA replication, Academic Press, New York, pp 91–111
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467
Sharp PA, Cohen SN, Davidson N (1973) Electron microscope heteroduplex studies of sequence relations among plasmids ofEscherichia coli. II Structure of drug resistance (R) factors and F factors. J Mol Biol 25: 235–255
Stougaard P, Light J, Molin S (1982) Convergent transcription interferes with expression of the copy number control gene,copA, from plasmid R1, EMBO J 1: 323–328
Stougaard P, Molin S, Nordstrom (1981) RNA molecules involved in copy number control and incompatibility of plasmid R1. Proc Natl Acad Sci USA 78: 6008–6012
Sutcliffe JG (1979) Complete nucleotide sequence of theEscherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43: 77–90
Tanimoto K, Iino T, Ohtsubo H, Ohtsubo E (1985) Identification of a genetir of R100 functionally homologous to the F3 gene in the transfer inhibition of RP4. Mol Gen Genet 198: 356–357
Taylor DP, Cohen SN (1979) Structural and functional analysis of cloned DNA segments containing the replication and incompatibility regions of a miniplasmid derived from a copy number mutant of NR1. J Bacteriol 137: 92–104
Tegtmeyer P, Schwartz M, Collins JK, Rundell K (1975) Regulation of tumor antigen synthesis by simian virus 40 gene A. J Virology 16: 168–170
Timmis KN, Andres I, Achtman M (1978) Fertility repression of F-like conjugative plasmids: Physical mapping of the R6-5finO andfinP cistrons and identification of thefinO protein. Proc Natl Acad Sci USA 75: 5836–5840
Watanabe T, Nishida H, Ogata C Arai T, Sato S (1964) Episomemediated transfer of drug resistance in Enterobacteriaceae. VII two types of naturally-occurring R-factors. J Bacteriol 88: 716–726
Willetts N, Skurray R (1980) The conjugation system of F-like plasmids. Ann Rev Genet 14: 41–76
Zenilman M, Machida Y, Ohtsubo H, and Ohtsubo E (1983) Inactivation of the replication genes of a pSC101 derivative by IS1-mediated integration of the plasmid pSM1. J Gen Appl Microbiol 29: 223–232
Author information
Authors and Affiliations
Additional information
Communicated by N.D.F. Grindley
Rights and permissions
About this article
Cite this article
Armstrong, K.A., Ohtsubo, H., Bauer, W.R. et al. Characterization of the gene products produced in minicells by pSM1, a derivative of R100. Molec. Gen. Genet. 205, 56–65 (1986). https://doi.org/10.1007/BF02428032
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02428032