Advertisement

Molecular and General Genetics MGG

, Volume 196, Issue 3, pp 482–487 | Cite as

Cloning and expression of the activity and immunity genes of colicins B and M on ColBM plasmids

  • Tobias Ölschläger
  • Edgar Schramm
  • Volkmar Braun
Article

Summary

The activity and immunity genes for colicins B and M on two conjugative ColBM plasmids, pCl139 and pF166, were cloned into pBR322 and pACYC184, respectively. The colicin regions on both recombinant plasmids were identical with regard to restriction endonuclease sites and the arrangement of the genes. They map close to each other in the order cmi cma cbi cba, where cmi denotes the locus that determines immunity to colicin M, cma the structural gene for colicin M, cbi immunity to colicin B, and cba the structural gene for colicin B. With the use of mutants obtained by insertion of the transposon Tn5, and by translation in minicells, the transcriptional polarity of cma and cba was found to be from right to left. cma and cba code for polypeptides with molecular weights of 27,000 and 58,000, respectively. No evidence of biosynthetic precursors was obtained.

Keywords

Molecular Weight Polypeptide Restriction Endonuclease Structural Gene Recombinant Plasmid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523Google Scholar
  2. Bolivar F (1978) Derivatives of plasmid pBR322 in genetic engineering. In: Boyer HW, Nicosia S (eds) Molecular cloning vectors. Elsevier/North-Holland Biomedical Press. Amsterdam, pp 59–63Google Scholar
  3. Braun V, Maas E (1984) Colicin B consists of a single polypeptide chain. FEMS Microbiol Lett 12:373–380Google Scholar
  4. Braun V, Schaller K, Wabl MR (1974) Isolation, characterization, and action of colicin M. Antimicrob Agents Chemother 5:520–533Google Scholar
  5. Braun V, Frenz S, Hantke K, Schaller K (1980) Penetration of colicin M into cells of Escherichia coli. J Bacteriol 142:162–168Google Scholar
  6. Braun V, Gross R, Köster W, Zimmermann L (1983) Plasmid and chromosomal mutants in the iron(III)-aerobactin transport system of Escherichia coli. Use of streptonigrin for selection. Mol Gen Genet 192:131–139Google Scholar
  7. Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 138:1141–1156Google Scholar
  8. Chou J, Casadaban MJ, Lemaux PG, Cohen SN (1979) Identification and characterization of a self-regulated repressor and translocation of the Tn3 element. proc Natl Acad Sci USA 76:4020–4024Google Scholar
  9. Dougan G, Saul M, Twigg A, Gill R, Sherratt D (1979) Polypeptides expressed in Escherichia coli K-12 minicells by transposition elements Tn1 and Tn3. J Bacteriol 138:48–54Google Scholar
  10. Dreher R, Höltje JV, Braun V (1983) Functional domains of colicin M. In: Hakenbeck R, Höltjje JV, Labischinski H (eds) The target of penicillin. de Gruyter, Berlin, pp 643–647Google Scholar
  11. Fecker L, Braun V (1983) Cloning and expression of the fhu genes involved in iron(III)-hydroxamate uptake of Escherichia coli. J Bacteriol 156:1301–1314Google Scholar
  12. Frédericq P, Smarda P (1970) Complexité du facteur colicinogene B. Ann Inst Pasteur 118:767–774Google Scholar
  13. Gutermann SK (1973) Colicin B: mode of action and inhibition by enterochelin. J Bacteriol 114:1217–1224Google Scholar
  14. Hakkaart MJJ, Veltkamp E, Nijkamp HJJ (1981) Protein H encoded by plasmid CloDF13 involved in lysis of the bacterial host. II. Functions and regulation of synthesis of the gene H product. Mol Gen Genet 183:326–332Google Scholar
  15. Harwood CR, Williams DM, Lovett PS (1983) Nucleotide sequence of a Bacillus pumilus gene specifying chloramphenicol acetyltransferase. Gene 24:163–169Google Scholar
  16. Hashimoto E, Horogome T, Kanabayashi M, Yoshida K, Sugano H (1983) An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 129:192–199Google Scholar
  17. Jakes KS, Zinder ND (1984) Plasmid ColE3 specifies a lysis protein. J Bacteriol 157:582–590Google Scholar
  18. Kado CJ, Liu ST (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365–1373Google Scholar
  19. Matsuhashi Y, Sawa T, Takenchi T, Umezawa H (1976) Purification of aminoglycoside 3′-phosphotransferase II. J Antibiotics 29:204–207Google Scholar
  20. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  21. Oudega B, Stegehuis F, van Tiel-Menkveld GJ, de Graaf FK (1982) Protein H encoded by plasmid CloDF13 is involved in excretion of cloacin DF13. J Bacteriol 150:1115–1121Google Scholar
  22. Pugsley AP, Reeves P (1977) Comparison of Colicins B-K260 and D-CA23: Purification and characterization of the colicins and examination of colicin immunity in the producing strains. Anti-microb Agents Chemoter 22:961–968Google Scholar
  23. Pugsley AP (1983) Obligatory coupling of colicin release and lysis in mitomycin-treated Col+ Escherichia coli. J Gen Microbiol 129:1921–1928Google Scholar
  24. Rothstein SJ, Joergensen RA, Postle K, Reznikoff WS (1980) The inverted repeats of Tn5 are functionally different. Cell 19:795–805Google Scholar
  25. Sasarman A, Massie B, Zollinger M, Gagne-Tellier H, Shareck F, Garzon S, Morrisset R (1980) Naturally occurring R. ColBM plasmids belonging to the IncFIII incompatibility group. J Gen Microbiol 119:475–483Google Scholar
  26. Schaller K, Dreher R, Braun V (1981a) Structural and functional properties of colicin M. J Bacteriol 146:54–63Google Scholar
  27. Schaller K, Krauel A, Braun V (1981b) Temperature-sensitive, colicin M-tolerant mutant of Escherichia coli. J Bacteriol 147:135–139Google Scholar
  28. Schaller K, Höltje JV, Braun V (1982) Colicin M is an inhibitor of murein biosynthesis. J Bacteriol 152:994–1000Google Scholar
  29. Suit JL, Fan MLJ, Sabik JF, Labarre R, Luria SE (1984) Alternative forms of lethality in mitomycin C-induced bacteria carrying ColE1 plasmids. Proc Natl Acad Sci USA 80:579–583Google Scholar
  30. Sutcliffe JG (1978) Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–90Google Scholar
  31. Timmis KN, Cabello F, Cohen SN (1978) Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6. Mol Gen Genet 162:121–137Google Scholar
  32. Zimmermann R, Henning B, Neupert W (1981) Different transport pathways of individual precursor proteins in mitochondria. Eur J Biochem 116:455–460Google Scholar

Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • Tobias Ölschläger
    • 1
  • Edgar Schramm
    • 1
  • Volkmar Braun
    • 1
  1. 1.Mikrobiologie IIUniversität TübingenTubingenFederal Republic of Germany

Personalised recommendations