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. 1988 Sep;170(9):4153–4160. doi: 10.1128/jb.170.9.4153-4160.1988

Effect of a mutation preventing lipid modification on localization of the pCloDF13-encoded bacteriocin release protein and on release of cloacin DF13.

J Luirink 1, S Hayashi 1, H C Wu 1, M M Kater 1, F K de Graaf 1, B Oudega 1
PMCID: PMC211422  PMID: 3045086

Abstract

The pCloDF13-encoded bacteriocin release protein (BRP; Mr 2,871) is essential for the translocation of cloacin DF13 across the cell envelope of producing Escherichia coli cells. Overproduction of this BRP provokes lysis (quasilysis) of cells. Construction and analysis of a hybrid BRP-beta-lactamase protein (BRP-Bla) demonstrated that the BRP contains a lipid modified cysteine residue at its amino terminus and is mainly located in the outer membrane. The significance of lipid modification for the localization and functioning of the BRP was investigated. Site-directed mutagenesis was used to substitute the cysteine residue for a glycine residue in the lipobox of the BRP and the BRP-Bla protein. The mutated BRP was unable to bring about the release of cloacin DF13 and could not provide the lysis (quasilysis) of host cells. However, the mutated BRP strongly inhibited the colony-forming ability of the cells, indicating that induction of the mutated protein still affected cell viability. In contrast to the wild-type BRP-Bla protein, the mutated BRP-Bla protein was mainly located in the cytoplasmic membrane, indicating that the mutation prevented the proper localization of the protein. The results indicated that lipid modification of the BRP is required for its localization and release of cloacin DF13, but not for its lethality to host cells.

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Selected References

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  1. Broome-Smith J. K., Spratt B. G. A vector for the construction of translational fusions to TEM beta-lactamase and the analysis of protein export signals and membrane protein topology. Gene. 1986;49(3):341–349. doi: 10.1016/0378-1119(86)90370-7. [DOI] [PubMed] [Google Scholar]
  2. Cavard D., Baty D., Howard S. P., Verheij H. M., Lazdunski C. Lipoprotein nature of the colicin A lysis protein: effect of amino acid substitutions at the site of modification and processing. J Bacteriol. 1987 May;169(5):2187–2194. doi: 10.1128/jb.169.5.2187-2194.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cavard D., Lloubès R., Morlon J., Chartier M., Lazdunski C. Lysis protein encoded by plasmid ColA-CA31. Gene sequence and export. Mol Gen Genet. 1985;199(1):95–100. doi: 10.1007/BF00327516. [DOI] [PubMed] [Google Scholar]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cole S. T., Saint-Joanis B., Pugsley A. P. Molecular characterisation of the colicin E2 operon and identification of its products. Mol Gen Genet. 1985;198(3):465–472. doi: 10.1007/BF00332940. [DOI] [PubMed] [Google Scholar]
  6. De Graaf F. K., Oudega B. Production and release of cloacin DF13 and related colicins. Curr Top Microbiol Immunol. 1986;125:183–205. doi: 10.1007/978-3-642-71251-7_11. [DOI] [PubMed] [Google Scholar]
  7. Hakkaart M. J., Veltkamp E., Nijkamp H. J. Protein H encoded by plasmid Clo DF13 involved in lysis of the bacterial host. I. Localisation of the gene and identification and subcellular localisation of the gene H product. Mol Gen Genet. 1981;183(2):318–325. doi: 10.1007/BF00270635. [DOI] [PubMed] [Google Scholar]
  8. Hayashi S., Chang S. Y., Chang S., Wu H. C. Modification and processing of Bacillus licheniformis prepenicillinase in Escherichia coli. Fate of mutant penicillinase lacking lipoprotein modification site. J Biol Chem. 1984 Aug 25;259(16):10448–10454. [PubMed] [Google Scholar]
  9. Hayashi S., Wu H. C. Biosynthesis of Bacillus licheniformis penicillinase in Escherichia coli and in Bacillus subtilis. J Bacteriol. 1983 Nov;156(2):773–777. doi: 10.1128/jb.156.2.773-777.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hussain M., Ichihara S., Mizushima S. Accumulation of glyceride-containing precursor of the outer membrane lipoprotein in the cytoplasmic membrane of Escherichia coli treated with globomycin. J Biol Chem. 1980 Apr 25;255(8):3707–3712. [PubMed] [Google Scholar]
  11. Inouye S., Franceschini T., Sato M., Itakura K., Inouye M. Prolipoprotein signal peptidase of Escherichia coli requires a cysteine residue at the cleavage site. EMBO J. 1983;2(1):87–91. doi: 10.1002/j.1460-2075.1983.tb01386.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. James R., Jarvis M., Barker D. F. Nucleotide sequence of the immunity and lysis region of the ColE9-J plasmid. J Gen Microbiol. 1987 Jun;133(6):1553–1562. doi: 10.1099/00221287-133-6-1553. [DOI] [PubMed] [Google Scholar]
  13. Kadonaga J. T., Gautier A. E., Straus D. R., Charles A. D., Edge M. D., Knowles J. R. The role of the beta-lactamase signal sequence in the secretion of proteins by Escherichia coli. J Biol Chem. 1984 Feb 25;259(4):2149–2154. [PubMed] [Google Scholar]
  14. Koshland D., Sauer R. T., Botstein D. Diverse effects of mutations in the signal sequence on the secretion of beta-lactamase in Salmonella typhimurium. Cell. 1982 Oct;30(3):903–914. doi: 10.1016/0092-8674(82)90295-1. [DOI] [PubMed] [Google Scholar]
  15. Kramer B., Kramer W., Fritz H. J. Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell. 1984 Oct;38(3):879–887. doi: 10.1016/0092-8674(84)90283-6. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Luirink J., Watanabe T., Wu H. C., Stegehuis F., de Graaf F. K., Oudega B. Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase. J Bacteriol. 1987 May;169(5):2245–2250. doi: 10.1128/jb.169.5.2245-2250.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Luirink J., de Graaf F. K., Oudega B. Uncoupling of synthesis and release of cloacin DF13 and its immunity protein by Escherichia coli. Mol Gen Genet. 1987 Jan;206(1):126–132. doi: 10.1007/BF00326547. [DOI] [PubMed] [Google Scholar]
  19. Luirink J., van der Sande C., Tommassen J., Veltkamp E., De Graaf F. K., Oudega B. Effects of divalent cations and of phospholipase A activity on excretion of cloacin DF13 and lysis of host cells. J Gen Microbiol. 1986 Mar;132(3):825–834. doi: 10.1099/00221287-132-3-825. [DOI] [PubMed] [Google Scholar]
  20. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  22. Oka A., Nomura N., Morita M., Sugisaki H., Sugimoto K., Takanami M. Nucleotide sequence of small ColE1 derivatives: structure of the regions essential for autonomous replication and colicin E1 immunity. Mol Gen Genet. 1979 May 4;172(2):151–159. doi: 10.1007/BF00268276. [DOI] [PubMed] [Google Scholar]
  23. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  24. Pugsley A. P., Cole S. T. An unmodified form of the ColE2 lysis protein, an envelope lipoprotein, retains reduced ability to promote colicin E2 release and lysis of producing cells. J Gen Microbiol. 1987 Sep;133(9):2411–2420. doi: 10.1099/00221287-133-9-2411. [DOI] [PubMed] [Google Scholar]
  25. Pugsley A. P., Schwartz M. Colicin E2 release: lysis, leakage or secretion? Possible role of a phospholipase. EMBO J. 1984 Oct;3(10):2393–2397. doi: 10.1002/j.1460-2075.1984.tb02145.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pugsley A. P. The ins and outs of colicins. Part I: Production, and translocation across membranes. Microbiol Sci. 1984 Oct;1(7):168–175. [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Van Tiel-Menkvled G. J., Rezee A., De Graaf F. K. Production and excretion of cloacin DF13 by Escherichia coli harboring plasmid CloDF13. J Bacteriol. 1979 Nov;140(2):415–423. doi: 10.1128/jb.140.2.415-423.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Watson R. J., Lau P. C., Vernet T., Visentin L. P. Characterization and nucleotide sequence of a colicin-release gene in the hic region of plasmid ColE3-CA38. Gene. 1984 Jul-Aug;29(1-2):175–184. doi: 10.1016/0378-1119(84)90178-1. [DOI] [PubMed] [Google Scholar]
  31. Witholt B., Boekhout M., Brock M., Kingma J., Heerikhuizen H. V., Leij L. D. An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli. Anal Biochem. 1976 Jul;74(1):160–170. doi: 10.1016/0003-2697(76)90320-1. [DOI] [PubMed] [Google Scholar]
  32. Wu H. C., Tokunaga M. Biogenesis of lipoproteins in bacteria. Curr Top Microbiol Immunol. 1986;125:127–157. doi: 10.1007/978-3-642-71251-7_9. [DOI] [PubMed] [Google Scholar]
  33. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]
  34. de Geus P., van Die I., Bergmans H., Tommassen J., de Haas G. Molecular cloning of pldA, the structural gene for outer membrane phospholipase of E. coli K12. Mol Gen Genet. 1983;190(1):150–155. doi: 10.1007/BF00330338. [DOI] [PubMed] [Google Scholar]
  35. van Embden J. D., Veltkamp E., Stuitje T., Andreoli P. M., Nijkamp H. J. Integration of a transposable DNA sequence which mediates ampicillin resistance into Clo DF13 plasmid DNA: determination of the site and orientation of TnA insertions. Plasmid. 1978 Feb;1(2):204–217. doi: 10.1016/0147-619x(78)90039-2. [DOI] [PubMed] [Google Scholar]
  36. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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