Skip to main content
SpringerLink
Log in

Overproduction, purification and characterization of the F traT protein

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

A lambda transducing phage (EDλ110) which carries the sex factor F surface exclusion genes, traS and traT, was characterized by both genetic and physiochemical techniques. The transducing segment consists of 5.2 kilobases of F tra DNA, and carries the carboxy-terminal onehalf of the upstream traG gene, as well as traS, traT, and the adjacent downstream gene traD. These tra proteins could be identified in infected UV-irradiated cells, and the major part of their synthesis was found to occur from the phage's late promoter pR under Q control.

Lysogens for EDλ110 were induced and found to greatly overproduce the traT gene product (TraTp), an outer membrane protein normally found in about 20,000 copies per cell, to levels which exceeded the major outer membrane proteins. This led to the development of a simple purification procedure for TraTp, the most important step of which was the construction of an appropriate ompB derivative to eliminate the major outer membrane porin proteins, which have several physical properties in common with TraTp.

Purified TraTp was added to mixtures of donor and recipient cells and found to inhibit mating. The specificity of this assay was demonstrated by using an R100-1 donor, which responds to a heterologous surface exclusion system, and by using an altered TraTp containing a missense amino acid substitution.

A mechanism by which TraTp mediates surface exclusion is proposed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Achtman M, Willetts N, Clark AJ (1971) Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherichia coli by isolation and characterization of transferdeficient mutants. J Bacteriol 106:529–538

    Google Scholar 

  • Achtman M, Willetts N, Clark AJ (1972) Conjugational complementation analysis of transfer-deficient mutants of Flac in Escherichia coli. J Bacteriol 110:831–842

    Google Scholar 

  • Achtman M, Kennedy N, Skurray R (1977) Cell-cell interactions in conjugating Escherichia coli: role of traT protein in surface exclusion. Proc Natl Acad Sci USA 74:5104–5108

    Google Scholar 

  • Achtman M, Morelli G, Schwuchow S (1978a) Cell-cell interactions in conjugating Escherichia coli: role of F pili and fate of mating aggregates. J Bacteriol 135:1053–1061

    Google Scholar 

  • Achtman M, Schwuchow S, Helmuth R, Morelli G, Manning PA (1978b) Cell-cell interactions in conjugating Escherichia coli: Con− mutants and stabilization of mating aggregates. Mol Gen Genet 164:171–183

    Google Scholar 

  • Achtman M, Skurray RA, Thompson R, Helmuth R, Hall S, Beutin L, Clark AJ (1978c) Assignment of tra cistrons to EcoRI fragments of F sex factor DNA. J Bacteriol 133:1383–1392

    Google Scholar 

  • Achtman M, Manning PA, Edelbluth C, Herrlich P (1979) Export without proteolytic processing of inner and outer membrane proteins encoded by F sex factor tra cistrons in Escherichia coli minicells. Proc Natl Acad Sci USA 76:4837–4841

    Google Scholar 

  • Achtman M, Manning PA, Kusecek B, Schwuchow S, Willetts N (1980) A genetic analysis of F sex factor cistrons needed for surface exclusion in Escherichia coli. J Mol Biol 138:779–795

    Google Scholar 

  • Daniels DL, Schroeder JL, Szybalski W, Sanger F, Blattner FR (1983) A molecular map of coliphage lambda. In: Hendrix, RW, Roberts JW, Stahl FW, Weisberg RA (eds) Lambda II. Cold Spring Harbor Laboratory, New York, pp 469–517

    Google Scholar 

  • Ferrazza D, Levy SB (1980) Biochemical and immunological characterization of an R plasmid-encoded protein with properties resembling those of major cellular outer membrane proteins. J Bacteriol 144:149–158

    Google Scholar 

  • Filip C, Fletcher G, Wulff JL, Earhart CF (1973) Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol 115:717–722

    Google Scholar 

  • Galanos C, Luderitz O, Westphal O (1969) A new method for the extraction of R lipopolysaccharides. Eur J Biochem 9:245–249

    Google Scholar 

  • Hansen BS, Manning PA, Achtman M (1982) Promoter-distal region of the tra operon of F-like sex factor R100 in Escherichia coli K-12. J Bacteriol 150:89–99

    Google Scholar 

  • Hantke K, Braun V (1973) Covalent binding of lipid to protein. Eur J Biochem 34:284–296

    Google Scholar 

  • Havekes L, Hoekstra W, Kempen H (1977) Relation Between F, R1, R100 and R144 Escherichia coli K-12 donor strains in mating. Mol Gen Genet 155:185–189

    Google Scholar 

  • Helmuth R, Achtman M (1975) Operon structure of DNA transfer cistrons on the F sex factor. Nature 257:652–656

    Google Scholar 

  • Hendrix RW (1971) Identification of proteins coded in phage lambda. In: Hershey AD (ed) The bacteriophage lambda. Cold Spring Harbor Laboratory, New York, pp 355–370

    Google Scholar 

  • Hindennach I, Henning U (1975) The major proteins of the Escherichia coli outer cell envelope membrane. Eur J Biochem 59:207–213

    Google Scholar 

  • Johnson DA, Willetts, NS (1980) Construction and characterization of multicopy plasmids containing the entire F transfer region. Plasmid 4:292–304

    Google Scholar 

  • Kennedy N, Beutin L, Achtman M, Skurray R, Rahmsdorf U, Herrlich P (1977) Conjugation proteins encoded by the F sex factor. Nature 270:580–585

    Google Scholar 

  • Krell K, Gottesman ME, Parks JS, Eisenberg MA (1972) Escape synthesis of the biotin operon in induced λb2 lysogens. J Mol Biol 68:69–82

    Google Scholar 

  • Liu T-Y, Chang YH (1971) Hydrolysis of proteins with p-toluenesufonic acid. J Biol Chem 246:2842–2848

    Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    Google Scholar 

  • Manning PA, Achman M (1979) Cell to cell interactions in conjugating Escherichia coli: the involvement of the cell envelope. In: Inouye M (ed) Bacterial outer membranes: biogenesis and functions. J Wiley & Sons, New York, pp 409–447

    Google Scholar 

  • Manning PA, Morelli G (1982) DNA homology of the promoterdistal regions of the tra operons of sex factors F and R100 in Escherichia coli K-12. J Bacteriol 150:389–394

    Google Scholar 

  • Manning PA, Beutin L, Achtman M (1980) Outer membrane of Escherichia coli: properties of the F sex factor traT protein which is involved surface exclusion. J Bacteriol 142:285–294

    Google Scholar 

  • Manning PA, Morelli G, Achtman M (1981) traG protein of the F sex factor of Escherichia coli K-12 and its role in conjugation. Proc Natl Acad Sci USA 78:7487–7491

    Google Scholar 

  • McIntire S, Willetts NS (1978) Plasmid cointegrates of Flac and lambda prophage. J Bacteriol 134:184–192

    Google Scholar 

  • McIntire S, Willetts NS (1980) Transfer-deficient cointegrates of Flac and lambda prophage. Mol Gen Genet 178:165–172

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, New York, p 433

    Google Scholar 

  • Minkley EG, Jr, Ippen-Ihler K (1977) Identification of a membrane protein associated with expression of the surface exclusion region of the F transfer operon. J Bacteriol 129:1613–1622

    Google Scholar 

  • Moll A, Manning PA, Timmis KN (1980) Plasmid-determined resistance to serum bactericidal activity: a major outer membrane protein, the traT gene product, is responsible for plasmid-specified serum resistance in Escherichia coli. Infect Immun 28:359–367

    Google Scholar 

  • Moore D, Sowa BA, Ippen-Ihler K (1981) The effect of tra mutations on the synthesis of the F-pilin membrane polypeptide. Mol Gen Genet 184:260–264

    Google Scholar 

  • Murialdo H, Siminovitch L (1972) The morphogenesis of bacteriophage lambda. IV. Identification of gene products and control of the expression of morphogenetic information. Virology 48:785–823

    Google Scholar 

  • Noel D, Nikaido K, Ames GF-L (1979) A single amino acid substitution in a histidine-transport protein drastically alters its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry 18:4159–4165

    Google Scholar 

  • Ogata RT, Winters C, Levine RP (1982) Nucleotide sequence analysis of the complement resistance gene from plasmid R100. J Bacteriol 151:819–827

    Google Scholar 

  • Rashtchian A, Crooks JH, Levy SB (1983) traJ independence in expression of traT on F. J Bacteriol 154:1009–1012

    Google Scholar 

  • Reeve JN, Shaw JE (1979) Lambda encodes an outer membrane protein: the lom gene. Mol Gen Genet 172:243–248

    Google Scholar 

  • Rosenbusch JP (1974) Characterization of the major envelope protein from Escherichia coli. J Biol Chem 249:8019–8029

    Google Scholar 

  • Sarma V, Reeves P (1977) Genetic locus (ompB) affecting a major outer membrane protein of Escherichia coli K-12. J Bacteriol 132:23–27

    Google Scholar 

  • Schweizer M, Henning U (1977) Action of a major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J Bacteriol 129:1651–1652

    Google Scholar 

  • Schweizer M, Hindennach I, Garten W, Henning U (1978) Major proteins of the Escherichia coli outer cell envelope membrane. Interaction of protein II* with lipopolysaccharide. Eur J Biochem 82:211–217

    Google Scholar 

  • Shimada K, Weisberg RA, Gottesman ME (1972) Prophage lambda at unusual chromosomal locations. I. Location of the secondary attachment sites and the properties of the lysogens. J Mol Biol 63:483–503

    Google Scholar 

  • Skurray RA, Hancock REW, Reeves P (1974) Con− mutants: class of mutants in Escherichia coli K-12 lacking a major cell wall protein, and defective in conjugation and adsorption of a bacteriophage. J Bacteriol 119:726–735

    Google Scholar 

  • Skurray RA, Nagaishi H, Clark AJ (1976) Molecular cloning of DNA from F sex factor of Escherichia coli K-12. Proc Natl Acad Sci USA 73:64–68

    Google Scholar 

  • Van Alphen L, Havekes L, Lugtenberg B (1977) Major outer membrane protein d of Escherichia coli K12. Purification and in vitro activity of bacteriophage k3 and f-pilus mediated conjugation. FEBS Lett 75:285–290

    Google Scholar 

  • Willetts N, Johnson G (1979) R. SmaI cleavage map of the transfer region of the E. coli K12 sex factor F. Genet Res 34:195–202

    Google Scholar 

  • Willetts N, Maule J (1974) Interactions between the surface exclusion systems of some F-like plasmids. Genet Res 24:81–89

    Google Scholar 

  • Willetts N, Maule J (1979) Investigation of the F conjugation gene traI: traI mutants and λtraI transducing phages. Mol Gen Genet 169:325–336

    Google Scholar 

  • Willetts N, Maule J (1980) Characterization of a λ transducing phage carrying the F conjugation gene traG. Mol Gen Genet 178:675–680

    Google Scholar 

  • Willetts NS, McIntire S (1978) Isolation and characterization of λtra transducing phages from EDFL223 (Flac traB::EDλ4). J Mol Biol 126:525–549

    Google Scholar 

  • Willetts N, Skurray R (1980) The conjugation system of F-like plasmids. Annu Rev Genet 14:41–76

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biological Sciences, Carnegie-Mellon University, 15213, Pittsburgh, PA, USA

    Edwin G. Minkley Jr.

  2. Department of Molecular Biology, University of Edinburgh, EH9 3JR, Edinburgh, Scotland

    Neil S. Willetts

Authors
  1. Edwin G. Minkley Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Neil S. Willetts
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by W. Arber

Rights and permissions

Reprints and permissions

About this article

Cite this article

Minkley, E.G., Willetts, N.S. Overproduction, purification and characterization of the F traT protein. Mol Gen Genet 196, 225–235 (1984). https://doi.org/10.1007/BF00328054

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00328054

Keywords

Search

Navigation