Summary
We have devised a general, one-step technique for isolation of strains in which the gene coding for an exported protein is fused to the gene for β-galactosidase (lacZ). These fusions specify a hybrid protein comprised of an NH2-terminal portion of the exported protein and a large functional COOH-terminal portion of β-galactosidase. The fusions are constructed with a derivative of the MudII(lac, Ap) phage. To overcome the lethality that is often associated with the expression of such a hybrid gene, we have recombined an early lacZ nonsense mutation onto this phage. With the use of strains that carry a temperaturesensitive nonsense suppressor, expression of the full-length hybrid protein can be controlled by varying the growth temperature. We demonstrated the utility of this technique by isolating a series of fusions to a gene, ompA, coding for a major outer membrane protein. As expected, strains containing these fusions are not viable under conditions that permit synthesis of a functional nonsense suppressor. Accordingly, this method should also be useful for direct selection of export-defective mutants.
Similar content being viewed by others
References
Bassford PJ Jr, Silhavy TJ, Beckwith J (1979) Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol 139:19–31
Benson SA, Silhavy TJ (1983) Information within the mature LamB protein necessary for localization to the outer membrane of E. coli K12. Cell 32:1325–1335
Bremer E, Cole ST, Hindennach I, Henning U, Beck E, Kurz C, Schaller H (1982) Export of a protein into the outer membrane of Escherichia coli K-12. Stable incorporation of the OmpA protein requires more than 133 amino terminal amino acids residues. Eur J Biochem 122:223–231
Casadaban MJ (1976) Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol 104:541–555
Casadaban MJ, Cohen SN (1979) Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: In vivo probe for transcriptional control sequences. Proc Natl Acad Sci USA 76:4530–4533
Casadaban MJ, Chou J (1984) In vivo formation of hybrid protein β-galactosidase gene fusions in one step with a new transposable Mu-lac transducing phage. Proc Natl Acad Sci USA 81: 535–539
Emr S, Silhavy TJ (1980) Mutations affecting localization of an Escherichia coli outer membrane protein, the bacteriophage λ receptor. J Mol Biol 141:63–90
Fairbanks G, Steck TL, Wallach DFG (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617
Foulds J (1972) Purification and particle characterization of a bacteriocin in Serratia marcescens. J Bacteriol 110:1001–1009
Hall MN, Silhavy TJ (1979) Transcriptional regulation of Escherichia coli K-12 major outer membrane protein 1b. J Bacteriol 140:342–350
Hall MN, Silhavy TJ (1981) The ompB locus and the regulation of the major outer membrane porin proteins of Escherichia coli K-12. J Mol Biol 146:23–43
Ito K, Bassford PJ Jr, Beckwith J (1981) Protein localization in E. coli: Is there a common step in the secretion of periplasmic and outer membrane proteins? Cell 24:707–717
Komeda Y, Iino T (1979) Regulation of expression of the flagellin gene (hag) in Escherichia coli K-12: analysis of hag-lac gene fusions. J Bacteriol 139:721–729
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (New Biol) 227:680–685
Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Silhavy TJ, Shuman HA, Beckwith J, Schwartz M (1977) Use of gene fusions to study outer membrane protein localization in Escherichia coli. Proc Natl Acad Sci USA 74:5411–5415
Silhavy TJ, Bassford PJ Jr, Beckwith J (1979) A genetic approach to the study of protein localization in Escherichia coli. In: Inouye M (ed) Bacterial outen membranes: biogenesis and functions. John Wiley & Sons, New York, pp 203–254
Welply JK, Fowler AV, Beckwith JR, Zabin I (1980) Positions of early nonsense and deletion mutations in lacZ. J Bacteriol 142:732–734
Author information
Authors and Affiliations
Additional information
Communicated by K. Isono
Rights and permissions
About this article
Cite this article
Palva, E.T., Silhavy, T.J. lacZ fusions to genes that specify exported proteins: A general technique. Molec. Gen. Genet. 194, 388–394 (1984). https://doi.org/10.1007/BF00425549
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00425549