Abstract
Operon and gene fusions between lacZ and the hemolysin genes, hlyC and hlyA, were performed. These two genes are essential for the synthesis of active hemolysin and are transcribed from a common promoter (p1). Whereas the amount of hemolysin produced in Escherichia coli is not changed by altering the hly gene dose, beta-galactosidase activity follows the gene dosage in both types of fusions when lacZ comes under the control of p1. This indicates that hemolysin is not negatively regulated on the transcription or translation level. The products of the gene fusions hlyC::lacZ and hlyA::lacZ were identified in maxicells as stable proteins of 146,000 and 220,000 daltons, respectively. Both fusion proteins possess beta-galactosidase activity indicating that the performed fusions of lacZ to the hly genes do not destroy the reading frame of hlyC and hlyA. The fusion proteins HlyC-beta-gal and HlyA-beta-gal were predominantly detected in the cytoplasm, confirming previous data which suggested that the primary gene products of hlyC and hlyA are not transported across the cytoplasmic membrane.
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- Berger H., Hacker J., Juarez A., Hughes C., Goebel W. Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli. J Bacteriol. 1982 Dec;152(3):1241–1247. doi: 10.1128/jb.152.3.1241-1247.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J., Cohen S. N. 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 U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Cohen S. N., Chang A. C. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol. 1977 Nov;132(2):734–737. doi: 10.1128/jb.132.2.734-737.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Collins J., Brüning H. J. Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage lambda: "cosmids". Gene. 1978 Oct;4(2):85–107. doi: 10.1016/0378-1119(78)90023-9. [DOI] [PubMed] [Google Scholar]
- Goebel W., Hedgpeth J. Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol. 1982 Sep;151(3):1290–1298. doi: 10.1128/jb.151.3.1290-1298.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Härtlein M., Schiessl S., Wagner W., Rdest U., Kreft J., Goebel W. Transport of hemolysin by Escherichia coli. J Cell Biochem. 1983;22(2):87–97. doi: 10.1002/jcb.240220203. [DOI] [PubMed] [Google Scholar]
- Koshland D., Botstein D. Secretion of beta-lactamase requires the carboxy end of the protein. Cell. 1980 Jul;20(3):749–760. doi: 10.1016/0092-8674(80)90321-9. [DOI] [PubMed] [Google Scholar]
- Mackman N., Holland I. B. Secretion of a 107 K dalton polypeptide into the medium from a haemolytic E. coli K12 strain. Mol Gen Genet. 1984;193(2):312–315. doi: 10.1007/BF00330686. [DOI] [PubMed] [Google Scholar]
- Michaelis S., Guarente L., Beckwith J. In vitro construction and characterization of phoA-lacZ gene fusions in Escherichia coli. J Bacteriol. 1983 Apr;154(1):356–365. doi: 10.1128/jb.154.1.356-365.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Müller D., Hughes C., Goebel W. Relationship between plasmid and chromosomal hemolysin determinants of Escherichia coli. J Bacteriol. 1983 Feb;153(2):846–851. doi: 10.1128/jb.153.2.846-851.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Noegel A., Rdest U., Goebel W. Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli. J Bacteriol. 1981 Jan;145(1):233–247. doi: 10.1128/jb.145.1.233-247.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruscetti S. K., Feild J. A., Scolnick E. M. Polycythaemia- and anaemia-inducing strains of spleen focus-forming virus differ in post-translational processing of envelope-related glycoproteins. Nature. 1981 Dec 17;294(5842):663–665. doi: 10.1038/294663a0. [DOI] [PubMed] [Google Scholar]
- SMITH H. W. The haemolysins of Escherichia coli. J Pathol Bacteriol. 1963 Jan;85:197–211. doi: 10.1002/path.1700850119. [DOI] [PubMed] [Google Scholar]
- Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Short E. C., Kurtz H. J. Properties of the Hemolytic Activities of Escherichia coli. Infect Immun. 1971 May;3(5):678–687. doi: 10.1128/iai.3.5.678-687.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shuman H. A., Silhavy T. J., Beckwith J. R. Labeling of proteins with beta-galactosidase by gene fusion. Identification of a cytoplasmic membrane component of the Escherichia coli maltose transport system. J Biol Chem. 1980 Jan 10;255(1):168–174. [PubMed] [Google Scholar]
- Shuman H. A., Silhavy T. J. Identification of the malK gene product. A peripheral membrane component of the Escherichia coli maltose transport system. J Biol Chem. 1981 Jan 25;256(2):560–562. [PubMed] [Google Scholar]
- Springer W., Goebel W. Synthesis and secretion of hemolysin by Escherichia coli. J Bacteriol. 1980 Oct;144(1):53–59. doi: 10.1128/jb.144.1.53-59.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wagner W., Vogel M., Goebel W. Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol. 1983 Apr;154(1):200–210. doi: 10.1128/jb.154.1.200-210.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de la Cruz F., Müller D., Ortiz J. M., Goebel W. Hemolysis determinant common to Escherichia coli hemolytic plasmids of different incompatibility groups. J Bacteriol. 1980 Aug;143(2):825–833. doi: 10.1128/jb.143.2.825-833.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van den Bosch J. F., Postma P., de Graaff J., MacLaren D. M. Haemolysis by urinary Escherichia coli and virulence in mice. J Med Microbiol. 1981 Aug;14(3):321–331. doi: 10.1099/00222615-14-3-321. [DOI] [PubMed] [Google Scholar]