Summary
We have identified gene products corresponding to hlyC, hlyA and hlyD encoded by the Escherichia coli haemolytic determinant 2001 of human origin cloned into the recombinant plasmid pLG570. The product of hlyC is required for the “activation” of the inactive 107K polypeptide encoded by the hlyA gene. the activated 107K protein constitutes the active haemolysin secreted into the medium. hlyB and hlyD are separate regions defined by complementation studies and encode functions essential for the export of haemolysin with hlyD encoding a 53K protein. Complementation studies using subclones and Tn5 insertions into pLG570 have revealed the presence of two major promoters upstream of hlyC and hlyD which transcribe the four hly genes in the same direction. Finally, we were able to reconstitute the complete haemolysin system from three different plasmids encoding hlyC, hlyA and hlyB+hlyD, respectively.
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References
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Goebel W, Hedgpeth J (1982) Cloning and functional characterisation of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol 151:1290–1298
Hartlein M, Schiessel S, Wagner W, Rdest U, Kreft J, Goebel W (1983) Transport of hemolysin by Escherichia coli. J Cell Biochem 22:87–97
Juarez A, Hughes C, Vogel M, Goebel W (1984) Expression and regulation of the plasmid-encoded hemolysin determinant of Escherichia coli. Mol Gen Genet 197:196–203
Larsen H, Gerdes K, Light J, Molin S (1984) Low copy number plasmid vectors amplifiable by derepression of an inserted foreign promoter. Gene 28:45–54
Mackman N, Holland IB (1984a) Secretion of a 107K dalton polypeptide into the medium from a haemolytic E. coli K12 strain. Mol Gen Genet 193:312–315
Mackman N, Holland IB (1984b) Functional characterisation of a cloned haemolysin determinant from E. coli of human origin, encoding information for the secretion of a 107K polypeptide. Mol Gen Genet 196:129–134
Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:154
Muller D, Hughes C, Goebel W (1983) Relationship between plasmid and chromosomal hemolysin determinants of Escherichia coli. J Bacteriol 153:846–851
Nicaud J-M, Mackman N, Gray L, Holland IB (1985) Regulation of haemolysin synthesis in E. coli determined by Hly genes of human origin. Mol Gen Genet 199:111–116
Noegel A, Rdest U, Springer W, Goebel W (1979) Plasmid cistrons controlling synthesis and excretion of the exotoxin α-haemolysin of Escherichia coli. Mol Gen Genet 175:343–350
Noegel A, Rdest U, Goebel W (1981) Determination of the functions of hemolytic plasmid pHly 152 of Escherichia coli. J Bacteriol 145:233–247
Pratt JM (1984) Coupled transcription-translation in prokaryotic cell-free systems. In: Hames BD, Higgins SJ (eds) Transcription and translation — A practical approach. IRL Press, Oxford, Washington DC, pp 179–209
Smith HW (1963) The haemolysins of Escherichia coli. J Pathol Bacteriol 85:197–211
Stark JM, Shuster CW (1983) The structure of cloned hemolysin DNA from plasmid pHly185. Plasmid 10:45–54
Stoker NG, Fairweather NF, Spratt BG (1982) Veraatile low-copynumber plasmid vectors for cloning in Escherichia coli. Gene 18:335–341
Stuber D, Bujard H (1981) Organisation of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci USA 78:167–171
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Wagner W, Vogel M, Goebel W (1983) Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol 154:200–210
Welch RA, Patchen-Dellinger E, Minshew B, Falkow S (1981) Haemolysin contributes to virulence of extraintestinal E. coli infections. Nature 294:665–667
Welch RA, Hall R, Falkow S (1983) Molecular cloning and physical characterisation of a chromosomal hemolysin from Escherichia coli. Infect Immunol 42:178–186
Zabala JC, Garcia-Lobo JM, Diaz-Aroca E, de la Cruz F, Ortiz JM (1984) Escherichia coli alpha-haemolysin synthesis and export genes are flanked by a direct repetition of IS91-like elements. Mol Gen Genet 197:90–97
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Mackman, N., Nicaud, J.M., Gray, L. et al. Genetical and functional organisation of the Escherichia coli haemolysin determinant 2001. Molec. Gen. Genet. 201, 282–288 (1985). https://doi.org/10.1007/BF00425672
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DOI: https://doi.org/10.1007/BF00425672