Summary
We have recently reported the secretion of a 107K polypeptide by an E. coli strain containing the haemolytic plasmid pHly167 (Mackman and Holland 1984). In this paper we show that a large number of haemolytic E. coli strains, apparently including both plasmid and chromosomally located haemolysin genes, secrete similar large molecular weight proteins. Partial purification of one haemolysin suggests that activity co-purifies with a 107K polypeptide. These results were confirmed by cloning the corresponding haemolysin determinant in the form of a recombinant plasmid pLG570, containing chromosomal DNA prepared from a human isolate of E. coli, LE2001. Tn5 was used as a mutagen to localize the haemolysin genes to a 7-kilobase region of pLG570. Structural and export functions were identified by assaying cell sonicates of non-haemolytic mutants. At least one structural gene was identified which coded for a 107K polypeptide. Insertions into this gene completely eliminated haemolysin activity and resulted in truncation of the 107K protein whereas insertions into the adjacent 4-kb region resulted in intracellular haemolytic activity. This internal haemolysin appeared to accumulate in the periplasm which suggests that factors encoded by the 4-kb region are involved in exporting the 107K polypeptide across the outer membrane.
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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
Chou L, Kahmann R, Kamp D (1977) Electron microscope characteristics of DNA's of non-defective deletion mutants of bacteriophage Mu. J Mol Biol 113:591–609
Goebel W, Royer-Pokora B, Lindenmaier W, Bujard H (1974) Plasmids controlling synthesis of hemolysin in Escherichia coli: molecular properties. J Bacteriol 118:964–973
Goebel W, Hedgpeth J (1982) Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol 151:1290–1298
Lory S, Tai P, Davis B (1983) Mechanism of protein excretion by gram-negative bacteria: Pseudomonas aeruginosa exotoxin A. J Bacteriol 156:695–702
Mackman N, Holland IB (1984) Secretion of a 107K dalton polypeptide into the medium from a haemolytic E. coli K12 strain. Mol Gen Genet 193:312–215
Mooi F, Wijfjes A, De Graff F (1983) Identification and characterization of precursors in the biosynthesis of the K88ab fimbria of Escherichia coli. J Bacteriol 154:41–49
Muller D, Hughes C, Goebel W (1983) Relationship between plasmid and chromosomal hemolysin determinants of Escherichiacoli. J Bacteriol 153:846–851
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 pHly152 of Escherichia coli. J Bacteriol 145:233–247
Nossal N, Heppel L (1966) The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 241:3055–3062
Pratt J, Boulnois G, Darby V, Orr E, Wahle E, Holland IB (1981) Identification of gene products programmed by restriction endonuclease DNA fragments using an E. coli in vitro system. Nucl Acids Res 9:4459–4474
Smith HW (1963) The haemolysins of Escherichia coli. J Pathol Bacteriol 85:197–211
Smith HW, Hall S (1967) The transmissible nature of the genetic factor in Escherichia coli that controls haemolysin production. J Gen Microbiol 47:153–161
Springer W, Goebel W (1980) Synthesis and secretion of hemolysin by Escherichia coli. J Bacteriol 144:53–59
Stark J, Shuster C (1983) The structure of cloned hemolysin DNA from plasmid pHly185. Plasmid 10:45–54
Wagner W, Voegel M, Goebel W (1983) Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol 154:200–210
Welch R, Hull R, Falkow S (1983) Molecular cloning and physical characterization of a chromosomal hemolysin from Escherichia coli. Infect Immun 42:178–186
Williams P (1979) Determination of the molecular weight of Escherichia coli α-haemolysin. FEMS Microbiol Lett 5:21–24
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Mackman, N., Holland, I.B. Functional characterization 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 (1984). https://doi.org/10.1007/BF00334104
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DOI: https://doi.org/10.1007/BF00334104