Summary
As a first approach towards studying the regulation of hemolysin synthesis in Escherichia coli, we have fused lacZ into the four hly genes (hlyC, hlyA, hlyB a and hlyB b) using the Mud-1 (Mu::lacZ, Y, Apr) phage. The sites of insertion of Mud-1 within the various hly genes of the Hly plasmid pHly152 were determined by the hemolytic phenotype of the Hly- mutants (Hly -ex /Hly -in or Hly -ex /Hly +in ) and by complementation of these Hly- mutants with recombinant plasmids carrying cloned hly genes. It was found that hlyC, hlyA and hlyB a are transcribed from a relatively weak promoter (hlypL) located in front of hlyC. The activity of β-galactosidase is considerably lower when Mud-1 is integrated in hlyB a than when it is inserted in hlyC, suggesting a considerable decline in hly gene expression from hlyC to hlyB a. The DNA sequence upstream of the coding region of hlyC was found to promote galK gene expression when a fragment covering this region was inserted into the promoter-probe vector pKO-11. A putative promoter sequence, which could correspond to hlypL, was identified in this sequence.
The hlyB b gene appears to be transcribed from a different promoter and the direction of transcription seems to be opposite to that of the hlyC, A, B a operon. The strength of this promoter (hlypR), based on the level of β-galactosidase activity of Mud-1 insertion mutants in hlyB b, is considerably higher than that of hlypL.
The hly::lacZ fusions were also used to study a possible regulatory control of the hly genes on the transcriptional level by various physiological conditions (cAMP, media composition, growth phase), which have been shown to affect the synthesis of hemolysin. It was found that none of these conditions influence the expression of β-galactosidase, ruling out the possibility that transcription of the hly genes is modulated by these conditions.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Berger H, Hacker J, Juarez A, Hughes C, Goebel W (1982) Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli. J Bacteriol 152:1241–1247
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1522
Casadaban MJ, Cohen S (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
De la Cruz F, Zabala JC, Ortiz JM (1979) Incompatibility among α-hemolysin genes by transposition of Tn802. Plasmid 2:507–519
De la Cruz F, Müller D, Ortiz JM, Goebel W (1980) Hemolysis determinant common to Escherichia coli hemolytic plasmids of different incompatibility groups. J Bacteriol 143:825–833
Dorner F, Hughes C, Nahler G, Högenauer G (1979) Escherichia coli heat-labile enterotoxin: DNA directed in vitro synthesis and structure. Proc Natl Acad Sci USA 76:4832–4836
Goebel W, Hedgpeth J (1982) Cloning and characterizatin of the entire plasmid-encoded hemolysin determinant of Escherichia coli. J Bacteriol 151:1290–1298
Goebel W, Royer-Pokora B, Lindenmaier W, Bujard H (1973) Plasmids controlling synthesis of hemolysin in Escherichia coli I. Molecular properties. J Bacteriol 118:964–973
Gowrishankar J, Pittard J (1982) Construction from Mud1 (lacApr) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of λpphoA-lac. J Bacteriol 150:1222–1229
Guarante L, Lauer G, Roberts R, Ptashne M (1980) Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit β-globin. Cell 20:543–553
Hacker J, Hughes C, Hof H, Goebel W (1983) Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice. Infect Immun 42:57–63
Härtlein H, Schießl S, Wagner W, Rdest U, Kreft J, Goebel W (1983) Transport of hemolysin by Escherichia coli. J Cell Biochem 22:87–97
Hu N, Messing J (1982) The making of stand-specific M13 probes. Gene 17:271–277
Hughes C, Phillips R, Roberts AP (1982) Serum resistance among Escherichia coli strains causing urinary tract infection in relation to O type and the carriage of hemolysin, colicin, and antibiotic resistance detrminants. Infect Immun 35:270–275
Lee JH, Hefferman L, Wilcox G (1980) Isolation of ara-lac gene fusions in Salmonella typhimurium LT2 by using transducing bacteriophage Mud(Apr, lac). J Bacteriol 143:1325–1331
Le Minor S, Le Coueffic E (1975) Studies on haemolysins of enterobacteriaceae. Ann Microbiol (Paris) 126:313–332
Miller JA (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY
Minshew BH, Jorgenson J, Counts GW, Falkow S (1980) Association of hemolysin production, hemagglutination of human erythrocytes, and virulence for chicken embryos of extraintestinal Escherichia coli isolates. Infect Immun 20:50–54
Noegel A, Rdest U, Goebel W (1981) Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli. J Bacteriol 145:233–247
Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353
Rosenberg M, Chepelinky AB, McKenney K (1983) Studying promoters and terminators by gene fusion. Science 222:734–739
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Springer W, Goebel W (1980) Synthesis and secretion of hemolysin by Escherichia coli. J Bacteriol 144:53–59
Wagner W, Vogel M, Goebel W (1983) Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Baceriol 154:200–210
Wanner BW, McSharry R (1981) Phosphate-controlled gene expression in Escherichic coli K12 using Mud1-directed lacZ fusions. J Mol Biol 158:347–363
Wanner BW, Wieder S, McSharry R (1981) Use of bacteriophage transposon Mud-1 to determine the orientation for three proC-linked phosphate-starvation inducible (psi) genes in Escherichia coli K12. J Bacteriol 146:93–101
Welch RA, Dellinger EP, Minshew B, Falkow S (1981) Haemolysin contributes to virulence of extraintestinal E. coli infections. Nature (London) 294:665–667
Author information
Authors and Affiliations
Additional information
Communicated by E. Bautz
Rights and permissions
About this article
Cite this article
Juarez, A., Hughes, C., Vogel, M. et al. Expression and regulation of the plasmid-encoded hemolysin determinant of Escherichia coli . Mol Gen Genet 197, 196–203 (1984). https://doi.org/10.1007/BF00330963
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00330963