Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or iron-regulated promoter systems

Infect Immun. 1993 Mar;61(3):1098-104. doi: 10.1128/iai.61.3.1098-1104.1993.

Abstract

Shiga-like toxin I (SLT-I) B-subunit expression was examined by using the trc promoter in two different constructs, pSBC32 and pSBC54, in which 710 bp of DNA downstream of the B subunit in pSBC32 was deleted. The trc promoter in pSBC54 was replaced also with the SLT-I iron-regulated promoter to create a third plasmid, pSBC61. SLT-I B-subunit expression was examined from all three plasmids following transfer into Escherichia coli JM105 and the cholera toxin A-subunit gene deletion mutant Vibrio cholerae 0395-N1. The SLT-I B subunit was expressed from all constructs. pSBC61 was regulated by elemental iron and produced equivalent amounts of SLT-I B subunit from both E. coli and V. cholerae. In contrast to the cholera toxin B subunit, virtually all released into the medium, the SLT-I B subunit was predominantly cell associated in the pSBC61 constructs. Both pSBC32 and pSBC54 were inducible with isopropyl-beta-D-thiogalactopyranoside (IPTG) in the E. coli background but not the V. cholerae background; however, when E. coli cultures were allowed to grow for 24 h, the yield of SLT-I B subunit was not increased by IPTG induction. Both pSBC32 and -54 expressed more SLT-I B subunit in the V. cholerae host than in the E. coli host. Scale-up to a 9.9-liter fermentor culture of V. cholerae 0395 N1 (pSBC32) resulted in the isolation of 220 mg of SLT-I B. The purified B subunit was identical, in terms of binding to Vero cells, stoichiometry after chemical cross-linking, and ability to inhibit cytotoxicity of intact Shiga toxin, to native SLT-I B subunit from E. coli O157:H7.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bacterial Toxins / genetics*
  • Bacterial Toxins / isolation & purification
  • Bacterial Toxins / toxicity
  • Base Sequence
  • Biological Transport
  • Cell Compartmentation
  • Cell Line
  • Cell Survival / drug effects
  • Chlorocebus aethiops
  • Cloning, Molecular
  • Escherichia coli / metabolism*
  • Gene Expression
  • Genetic Vectors
  • In Vitro Techniques
  • Iron / metabolism
  • Macromolecular Substances
  • Molecular Sequence Data
  • Promoter Regions, Genetic
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Shiga Toxin 1
  • Vibrio cholerae / metabolism*

Substances

  • Bacterial Toxins
  • Macromolecular Substances
  • Recombinant Proteins
  • Shiga Toxin 1
  • Iron