title: 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.
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.
authors.affiliation: Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Boston, Massachusetts 02111.
authors.forename: D W
authors.initials: DW
authors.lastname: Acheson
authors.forename: S B
authors.initials: SB
authors.lastname: Calderwood
authors.forename: S A
authors.initials: SA
authors.lastname: Boyko
authors.forename: L L
authors.initials: LL
authors.lastname: Lincicome
authors.forename: A V
authors.initials: AV
authors.lastname: Kane
authors.forename: A
authors.initials: A
authors.lastname: Donohue-Rolfe
authors.forename: G T
authors.initials: GT
authors.lastname: Keusch
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D066298:In Vitro Techniques; D007501:Iron / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D022622:Shiga Toxin 1; D014734:Vibrio cholerae / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D007501:Iron