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The smtB gene of Synechococcus PCC 7942 encodes a trans-acting repressor of the metal-regulated smtA gene that encodes a class II metallothionein. Recombinant SmtB has been expressed in Escherichia coli and purified. Electrophoretic mobility shift assays using recombinant SmtB or a protein extract from Synechococcus PCC 6301 reveal the concentration-dependent formation of three specific complexes with the smt operator/promoter. SmtB is also capable of direct interaction with metals as evidenced by 65Zn binding to the SmtB protein as well as the inhibition of repressor-DNA complex formation in the presence of various metal ions. Methylation interference analysis of such complexes identifies four protein contact points within the smt operator/promoter DNA. The points of contact appear to represent two pairs of binding sites, one pair in each of two inverted repeats (nt 548-563, 589-602). The contact points within each pair lie on opposing DNA strands and are separated by 10 bp, placing the repressor binding sites on opposite sides of the DNA helix. Based on electrophoretic mobility shift assays, methylation interference and molecular size calculations we propose that recombinant SmtB binds to the smt operator/promoter in multimeric fashion.

This is a follow-up to our previous study using an intranasal vaccine composed of MrpH, the tip adhesin of the MR/P fimbria, and cholera toxin to prevent urinary tract infection by Proteus mirabilis (X. Li, C. V. Lockatell, D. E. Johnson, M. C. Lane, J. W. Warren, and H. L. Mobley, Infect. Immun. 72:66-75, 2004). Here, we have expressed a cholera toxin-like chimera in which the MrpH adhesin-binding domain (residues 23 to 157) replaces the cholera toxin A1 ADP-ribosyltransferase domain. This chimera, when administered intranasally without additional adjuvant, is sufficient to induce protective immunity in mice

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Cholera toxin (CT) is an AB 5 toxin responsible for the profuse secretory diarrhea resulting from Vibrio cholerae infection. CT consists of a pentameric, receptor-binding B subunit (CTB) and a monomeric A subunit (CTA) that has latent enzymatic activity. In addition to its enterotoxicity, CT has potent mucosal adjuvant activity and can also function as a carrier molecule with many potential applications in cell biology. In earlier studies, the toxic CTA 1 domain was replaced by several other antigenic protein domains to produce holotoxinlike chimeras for use as potential mucosal vaccines. In the present study we utilized the twin arginine translocation (tat) system to produce fluorescent CT chimeras, as well as fluorescent chimeras of Escherichia coli heat-labile toxins LTI and LTIIb. Fusion proteins containing either green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP) and the A 2 domain of CT, LTI, or LTIIb were transported to the periplasm of E. coli by the tat system, and the corresponding B polypeptides of CT, LTI, and LTIIb were transported to the periplasm by the sec system. The fluorescent fusion proteins were shown to assemble spontaneously and efficiently with the corresponding B polypeptides in the periplasm to form chimeric holotoxin-like molecules, and these chimeras bound to and entered cultured cells in a manner similar to native CT, LTI, or LTIIb. The GFP and mRFP derivatives of CT, LT, and LTIIb developed here are useful tools for studies on the cell biology of trafficking of the CT/LT family of bacterial enterotoxins. In addition, these constructs provide proof in principle for the development of novel chimeric CT-like or LT-like vaccine candidates containing CTA 2 fusion proteins that cannot be delivered to the periplasm of E. coli by use of the sec secretion pathway.The AB 5 -type enterotoxins produced by Vibrio cholerae and Escherichia coli have generated interest as potent mucosal adjuvants and immunomodulators, as well as molecular tools to study endocytosis and trafficking from the cell surface to the endoplasmic reticulum (ER) (14,25). Cholera toxin (CT) and E. coli type I heat-labile enterotoxin (LTI) are approximately 80% identical at the amino acid level and consist of an enzymatically active A subunit and a pentameric, receptor-binding B subunit. The B subunit of CT (CTB) binds to ganglioside GM 1 and triggers uptake of the toxin into epithelial cells by endocytosis. Subsequent reduction of the proteolytically nicked A subunit (CTA) releases the active CTA 1 fragment within the endoplasmic reticulum, and CTA 1 is then translocated to the host cytoplasm to catalyze ADP-ribosylation of Gs␣ (25). The E. coli type II enterotoxins, LTIIa and LTIIb, are structurally and functionally homologous to CT and LTI; however, they are more divergent at the amino acid sequence level, and their pentameric B subunits bind preferentially to different ganglioside receptors.Our laboratory and others have constructed stable holotoxin-like molecules with another protein of interest r...