Modulation of gene expression by promoter mutants of the lambdacI857/pRM/pR system.

Gene expression driven by the p(R) promoter of the lambdacI857/p(RM)/p(R) system results from inactivation of the temperature-sensitive CI857 repressor. The CI857 repressor, whose gene is transcribed by the divergently orientated p(RM) promoter, is destabilised at temperatures above 30 degrees C. In this study, the lambdacI857/p(RM)/p(R) system was modified by the introduction of a single (A-32G) and a double mutation (A-32G and T-41C). The mutated lambdap(R) expression modules, 32G and 32G/41C, tightly repressed the highly lethal phage PhiX174 lysis gene E at temperatures up to 37 and 39 degrees C, respectively. Expression of protein E and subsequent lysis of Escherichia coli was still induced by a temperature up-shift to 42 degrees C. The impact of the mutations on gene expression levels driven by the lambdap(R) and p(RM) promoters was evaluated at various temperatures using the lacZ reporter gene. Results indicate that the A-32G mutation confers a lambdap(R) promoter-down phenotype. The additional increase in the temperature stability of the 32G/41C expression system is due to the T-41C mutation leading to a higher p(RM) activity. The described lambdap(R) expression modules can be used to obtain a defined expression level at a given temperature and to tightly repress in particular highly lethal genes at different bacterial growth temperatures.

Generation of Helicobacter pylori ghosts by PhiX protein E-mediated inactivation and their evaluation as vaccine candidates.

Bacterial ghosts are empty cell envelopes, which may be generated by the controlled expression of the PhiX174 lysis gene E in gram-negative bacteria to obtain vaccine candidates. We describe here the application of this technology to Helicobacter pylori. The lysis gene cassette was cloned into an Escherichia coli-Helicobacter pylori shuttle vector and introduced into an H. pylori recipient strain by bacterial conjugation. Temperature induction of the lysis gene cassette revealed a quantitative killing of the H. pylori culture without induction of lysis-resistant bacteria. Biochemical and transmission electron microscopic studies identified structurally intact H. pylori. Prophylactic oral vaccination experiments using these H. pylori ghosts in the BALB/c mouse model showed a significant reduction of the bacterial load in the ghost group, as measured by a quantitative bacterial reisolation procedure. Ten of 10 and 5 of 10 mice were protected, respectively, without the use of a mucosal adjuvant. Coadministration of ghosts with cholera toxin as mucosal adjuvant resulted in a complete protection of 10 of 10 and 8 of 8 mice against H. pylori challenge, with three animals showing a sterile immunity.