Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori

Abstract

The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.

Genome Sequence of Helicobacter pylori hpEurope Strain N6

“…The strain exhibited a very high transformation efficiency when plasmids or DNA fragments were introduced (12,13), and it has provided important data about mechanisms introducing genetic flexibility in the species. N6 was later employed to develop novel genetic tools for the construction of conditional H. pylori mutants in essential genes (3). In addition to being naturally transformable, N6 is capable of infecting gnotobiotic piglets, allowing the demonstration that urease is essential for H. pylori colonization (5,6).…”

Employing Site-Specific Recombination for Conditional Genetic Analysis in Sinorhizobium meliloti

“…Historically, essential genes have been studied with the use of temperature-sensitive alleles or by placing the gene of interest under the control of a promoter that is inducible by an exogenous chemical (3,4,9,15,30,31). In these systems, the manipulation of either the temperature or chemical inducer produces a reversible change in phenotype, while the genetic status of the organism remains unchanged.…”

Recombination-Based In Vivo Expression Technology IdentifiesHelicobacter pyloriGenes Important for Host Colonization

“…Although analysis of essential genes to determine their contributions to H. pylori colonization and virulence poses a unique set of challenges, these genes may produce a novel set of H. pylori virulence factors. The recent development of an inducible gene expression system for H. pylori should expedite the study of these essential genes (10). In summary, development and use of RIVET for H. pylori identified six sets of genes, two of which are important in animal colonization, as shown here.…”