title: Expansion of the tetracycline-dependent regulation toolbox for Helicobacter pylori.
abstract: In an effort to gain greater understanding of the biology and infection processes of Helicobacter pylori, we have expanded the functionality of the tetracycline-dependent gene regulation (tet) system to provide more improved and versatile genetic control and facilitate the generation of conditional mutants to study essential genes. Second-generation tetracycline-responsive H. pylori uPtetO5 promoters were based on the mutated core ureA promoter. Single point mutations at either the ribosomal binding site or the start codon were introduced to shift the regulatory range of three uPtetO5 derivatives. All promoters were tested for regulation by TetR and revTetR using dapD, a gene essential to peptidoglycan biosynthesis, as a reporter. All tet promoters were effectively regulated by both TetR and revTetR, and their regulation windows overlapped so as to cover a broad range of expression levels. tet promoters uPtetO5m1 and uPtetO5m2 could be sufficiently silenced by both TetR and revTetR so that the conditional mutants could not grow in the absence of diaminopimelic acid (DAP). Furthermore, through the use of these inducible promoters, we reveal that insufficient DAP biosynthesis results in viable cells with altered morphology. Overall, the development and optimization of tet regulation for H. pylori will not only permit the study of essential genes but also facilitate investigations into gene dosage effects on H. pylori physiology.
authors.affiliation: Helicobacter pylori Research Laboratory and Ondek Pty. Ltd., Marshall Centre for Infectious Disease Research and Training, School of Pathology & Laboratory Medicine, University of Western Australia, Nedlands, Western Australia, Australia School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, Australia aleksandra.debowski@uwa.edu.au.
authors.forename: Aleksandra W
authors.initials: AW
authors.lastname: Debowski
authors.affiliation: Helicobacter pylori Research Laboratory and Ondek Pty. Ltd., Marshall Centre for Infectious Disease Research and Training, School of Pathology & Laboratory Medicine, University of Western Australia, Nedlands, Western Australia, Australia.
authors.forename: Miriam
authors.initials: M
authors.lastname: Sehnal
authors.affiliation: Helicobacter pylori Research Laboratory and Ondek Pty. Ltd., Marshall Centre for Infectious Disease Research and Training, School of Pathology & Laboratory Medicine, University of Western Australia, Nedlands, Western Australia, Australia.
authors.forename: Tingting
authors.initials: T
authors.lastname: Liao
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Stubbs
authors.affiliation: Helicobacter pylori Research Laboratory and Ondek Pty. Ltd., Marshall Centre for Infectious Disease Research and Training, School of Pathology & Laboratory Medicine, University of Western Australia, Nedlands, Western Australia, Australia.
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Marshall
authors.affiliation: Helicobacter pylori Research Laboratory and Ondek Pty. Ltd., Marshall Centre for Infectious Disease Research and Training, School of Pathology & Laboratory Medicine, University of Western Australia, Nedlands, Western Australia, Australia.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Benghezal
mesh_terms: D001483:Base Sequence; D003960:Diaminopimelic Acid / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects*; D020043:Genes, Essential; D005827:Genetics, Microbial / Q000379:methods*; D016480:Helicobacter pylori / Q000235:genetics* / Q000502:physiology; D008967:Molecular Biology / Q000379:methods*; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D013752:Tetracycline / Q000378:metabolism*
chemical_list: D003960:Diaminopimelic Acid; D013752:Tetracycline