Expansion of the Tetracycline-Dependent Regulation Toolbox for Helicobacter pylori

Abstract

bIn 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. Helicobacter pylori is an ancient member of the human microbiota that has coevolved with humans to dominate the gastric niche (1, 2), and its infection is ubiquitous in all human populations. However, the prevalence of H. pylori infection in adult populations can vary from more than 80% in many developing countries to significantly lower rates, ranging between 20% and 40%, in industrialized countries (3, 4). Infection by this bacterium causes chronic active gastritis, which may progress further into a gastric malady, such as peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer (5, 6). Consequently, H. pylori infection represents a significant burden on global health, and with increased rates of antibiotic resistance and declining eradication rates (7-9), there is an increasing pressure to identify new therapeutic targets. This will best be achieved by gaining a greater understanding of not only the pathogen's biology but also its infection processes.Much of our current knowledge about bacterial biology and pathogenesis has been attained through the use of deletion mutants in concert with animal models. However, gene deletion limits the study to loss-of-function knockout mutants; thus, it is not suitable for the study of essential genes, and this technique does not allow for investigating the temporal requirement of specific genes during different stages of infection. As such, there has been increasing interest in generating conditional knockouts, based on the inducible expression of the target gene, to address these important questions (10-18). Unli...

Lipoprotein Processing and Sorting in Helicobacter pylori

“…To place lgt under TetR regulation, tetR linked to a kanamycin resistance determinant was inserted into the ureA locus of strain 26695 to yield strain VM165. Then, a synthetic construct (GenScript) in which a chloramphenicol resistance determinant and 3 copies of tetO linked to the promoter of cagUT (65) placed upstream of HP0955 (and changing the ATG start codon to a TTG start codon [85]) was introduced into the intergenic region between HP0630 and HP0631 to yield strain HP173. Finally, the endogenous lgt locus was deleted using the lgt deletion construct described above to yield strain VM176.…”

“…To place lspA, lolA, or lolF under TetR regulation, strain VM124 (which contains TetR in the ureA locus) (65) was transformed with synthetic constructs in which a kanamycin resistance determinant and 3 copies of tetO linked to the promoter of cagUT (65) were placed upstream of lspA, lolA, and lolF to yield strains VM183, VM189, and VM191, respectively. The start codons for lolA (ATG) and lspA (GTG) were changed to TTG (85); lolF naturally begins with a TTG start codon.…”

Status quo of tet regulation in bacteria

“…Phenotypes attributed to gene silencing by revTetR were observed in Mycobacterium smegmatis after about 4 h by Western blotting against the target proteins, with only faint signals visible after 12 h (Guo et al ., 2007 ). Comparably, shutting off conditional complementation of the dap gene by revTetR in Helicobacter pylori resulted in growth retardation beginning 10 h after addition of ATc (Debowski et al ., 2015 ). Combined rapid ON and OFF kinetics can be realized by toggle switches.…”

“…A tet ‐controlled reporter gene was also inducible in a mouse model of infection. Second‐generation tet promoters for H. pylori are characterized by a tetO site in between −35 and −10 and the addition of a second tet operator upstream of −35 (Debowski et al ., 2015 ). This study also introduced regulation by revTetR‐r1.7 in H. pylori .…”