title: Mutations to essential orphan response regulator HP1043 of Helicobacter pylori result in growth-stage regulatory defects.
abstract: Helicobacter pylori establishes lifelong infections of the gastric mucosa, a niche considered hostile to most microbes. While responses to gastric acidity and local inflammation are understood, little is known as to how they are integrated into homeostatic control of cell division and growth-stage gene expression. Here we investigate the essential orphan response regulator HP1043, a member of the OmpR/PhoB subfamily of transcriptional regulators that is unique to the Epsilonproteobacteria and that lacks phosphorylation domains. To test the hypothesis that conformational changes in the homodimer might lead to defects in gene expression, we sought mutations that might alter DNA-binding efficiency. Two introduced mutations (C215S, C221S) C terminal to the DNA-binding domain of HP1043 (HP1043CC11) resulted in a 2-fold higher affinity for its own promoter by footprinting. Modeling studies with the crystal structure of HP1043 suggested that C215S might affect the helix-turn-helix domain. Genomic replacement of the hp1043 allele with the hp1043CC11 mutant allele resulted in a 2-fold decrease in protein levels, despite a dramatic increase in mRNA. The mutations did not affect in vitro growth rates or colonization efficiency in a mouse model. Proteomic profiling (CC11 mutant strain versus wild type) identified many expression differences, and quantitative PCR further revealed that 11 out of 12 examined genes had lost growth-stage regulation and that 6 of the genes contained HP1043 binding consensus sequences within the promoter regions (fur, cagA, cag23, flhA, flip, and napA). Our studies show that mutations that affect DNA-binding affinity can be used to identify new members of the HP1043 regulon.
authors: Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia, USA. io4n@virginia.edu
authors: Igor N
authors: IN
authors: Olekhnovich
authors: Serhiy
authors: S
authors: Vitko
authors: Olga
authors: O
authors: Chertihin
authors: Raquel
authors: R
authors: Hontecillas
authors: Monica
authors: M
authors: Viladomiu
authors: Josep
authors: J
authors: Bassaganya-Riera
authors: Paul S
authors: PS
authors: Hoffman
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D016480:Helicobacter pylori / Q000235:genetics* / Q000254:growth & development; D051379:Mice; D008810:Mice, Inbred C57BL; D050296:Microbial Viability; D009154:Mutation; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology
chemical_list: D004269:DNA, Bacterial; C501059:HP1043 protein, Helicobacter pylori; D014157:Transcription Factors