Molecular dissection of Helicobacter pylori Topoisomerase I reveals an additional active site in the carboxyl terminus of the enzyme.

DNA topoisomerases play a crucial role in maintaining DNA superhelicity, thereby regulating various cellular processes. Unlike most other species, the human pathogen Helicobacter pylori has only two topoisomerases, Topoisomerase I and DNA gyrase, the physiological roles of which remain to be explored. Interestingly, there is enormous variability among the C-terminal domains (CTDs) of Topoisomerase I across bacteria. H. pylori Topoisomerase I (HpTopoI) CTD harbors four zinc finger motifs (ZFs). We show here that sequential deletion of the third and/or fourth ZFs had only a marginal effect on the HpTopoI activity, while deletion of the second, third and fourth ZFs severely reduced DNA relaxation activity. Deletion of all ZFs drastically hampered DNA binding and thus abolished DNA relaxation. Surprisingly, mutagenesis of the annotated active site tyrosine residue (Y297 F) did not abrogate the enzyme activity and HpTopoI CTD alone (spanning the four ZFs) showed DNA relaxation activity. Additionally, a covalent linkage between the DNA and HpTopoI CTD was identified. The capacity of HpTopoI CTD to complement Escherichia coli topA mutant strains further supported the in vitro observations. Collectively these results imply that not all ZFs are dispensable for HpTopoI activity and unveil the presence of additional non-canonical catalytic site(s) within the enzyme.

Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori.

Horizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of Gram-negative bacteria, and in particular of Helicobacter pylori, the mechanisms underlying the handling of the incoming DNA within the periplasm are poorly understood. Here we identify the protein ComH as the periplasmic receptor for the transforming DNA during natural transformation in H. pylori. ComH is a DNA-binding protein required for the import of DNA into the periplasm. Its C-terminal domain displays strong affinity for double-stranded DNA and is sufficient for the accumulation of DNA in the periplasm, but not for DNA internalisation into the cytoplasm. The N-terminal region of the protein allows the interaction of ComH with a periplasmic domain of the inner-membrane channel ComEC, which is known to mediate the translocation of DNA into the cytoplasm. Our results indicate that ComH is involved in the import of DNA into the periplasm and its delivery to the inner membrane translocator ComEC.