title: Composite system mediates two-step DNA uptake into Helicobacter pylori.
abstract: The Gram-negative gastric pathogen Helicobacter pylori depends on natural transformation for genomic plasticity, which leads to host adaptation and spread of resistances. Here, we show that H. pylori takes up covalently labeled fluorescent DNA preferentially at the cell poles and that uptake is dependent on the type IV secretion system ComB. By titration of external pH and detection of accessibility of the fluorophor by protons, we localized imported fluorescent DNA in the periplasm. Single molecule analysis revealed that outer membrane DNA transport occurred at a velocity of 1.3 kbp x s(-1) and that previously imported DNA was reversibly extracted from the bacterium at pulling forces exceeding 23 pN. Thus, transport velocities were 10-fold higher than in Bacillus subtilis, and stalling forces were substantially lower. dsDNA stained with the intercalator YOYO-1 was transiently detected in the periplasm in wild-type H. pylori but was periplasmatically trapped in a mutant lacking the B. subtilis membrane-channel homolog ComEC. We conclude that H. pylori uses a two-step DNA uptake mechanism in which ComB transports dsDNA across the outer membrane at low force and poor specificity for DNA structure. Subsequently, Hp-ComEC mediates transport into the cytoplasm, leading to the release of the noncovalently bound DNA dye. Our findings fill the gap to propose a model for composite DNA uptake machineries in competent bacteria, all comprising the conserved ComEC channel for cytoplasmic membrane transport in combination with various transporters for access of external DNA to the cytoplasmic membrane.
authors.affiliation: Westfälische Wilhelms-Universität, Institut für Allgemeine Zoologie und Genetik, 48149 Münster, Germany. k.stingl@web.de
authors.forename: Kerstin
authors.initials: K
authors.lastname: Stingl
authors.forename: Stephanie
authors.initials: S
authors.lastname: Müller
authors.forename: Gerda
authors.initials: G
authors.lastname: Scheidgen-Kleyboldt
authors.forename: Martin
authors.initials: M
authors.lastname: Clausen
authors.forename: Berenike
authors.initials: B
authors.lastname: Maier
mesh_terms: D001692:Biological Transport; D004247:DNA / Q000378:metabolism*; D005456:Fluorescent Dyes; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D009154:Mutation
chemical_list: D005456:Fluorescent Dyes; D004247:DNA