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·sand 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.genome plasticity | horizontal gene transfer | molecular motor | natural transformation | single-cell analysis

Within the field of DNA nanotechnology, numerous methods were developed to produce complex two- and three-dimensional DNA nanostructures for many different emerging applications. These structures typically suffer from a low...

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The development of a sensitive screening procedure for mutants of Tetrahymena thermophila blocked in secretion of lysosomal enzymes is described. By means of this procedure a mutant blocked in secretion of lysosomal enzymes has been isolated. This sec- mutant, MS-1, is constitutively blocked in release of at least six lysosomal enzymes, under both nutrient and non-nutrient conditions. MS-1 possesses, bound within the cell, the same amount of active lysosomal enzymes as the wild type. During starvation in media of low ionic strength MS-1 develops a highly vacuolated phenotype. This phenotype is caused by the sec- allele. It is reversed to a normal cell shape when the mutant is transferred to isotonic medium. The sec- mutant MS-1 contains mucocysts and is capable of inducing exocytosis of these secretory organelles, suggesting that Tetrahymena possesses at least two independent protein-secreting organelles.

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