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

The type IV pilus (T4P) system of Neisseria gonorrhoeae is the strongest linear molecular motor reported to date, but it is unclear whether high-force generation is conserved between bacterial species. Using laser tweezers, we found that the average stalling force of single-pilus retraction in Myxococcus xanthus of 149 ؎ 14 pN exceeds the force generated by N. gonorrhoeae. Retraction velocities including a bimodal distribution were similar between M. xanthus and N. gonorrhoeae, but force-dependent directional switching was not. Force generation by pilus retraction is energized by the ATPase PilT. Surprisingly, an M. xanthus mutant lacking PilT apparently still retracted T4P, although at a reduced frequency. The retraction velocity was comparable to the high-velocity mode in the wild type at low forces but decreased drastically when the force increased, with an average stalling force of 70 ؎ 10 pN. Thus, M. xanthus harbors at least two different retraction motors. Our results demonstrate that the major physical properties are conserved between bacteria that are phylogenetically distant and pursue very different lifestyles.Type IV pili (T4P) are among the most widespread cell surface appendages in bacteria and have been found in beta-, gamma-, delta-, and epsilonproteobacteria and cyanobacteria, as well as in firmicutes (27). As opposed to other filamentous surface structures, T4P are highly dynamic structures and undergo cycles of extension and retraction (22,30,34). During the retraction step, sufficient force is generated to pull a bacterial cell forward in a type of surface movement referred to as twitching motility (30). The dynamic behavior is central to most of the functions of T4P, which in addition to cell motility, include surface adhesion, horizontal gene transfer, biofilm formation, and protein secretion (3).T4P are thin (5-to 8-nm) flexible filaments with a length of several micrometers (7). A core set of 10 proteins is conserved between different T4P systems and is required for T4P dynamics in Myxococcus xanthus, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, and Synechocystis sp. strain PCC6803 (24,27). Genetic and biochemical data suggest that the proteins required for T4P function interact to form a complex that spans the cell envelope (2, 9, 10, 14, 28). The molecular mechanism underlying the assembly of T4P involves the incorporation of pilin subunits in the base of the pilus (8) from a reservoir in the cytoplasmic membrane (15, 30), and retraction involves the removal and transfer of pilin subunits from the pilus base into the cytoplasmic membrane (23). Genetic and biochemical evidence suggest that assembly of T4P is energized by ATP hydrolysis by the assembly ATPase PilB (PilF in Neisseria spp.) (15,29) and that T4P retraction is energized by ATP hydrolysis by the retraction ATPase PilT (5, 11, 15).The soil-dwelling bacterium M. xanthus (a rod-shaped bacterium belonging to the deltaproteobacteria) requires T4P-dependent motility for the formation of spreading colonies i...

Type IV pili are major bacterial virulence factors supporting adhesion, surface motility, and gene transfer. The polymeric pilus fiber is a highly dynamic molecular machine that switches between elongation and retraction. We used laser tweezers to investigate the dynamics of individual pili of Neisseria gonorrheae at clamped forces between 8 pN and 100 pN and at varying concentration of the retraction ATPase PilT. The elongation probability of individual pili increased with increasing mechanical force. Directional switching occurred on two distinct timescales, and regular stepping was absent on a scale > 3 nm. We found that the retraction velocity is bimodal and that the bimodality depends on force and on the concentration of PilT proteins. We conclude that the pilus motor is a multistate system with at least one polymerization mode and two depolymerization modes with the dynamics fine-tuned by force and PilT concentration.

SummaryType IV pilus (T4P) dynamics is important for various bacterial functions including host cell interaction, surface motility, and horizontal gene transfer. T4P retract rapidly by depolymerization, generating large mechanical force. The gene that encodes the pilus retraction ATPase PilT has multiple paralogues, whose number varies between different bacterial species, but their role in regulating physical parameters of T4P dynamics remains unclear. Here, we address this question in the human pathogen Neisseria gonorrhoeae, which possesses two pilT paralogues, namely pilT2 and pilU. We show that the speed of twitching motility is strongly reduced in a pilT2 deletion mutant, while directional persistence time and sensitivity of speed to oxygen are unaffected. Using laser tweezers, we found that the speed of single T4P retraction was reduced by a factor of ª 2 in a pilT2 deletion strain, whereas pilU deletion showed a minor effect. The maximum force and the probability for switching from retraction to elongation under application of high force were not significantly affected. We conclude that the physical parameters of T4P are fine-tuned through PilT2.

We present an approach to measure the angular dependence of the diffusely scattered intensity of a multiple scattering sample in backscattering geometry. Increasing scattering strength give rise to an increased width of the coherent backscattering and sets higher demands on the angular detection range. This is of particular interest in the search for the transition to Anderson localization of light. To cover a range of −60°to +85°from direct back-reflection, we introduced a new parallel intensity recording technique. This allows one-shot measurements, with fast alignment and short measuring time, which prevents the influence of illumination variations. Configurational average is achieved by rotating the sample and singly scattered light is suppressed with the use of circularly polarized light up to 97%. This implies that backscattering enhancements of almost two can be achieved. In combination with a standard setup for measuring small angles up to ±3°, a full characterization of the coherent backscattering cone can be achieved. With this setup we are able to accurately determine transport mean free paths as low as 235 nm.