Berenike Maier

Single pilus motor forces exceed 100 pN

Force production by type IV pilus retraction is critical for infectivity of Neisseria gonorrhoeae and DNA transfer. We investigated the roles of pilus number and the retraction motor, PilT, in force generation in vivo at the single-molecule level and found that individual retraction events are generated by a single pilus fiber, and only one PilT complex powers retraction. Retraction velocity is constant at low forces but decreases at forces greater than 40 pN, giving a remarkably high average stall force of 110 ± 30 pN. Further insights into the molecular mechanism of force generation are gained from the effect of ATP-depletion, which reduces the rate of retraction but not the stall force. Energetic considerations suggest that more than one ATP is involved in the removal of a single pilin subunit from a pilus. The results are most consistent with a model in which the ATPase PilT forms an oligomer that disassembles the pilus by a cooperative conformational change.

Transformation Proteins and DNA Uptake Localize to the Cell Poles in Bacillus subtilis

The Gram-positive, rod-forming bacterium Bacillus subtilis efficiently binds and internalizes transforming DNA. The localization of several competence proteins, required for DNA uptake, has been studied using fluorescence microscopy. At least three proteins (ComGA, ComFA, and YwpH) are preferentially associated with the cell poles and appear to colocalize. This association is dynamic; the proteins accumulate at the poles as transformability develops and then delocalize as transformability wanes. DNA binding and uptake also occur preferentially at the cell poles, as shown using fluorescent DNA and in single-molecule experiments with laser tweezers. In addition to the prominent polar sites, the competence proteins also localize as foci in association with the lateral cell membrane, but this distribution does not exhibit the same temporal changes as the polar accumulation. The results suggest the regulated assembly and disassembly of a DNA-uptake machine at the cell poles.

Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins

Type IV pili (T4P) are surface structures that undergo extension/retraction oscillations to generate cell motility. In Myxococcus xanthus, T4P are unipolarly localized and undergo pole‐to‐pole oscillations synchronously with cellular reversals. We investigated the mechanisms underlying these oscillations. We show that several T4P proteins localize symmetrically in clusters at both cell poles between reversals, and these clusters remain stationary during reversals. Conversely, the PilB and PilT motor ATPases that energize extension and retraction, respectively, localize to opposite poles with PilB predominantly at the piliated and PilT predominantly at the non‐piliated pole, and these proteins oscillate between the poles during reversals. Therefore, T4P pole‐to‐pole oscillations involve the disassembly of T4P machinery at one pole and reassembly of this machinery at the opposite pole. Fluorescence recovery after photobleaching experiments showed rapid turnover of YFP–PilT in the polar clusters between reversals. Moreover, PilT displays bursts of accumulation at the piliated pole between reversals. These observations suggest that the spatial separation of PilB and PilT in combination with the noisy PilT accumulation at the piliated pole allow the temporal separation of extension and retraction. This is the first demonstration that the function of a molecular machine depends on disassembly and reassembly of its individual parts.

Composite system mediates two-step DNA uptake into Helicobacter pylori

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·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.

High-Force Generation Is a Conserved Property of Type IV Pilus Systems

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.

Searching for dark matter with the enriched Ge detectors of the Heidelberg-Moscow ββ experiment

Abstract For the first time a search for dark matter with isotopically enriched material is done, by using the Ge detectors of the Heidelberg-Moscow experiment. A measuring time of 165.6 kg·d is used to set limits on the spin-independent cross section of weakly interacting massive particles (WIMPs). A background level of 0.102±0.005 events/(kg·d·keV) was achieved (average value between 11 keV and 30 keV). It was possible to extend the exclusion range for Dirac neutrino masses up to 4.7 TeV.

Full 3D translational and rotational optical control of multiple rod‐shaped bacteria

The class of rod-shaped bacteria is an important example of non-spherical objects where defined alignment is desired for the observation of intracellular processes or studies of the flagella. However, all available methods for orientational control of rod-shaped bacteria are either limited with respect to the accessible rotational axes or feasible angles or restricted to one single bacterium. In this paper we demonstrate a scheme to orientate rod-shaped bacteria with holographic optical tweezers (HOT) in any direction. While these bacteria have a strong preference to align along the direction of the incident laser beam, our scheme provides for the first time full rotational control of multiple bacteria with respect to any arbitrary axis. In combination with the translational control HOT inherently provide, this enables full control of all three translational and the two important rotational degrees of freedom of multiple rod-shaped bacteria and allows one to arrange them in any desired configuration.

Stochastic switching to competence

Distinct modes of gene expression enable isogenic populations of bacteria to maintain a diversity of phenotypes and to rapidly adapt to environmental changes. Competence development for DNA transformation in Bacillus subtilis has become a paradigm for a multimodal system which implements a genetic switch through a nonlinear positive feedback of a transcriptional master regulator. Recent advances in quantitative analysis at the single cell level in conjunction with mathematical modeling allowed a molecular level understanding of the switching probability between the noncompetent state and the competent state. It has been discovered that the genetic switching probability may be tuned by controlling noise in the transcription of the master regulator of competence, by timing of its expression, and by rewiring of the control circuit.

How Bacteria Use Type IV Pili Machinery on Surfaces

The bacterial type IV pilus (T4P) is a versatile molecular machine with a broad range of functions. Recent advances revealed that the molecular components and the biophysical properties of the machine are well conserved among phylogenetically distant bacterial species. However, its functions are diverse, and include adhesion, motility, and horizontal gene transfer. This review focusses on the role of T4P in surface motility and bacterial interactions. Different species have evolved distinct mechanisms for intracellular coordination of multiple pili and of pili with other motility machines, ranging from physical coordination to biochemical clocks. Coordinated behavior between multiple bacteria on a surface is achieved by active manipulation of surfaces and modulation of pilus-pilus interactions. An emerging picture is that the T4P actively senses and responds to environmental conditions.

Dynamics of Type IV Pili Is Controlled by Switching Between Multiple States

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.