Andreas Gahlmann

Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging.

The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field.

Ultrashort electron pulses for diffraction, crystallography and microscopy: theoretical and experimental resolutions

Pulsed electron beams allow for the direct atomic-scale observation of structures with femtosecond to picosecond temporal resolution in a variety of fields ranging from materials science to chemistry and biology, and from the condensed phase to the gas phase. Motivated by recent developments in ultrafast electron diffraction and imaging techniques, we present here a comprehensive account of the fundamental processes involved in electron pulse propagation, and make comparisons with experimental results. The electron pulse, as an ensemble of charged particles, travels under the influence of the space-charge effect and the spread of the momenta among its electrons. The shape and size, as well as the trajectories of the individual electrons, may be altered. The resulting implications on the spatiotemporal resolution capabilities are discussed both for the N-electron pulse and for single-electron coherent packets introduced for microscopy without space-charge.

Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions.

We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.

Bacterial scaffold directs pole-specific centromere segregation

Significance Bacteria use molecular partitioning systems based on the ATPase ParA to segregate chromosome centromeres before cell division, but how these machines target centromeres to specific locations is unclear. This study shows that, in Caulobacter crescentus, a multimeric complex composed of the PopZ protein directs the ParA machine to transfer centromeres to the cell pole. Spent ParA subunits released from the mitotic apparatus during segregation are recruited throughout a 3D PopZ matrix at the pole. ParA recruitment and sequestration by PopZ stimulates the cell-pole proximal recycling of ParA into a nucleoid-bound complex to ensure pole-specific centromere transfer. PopZ therefore utilizes a 3D scaffolding strategy to create a subcellular microdomain that directly regulates the function of the bacterial centromere segregation machine. Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.

Ultrafast Electron Diffraction Reveals Dark Structures of the Biological Chromophore Indole

With UED, as detailed in the experimental section, we are able to determine both ground- and excited-state structures and obtain the temporal behavior for excitation of indole at 267 nm. For the ground-state structure (see Scheme SI1 in the Supporting Information), the experimental and the theoretical molecular scattering function, sM(s), together with the radial distributions, f(r), are shown in Figure 1. The refined structural parameters are listed in Table SI1 in the Supporting Information, together with values obtained from density functional theory (DFT) calculations. The satisfactory agreement between experiment and theory gives the refined structural parameters, which were found to have discrepancies at most within 0.007 A and 0.18° for bond lengths and angles, respectively.

Structure of Isolated Biomolecules by Electron Diffraction−Laser Desorption: Uracil and Guanine

We report the structure of isolated biomolecules, uracil and guanine, demonstrating the capability of a newly developed electron diffraction apparatus augmented with surface-assisted IR laser desorption. This UED-4 apparatus provides a pulsed, dense molecular beam, which is stable for many hours and possibly days. From the diffraction patterns, it is evident that the plume composition is chemically pure, without detectable background from ions, fragmentation products, or molecular aggregates. The vibrational temperature deduced is indeed lower than the translational temperature of the plume indicating that the molecules are intact on such short time scales. The structures of uracil and guanine were refined at the deduced internal temperatures, and we compare the results with those predicted by density functional theory. Such experimental capability opens the door for many other studies of the structure (and dynamics) of biomolecules.

Direct Structural Determination of Conformations of Photoswitchable Molecules by Laser Desorption-Electron Diffraction

Anfractuous paths: Electron diffraction reveals the involvement of multiple structures in the complex photochemistry of photoswitchable nitro-substituted 1,3,3-trimethylindolinobenzospiropyran. The spiropyran-to-merocyanine isomerization due to ring opening produces primarily the cis–trans–cis structure (see picture; red O, blue N, yellow C), while competing nonradiative pathways lead to other structures, namely the closed forms in their triplet and singlet ground states.

Structural Dynamics of Free Amino Acids in Diffraction

The amino acid tryptophan exhibits complex structural changes both in the ground state, owing to multiple conformations, and upon excitation, because of the involvement of nonradiative pathways. The first report of structural dynamics using combined ultrafast electron diffraction and laser desorption methods is presented.