Eva Potthoff

Functional overlap of the Arabidopsis leaf and root microbiota

Roots and leaves of healthy plants host taxonomically structured bacterial assemblies, and members of these communities contribute to plant growth and health. We established Arabidopsis leaf- and root-derived microbiota culture collections representing the majority of bacterial species that are reproducibly detectable by culture-independent community sequencing. We found an extensive taxonomic overlap between the leaf and root microbiota. Genome drafts of 400 isolates revealed a large overlap of genome-encoded functional capabilities between leaf- and root-derived bacteria with few significant differences at the level of individual functional categories. Using defined bacterial communities and a gnotobiotic Arabidopsis plant system we show that the isolates form assemblies resembling natural microbiota on their cognate host organs, but are also capable of ectopic leaf or root colonization. While this raises the possibility of reciprocal relocation between root and leaf microbiota members, genome information and recolonization experiments also provide evidence for microbiota specialization to their respective niche.

Force-controlled manipulation of single cells: from AFM to FluidFM

The ability to perturb individual cells and to obtain information at the single-cell level is of central importance for addressing numerous biological questions. Atomic force microscopy (AFM) offers great potential for this prospering field. Traditionally used as an imaging tool, more recent developments have extended the variety of cell-manipulation protocols. Fluidic force microscopy (FluidFM) combines AFM with microfluidics via microchanneled cantilevers with nano-sized apertures. The crucial element of the technology is the connection of the hollow cantilevers to a pressure controller, allowing their operation in liquid as force-controlled nanopipettes under optical control. Proof-of-concept studies demonstrated a broad spectrum of single-cell applications including isolation, deposition, adhesion and injection in a range of biological systems.

Rapid and Serial Quantification of Adhesion Forces of Yeast and Mammalian Cells

Cell adhesion to surfaces represents the basis for niche colonization and survival. Here we establish serial quantification of adhesion forces of different cell types using a single probe. The pace of single-cell force-spectroscopy was accelerated to up to 200 yeast and 20 mammalian cells per probe when replacing the conventional cell trapping cantilever chemistry of atomic force microscopy by underpressure immobilization with fluidic force microscopy (FluidFM). In consequence, statistically relevant data could be recorded in a rapid manner, the spectrum of examinable cells was enlarged, and the cell physiology preserved until approached for force spectroscopy. Adhesion forces of Candida albicans increased from below 4 up to 16 nN at 37°C on hydrophobic surfaces, whereas a Δhgc1-mutant showed forces consistently below 4 nN. Monitoring adhesion of mammalian cells revealed mean adhesion forces of 600 nN of HeLa cells on fibronectin and were one order of magnitude higher than those observed for HEK cells.

Toward a Rational Design of Surface Textures Promoting Endothelialization

The safe integration of cardiovascular devices requires the sustainable coverage of their luminal surface by endothelial cells (ECs). The engineering of active surface textures has the potential to coordinate cellular adhesion and migration under the action of hemodynamic forces. We define a paradigm to rationally design textures maximizing EC activities as a function of the applied stresses. This is based on harnessing the adhesions established by ECs through fine-tuning of the vertical extend of the underlying surface nanotopography.

Spatial distribution analyses of natural phyllosphere-colonizing bacteria on Arabidopsis thaliana revealed by fluorescence in situ hybridization

Bacterial colonizers of the aerial parts of plants, or phyllosphere, have been identified on a number of different plants using cultivation-dependent and independent methods. However, the spatial distribution at the micrometer scale of different main phylogenetic lineages is not well documented and mostly based on fluorescence-tagged model strains. In this study, we developed and applied a spatial explicit approach that allowed the use of fluorescence in situ hybridization (FISH) to study bacterial phylloplane communities of environmentally grown Arabidopsis thaliana. We found on average 5.4 × 10(6) bacteria cm(-2) leaf surface and 1.5 × 10(8) bacteria g(-1) fresh weight. Furthermore, we found that the total biomass in the phylloplane was normally distributed. About 31% of the bacteria found in the phylloplane did not hybridize to FISH probes but exhibited infrared autofluorescence indicative for aerobic anoxygenic phototrophs. Four sets of FISH probes targeting Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes were sufficient to identify all other major contributors of the phylloplane community based on general bacterial probing. Spatial aggregation patterns were observed for all probe-targeted populations at distances up to 7 μm, with stronger tendencies to co-aggregate for members of the same phylogenetic group. Our findings contribute to a bottom-up description of leaf surface community composition.

SU-8 hollow cantilevers for AFM cell adhesion studies

A novel fabrication method was established to produce flexible, transparent, and robust tipless hollow atomic force microscopy (AFM) cantilevers made entirely from SU-8. Channels of 3 μm thickness and several millimeters length were integrated into 12 μm thick and 40 μm wide cantilevers. Connected to a pressure controller, the devices showed high sealing performance with no leakage up to 6 bars. Changing the cantilever lengths from 100 μm to 500 μm among the same wafer allowed the targeting of various spring constants ranging from 0.5 to 80 N m−1 within a single fabrication run. These hollow polymeric AFM cantilevers were operated in the optical beam deflection configuration. To demonstrate the performance of the device, single-cell force spectroscopy experiments were performed with a single probe detaching in a serial protocol more than 100 Saccharomyces cerevisiae yeast cells from plain glass and glass coated with polydopamine while measuring adhesion forces in the sub-nanoNewton range. SU-8 now offers a new alternative to conventional silicon-based hollow cantilevers with more flexibility in terms of complex geometric design and surface chemistry modification.

Cohesive Properties of the Caulobacter crescentus Holdfast Adhesin Are Regulated by a Novel c-di-GMP Effector Protein

ABSTRACT When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In Caulobacter crescentus, c-di-GMP regulates the synthesis of the polar holdfast adhesin during the cell cycle, yet the molecular and cellular details of this control are currently unknown. Here we identify HfsK, a member of a versatile N-acetyltransferase family, as a novel c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. We present indirect evidence that HfsK modifies the polysaccharide component of holdfast to buttress its cohesive properties. HfsK is a soluble protein but associates with the cell membrane during most of the cell cycle. Coincident with peak c-di-GMP levels during the C. crescentus cell cycle, HfsK relocalizes to the cytosol in a c-di-GMP-dependent manner. Our results indicate that this c-di-GMP-mediated dynamic positioning controls HfsK activity, leading to its inactivation at high c-di-GMP levels. A short C-terminal extension is essential for the membrane association, c-di-GMP binding, and activity of HfsK. We propose a model in which c-di-GMP binding leads to the dispersal and inactivation of HfsK as part of holdfast biogenesis progression. IMPORTANCE Exopolysaccharide (EPS) adhesins are important determinants of bacterial surface colonization and biofilm formation. Biofilms are a major cause of chronic infections and are responsible for biofouling on water-exposed surfaces. To tackle these problems, it is essential to dissect the processes leading to surface colonization at the molecular and cellular levels. Here we describe a novel c-di-GMP effector, HfsK, that contributes to the cohesive properties and stability of the holdfast adhesin in C. crescentus. We demonstrate for the first time that c-di-GMP, in addition to its role in the regulation of the rate of EPS production, also modulates the physicochemical properties of bacterial adhesins. By demonstrating how c-di-GMP coordinates the activity and subcellular localization of HfsK, we provide a novel understanding of the cellular processes involved in adhesin biogenesis control. Homologs of HfsK are found in representatives of different bacterial phyla, suggesting that they play important roles in various EPS synthesis systems. Exopolysaccharide (EPS) adhesins are important determinants of bacterial surface colonization and biofilm formation. Biofilms are a major cause of chronic infections and are responsible for biofouling on water-exposed surfaces. To tackle these problems, it is essential to dissect the processes leading to surface colonization at the molecular and cellular levels. Here we describe a novel c-di-GMP effector, HfsK, that contributes to the cohesive properties and stability of the holdfast adhesin in C. crescentus. We demonstrate for the first time that c-di-GMP, in addition to its role in the regulation of the rate of EPS production, also modulates the physicochemical properties of bacterial adhesins. By demonstrating how c-di-GMP coordinates the activity and subcellular localization of HfsK, we provide a novel understanding of the cellular processes involved in adhesin biogenesis control. Homologs of HfsK are found in representatives of different bacterial phyla, suggesting that they play important roles in various EPS synthesis systems.