The FluidFM enables the immobilization of single cells on a hollow cantilever using relative underpressure. In this study, we systematically optimize versatile measurement parameters (setpoint, z-speed, z-length, pause time, and relative underpressure) to improve the quality of force-distance curves recorded with a FluidFM. Using single bacterial cells (here the gram negative seawater bacterium Paracoccus seriniphilus and the gram positive bacterium Lactococcus lactis), we show that Single Cell Force Spectroscopy experiments with the FluidFM lead to comparable results to a conventional Single Cell Force Spectroscopy approach using polydopamine for chemical fixation of a bacterial cell on a tipless cantilever. Even for the bacterium Lactococcus lactis, which is difficult to immobilze chemically (like seen in an earlier study), immobilization and the measurement of force-distance curves are possible by using the FluidFM technology.
‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) has only one major membrane protein, the immunodominant membrane protein (Imp), which is regarded as being close to the ancestor of all phytoplasma immunodominant membrane proteins. Imp binds to actin and possibly facilitates its movement in the plant or insect host cells. However, protein sequences of Imp are quite diverse among phytoplasma species, thus resulting in difficulties in identifying conserved domains across species. In this work, we compare Imp protein sequences of ‘Ca. P. mali’ strain PM19 (Imp-PM19) with Imp of different strains of ‘Ca. P. mali’ and identify its actin-binding domain. Moreover, we show that Imp binds to the actin of apple (Malus x domestica), which is the host plant of ‘Ca. P. mali’. Using molecular and scanning force spectroscopy analysis, we find that the actin-binding domain of Imp-PM19 contains a highly positively charged amino acid cluster. Our result could allow investigating a possible correlation between Imp variants and the infectivity of the corresponding ‘Ca. P. mali’ isolates.
Microorganisms growing in biofilms might be possible biocatalysts for future biotechnological production processes. Attached to a surface and embedded in an extracellular polymeric matrix, they create their preferred environment and form robust cultures for continuous systems. With the objective of implementing highly efficient processes, productive biofilms need to be understood comprehensively. In this study, the influence of microstructured metallic surfaces on biofilm productivity was researched. To conduct this study, titanium and stainless steel sheets were polished, micromilled, as well as coated with particles. Subsequently, the metal sheets were exposed to the lactic acid producing Lactobacillus delbrueckii subsp. lactis under laminar and homogeneous flow conditions in a custom‐built flow cell. A proof‐of‐concept showed that biofilm formation in the system only occurred on the designated substratum. Following a 24‐h batch cultivation for primary biofilm development, the culture was continuously provided with glucose containing medium. As different experimental series have indicated, the process resulted to be stable for up to eleven days. Primary metabolite productivity averaged around 6–7 g/(L h). Interestingly, the productivity was shown to be affected neither by the type of metal, nor by the applied microstructures. Nevertheless, a higher dry biomass weight determined on micro‐milled substratum indicates a complementary differentiation of biofilm components in future experiments.
In nanobiotechnology, viral nanoparticles have come into focus as interesting nano building blocks. In this context, the formation of 2D and 3D structures is of particular interest. Here, we report on the creation of defined 2D patterns of an icosahedral plant virus, the tomato bushy stunt virus (TBSV) by means of different techniques: the top-down lithographies ebeam and focused ion beam (FIB) as well as the bottom-up Fluidic Force Microscope (FluidFM™) approach. The obtained layer structures were imaged by scanning force and scanning electron microscopy. The data show that a defined 2D structure can successfully be created either top-down by FIB or bottom-up by FluidFM. Electron beam lithography was not able to remove viruses from the substrate under the chosen conditions. FIB has an advantage if larger areas covered with viruses combined with smaller areas without being desired. FluidFM is advantageous if only small areas with viruses are required. A further benefit is, that the uncovered areas are not affected. The pattern formation in FluidFM is not only influenced by the spotting parameters but in particular by the drying process. It will be shown, that Deegan and Marangoni effects are playing a role if the spotted droplets are not very small.
The formation and colonization of biofilms inside the oral cavity are still not understood in detail, although biofilms inside the oral cavity can lead to expensive dental diseases like caries, periodontitis, or the detachment of implants. Such biofilms consist, to a large extent, of proteins, carbohydrates, and other macromolecules. Whereas the interaction of proteins with dental materials is widely studied, the literature does not report interactions of polysaccharides. Here, scanning force spectroscopy is used to investigate the adhesion forces of dextran as an abundant polysaccharide on different dental materials. The focus is dental titanium, accompanied by comparisons with dental gold and silicon as reference material. Different pH values and dental materials mimic representative conditions in the oral cavity. The main finding is that dextran adheres very well to dental materials, but with lower adhesion forces than proteins of similar mass, such as bovine serum albumin. As proteins, the adhesion forces increase with the contact time until a plateau is reached and the forces differ with the dental material. However, in contrast to proteins, pH does not play a role because dextran is uncharged over the measured range between pH 4.5 and pH 13.