Biological effects of Thymol loaded chitosan nanoparticles (TCNPs) on bacterial plant pathogen Xanthomonas campestris pv. campestris.

Engineered nanomaterials can provide eco-friendly alternatives for crop disease management. Chitosan based nanoparticles has shown beneficial applications in sustainable agricultural practices and effective healthcare. Previously we demonstrated that Thymol loaded chitosan nanoparticles (TCNPs) showed bactericidal activity against Xanthomonas campestris pv campestris (Xcc), a bacterium that causes black rot disease in brassica crops. Despite the progress in assessing the antibacterial action of TCNPs, the knowledge about the molecular response of Xcc when exposed to TCNPs is yet to be explored. In the present study, we combined physiological, spectroscopic and untargeted metabolomics studies to investigate the response mechanisms in Xcc induced by TCNPs. Cell proliferation and membrane potential assays of Xcc cells exposed to sub-lethal concentration of TCNPs showed that TCNPs affects the cell proliferation rate and damages the cell membrane altering the membrane potential. FTIR spectroscopy in conjunction with untargeted metabolite profiling using mass spectrometry of TCNPs treated Xcc cells revealed alterations in amino acids, lipids, nucleotides, fatty acids and antioxidant metabolites. Mass spectroscopy analysis revealed a 10-25% increase in nucleic acid, fatty acids and antioxidant metabolites and a 20% increase in lipid metabolites while a decrease of 10-20% in amino acids and carbohydrates was seen in in TCNP treated Xcc cells. Overall, our results demonstrate that the major metabolic perturbations induced by TCNPs in Xcc are associated with membrane damage and oxidative stress, thus providing information on the mechanism of TCNPs mediated cytotoxicity. This will aid towards the development of nano- based agrochemicals as an alternative to chemical pesticides in future.

Evaluating the Antibacterial Activity and Mode of Action of Thymol-Loaded Chitosan Nanoparticles Against Plant Bacterial Pathogen Xanthomonas campestris pv. campestris.

The bacterium Xanthomonas campestris pv. campestris (Xcc) causes black rot disease in cruciferous crops, resulting in severe yield loss worldwide. The excessive use of chemical pesticides in agriculture to control diseases has raised significant concern about the impact on the environment and human health. Nanoparticles have recently gained significant attention in agriculture owing to their promising application in plant disease control, increasing soil fertility and nutrient availability. In the current study, we synthesized thymol-loaded chitosan nanoparticles (TCNPs) and assessed their antibacterial activity against Xcc. The synthesis of TCNPs was confirmed by using ultraviolet-visible spectroscopy. Fourier-transform infrared spectroscopy, transmission electron microscopy, and scanning electron microscopy analysis revealed the functional groups, size, and shape of TCNPs, with sizes ranging from 54 to 250 nm, respectively. The antibacterial activity of TCNPs against Xcc was investigated in vitro by liquid broth, cell viability, and live dead staining assay, and all of them demonstrated the antibacterial activity of TCNPs. Furthermore, TCNPs were found to directly inhibit the growth of Xcc by suppressing the growth of biofilm formation and the production of exopolysaccharides and xanthomonadin. The ultrastructure studies revealed membrane damage in TCNP-treated Xcc cells, causing a release of intracellular contents. Headspace/gas chromatography (GC)-mass spectrometry (MS) analysis showed changes in the volatile profile of Xcc cells treated with TCNPs. Increased amounts of carbonyl components (mainly ketones) and production of new volatile metabolites were observed in Xcc cells incubated with TCNPs. Overall, this study reveals TCNPs as a promising antibacterial candidate against Xcc.

Biomimetic Autonomous Enzymatic Nanowalker of High Fuel Efficiency.

Replicating efficient chemical energy utilization of biological nanomotors is one ultimate goal of nanotechnology and energy technology. Here, we report a rationally designed autonomous bipedal nanowalker made of DNA that achieves a fuel efficiency of less than two fuel molecules decomposed per productive forward step, hence breaking a general threshold for chemically powered machines invented to date. As a genuine enzymatic nanomotor without changing itself nor the track, the walker demonstrates a sustained motion on an extended double-stranded track at a speed comparable to previous burn-bridge motors. Like its biological counterparts, this artificial nanowalker realizes multiple chemomechanical gatings, especially a bias-generating product control unique to chemically powered nanomotors. This study yields rich insights into how pure physical effects facilitate harvest of chemical energy at the single-molecule level and provides a rarely available motor system for future development toward replicating the efficient, repeatable, automatic, and mechanistically sophisticated transportation seen in biomotor-based intracellular transport but beyond the capacity of the current burn-bridge motors.

Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps.

Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor's mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A's molecular motility mechanism.

A bioinspired design principle for DNA nanomotors: mechanics-mediated symmetry breaking and experimental demonstration.

DNA nanotechnology is a powerful tool to fabricate nanoscale motors, but the DNA nanomotors to date are largely limited to the simplistic burn-the-bridge design principle that prevents re-use of a fabricated motor-track system and is unseen in biological nanomotors. Here we propose and experimentally demonstrate a scheme to implement a conceptually new design principle by which a symmetric bipedal nanomotor autonomously gains a direction not by damaging the traversed track but by fine-tuning the motor's size.

Autonomous synergic control of nanomotors.

Control is a hallmark of machines; effective control over a nanoscale system is necessary to turn it into a nanomachine. Nanomotors from biology often integrate a ratchet-like passive control and a power-stroke-like active control, and this synergic active-plus-passive control is critical to efficient utilization of energy. It remains a challenge to integrate the two differing types of control in rationally designed nanomotor systems. Recently a light-powered track-walking DNA nanomotor was developed from a bioinspired design principle that has the potential to integrate both controls. However, it is difficult to separate experimental signals for either control due to a tight coupling of both controls. Here we present a systematic study of the motor and new derivatives using different fluorescence labeling schemes and light operations. The experimental data suggest that the motor achieves the two controls autonomously through a mechanics-mediated symmetry breaking. This study presents an experimental validation for the bioinspired design principle of mechanical breaking of symmetry for synergic ratchet-plus-power stroke control. Augmented by mechanical and kinetic modeling, this experimental study provides mechanistic insights that may help advance molecular control in future nanotechnological systems.

Bipedal nanowalker by pure physical mechanisms.

Artificial nanowalkers are inspired by biomolecular counterparts from living cells, but remain far from comparable to the latter in design principles. The walkers reported to date mostly rely on chemical mechanisms to gain a direction; they all produce chemical wastes. Here we report a light-powered DNA bipedal walker based on a design principle derived from cellular walkers. The walker has two identical feet and the track has equal binding sites; yet the walker gains a direction by pure physical mechanisms that autonomously amplify an intrasite asymmetry into a ratchet effect. The nanowalker is free of any chemical waste. It has a distinct thermodynamic feature that it possesses the same equilibrium before and after operation, but generates a truly nonequilibrium distribution during operation. The demonstrated design principle exploits mechanical effects and is adaptable for use in other nanomachines.