On-Chip Fabrication of Colloidal Suprastructures by Assembly and Supramolecular Interlinking of Microgels.

In this report, a versatile method is demonstrated to create colloidal suprastructures by assembly and supramolecular interlinking of microgels using droplet-based microfluidics. The behavior of the microgels is systematically investigated to evaluate the influence of their concentration on their distribution between the continuous, the droplet phase, and the interface. At low concentrations, microgels are mainly localized at the water-oil interface whereas an excess of microgels results, following the complete coverage of the water-oil interface, in their distribution in the continuous phase. To stabilize the colloidal suprastructure, on-chip gelation is introduced by adding natural polyphenol tannic acid (TA) in the water phase. TA forms interparticle linking between the poly(N-vinylcaprolactam) (PVCL) microgels by supramolecular interactions. The combination of supramolecular interlinking with the variation of the microgel concentration in microfluidic droplets enables on-chip fabrication of defined colloidal suprastructures with morphologies ranging from colloidosomes to colloidal supraballs. The obtained supracolloidal structures exhibit a pH-responsive behavior with a disintegration at alkaline conditions within a scale of seconds. The destabilization process results from the deprotonation of phenolic groups and destruction of hydrogen bonds with PVCL chains at higher pH.

Engineering Fe(II)/α-Ketoglutarate-Dependent Halogenases and Desaturases.

Fe(II)/α-ketoglutarate-dependent dioxygenases (α-KGDs) are widespread enzymes in aerobic biology and serve a remarkable array of biological functions, including roles in collagen biosynthesis, plant and animal development, transcriptional regulation, nucleic acid modification, and secondary metabolite biosynthesis. This functional diversity is reflected in the enzymes' catalytic flexibility as α-KGDs can catalyze an intriguing set of synthetically valuable reactions, such as hydroxylations, halogenations, and desaturations, capturing the interest of scientists across disciplines. Mechanistically, all α-KGDs are understood to follow a similar activation pathway to generate a substrate radical, yet how individual members of the enzyme family direct this key intermediate toward the different reaction outcomes remains elusive, triggering structural, computational, spectroscopic, kinetic, and enzyme engineering studies. In this Perspective, we will highlight how first enzyme and substrate engineering examples suggest that the chemical reaction pathway within α-KGDs can be intentionally tailored using rational design principles. We will delineate the structural and mechanistic investigations of the reprogrammed enzymes and how they begin to inform about the enzymes' structure-function relationships that determine chemoselectivity. Application of this knowledge in future enzyme and substrate engineering campaigns will lead to the development of powerful C-H activation catalysts for chemical synthesis.

Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress.

Bacillus methanolicus MGA3 is a model facultative methylotroph of interest for fundamental research and biotechnological applications. Previous research uncovered a number of pathways potentially involved in one-carbon substrate utilization. Here, we applied dynamic (13) C labeling to elucidate which of these pathways operate during growth on methanol and to uncover potentially new ones. B. methanolicus MGA3 uses the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycles for conversion of the central but toxic intermediate formaldehyde. Additionally, the operation of two cofactor-dependent formaldehyde oxidation pathways with distinct roles was revealed. One is dependent on tri- and tetraglutamylated tetrahydrofolate (THF) and is involved in formaldehyde oxidation during growth on methanol. A second pathway was discovered that is dependent on bacillithiol, a thiol cofactor present also in other Bacilli where it is known to function in redox-homeostasis. We show that bacillithiol-dependent formaldehyde oxidation is activated upon an upshift in formaldehyde induced by a substrate switch from mannitol to methanol. The genes and the corresponding enzymes involved in the biosynthesis of bacillithiol were identified by heterologous production of bacillithiol in Escherichia coli. The presented results indicate metabolic plasticity of the methylotroph allowing acclimation to fluctuating intracellular formaldehyde concentrations.

Immunohistochemistry and real-time PCR as diagnostic tools for detection of Borrelia burgdorferi sensu lato in ticks collected from humans.

The objective of this study was to evaluate different methods used for detection of Borrelia burgdorferi sensu lato (s.l.) in ticks: immunohistochemistry followed by focus floating microscopy (FFM) and real-time polymerase chain reaction (real-time PCR) targeting the ospA and hbb genes. Additionally, an optimized ospA real-time PCR assay was developed with an integrated internal amplification control (IAC) for the detection of inhibition in the PCR assay and was validated as an improved screening tool for B. burgdorferi. One hundred and thirty-six ticks collected from humans in a hospital from Cluj-Napoca, Romania, were investigated regarding genus, stage of development and sex, and then tested by all three assays. A poor quality of agreement was found between FFM and each of the two real-time PCR assays, as assessed by concordance analysis (Cohen's kappa), whereas the agreement between the two real-time PCR assays was moderate. The present study argues for a low sensitivity of FFM and underlines that discordant results of different assays used for detection of B. burgdorferi in ticks are frequent.

DynaMet: a fully automated pipeline for dynamic LC-MS data.

Dynamic isotope labeling data provides crucial information about the operation of metabolic pathways and are commonly generated via liquid chromatography-mass spectrometry (LC-MS). Metabolome-wide analysis is challenging as it requires grouping of metabolite features over different samples. We developed DynaMet for fully automated investigations of isotope labeling experiments from LC-high-resolution MS raw data. DynaMet enables untargeted extraction of metabolite labeling profiles and provides integrated tools for expressive data visualization. To validate DynaMet we first used time course labeling data of the model strain Bacillus methanolicus from (13)C methanol resulting in complex spectra in multicarbon compounds. Analysis of two biological replicates revealed high robustness and reproducibility of the pipeline. In total, DynaMet extracted 386 features showing dynamic labeling within 10 min. Of these features, 357 could be fitted by implemented kinetic models. Feature identification against KEGG database resulted in 215 matches covering multiple pathways of core metabolism and major biosynthetic routes. Moreover, we performed time course labeling experiment with Escherichia coli on uniformly labeled (13)C glucose resulting in a comparable number of detected features with labeling profiles of high quality. The distinct labeling patterns of common central metabolites generated from both model bacteria can readily be explained by one versus multicarbon compound metabolism. DynaMet is freely available as an extension package for Python based eMZed2, an open source framework built for rapid development of LC-MS data analysis workflows.

Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair.

Engineering Escherichia coli for methanol conversion.

Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

Cell wall synthesizing complexes in Mycobacteriales.

Members of the order Mycobacteriales are distinguished by a characteristic diderm cell envelope, setting them apart from other Actinobacteria species. In addition to the conventional peptidoglycan cell wall, these organisms feature an extra polysaccharide polymer composed of arabinose and galactose, termed arabinogalactan. The nonreducing ends of arabinose are covalently linked to mycolic acids (MAs), forming the immobile inner leaflet of the highly hydrophobic MA membrane. The contiguous outer leaflet of the MA membrane comprises trehalose mycolates and various lipid species. Similar to all actinobacteria, Mycobacteriales exhibit apical growth, facilitated by a polar localized elongasome complex. A septal cell envelope synthesis machinery, the divisome, builds instead of the cell wall structures during cytokinesis. In recent years, a growing body of knowledge has emerged regarding the cell wall synthesizing complexes of Mycobacteriales., focusing particularly on three model species: Corynebacterium glutamicum, Mycobacterium smegmatis, and Mycobacterium tuberculosis.

Elucidating the Role of O2 Uncoupling for the Adaptation of Bacterial Biodegradation Reactions Catalyzed by Rieske Oxygenases.

Oxygenation of aromatic and aliphatic hydrocarbons by Rieske oxygenases is the initial step of various biodegradation pathways for environmental organic contaminants. Microorganisms carrying Rieske oxygenases are able to quickly adapt their substrate spectra to alternative carbon and energy sources that are structurally related to the original target substrate, yet the molecular events responsible for this rapid adaptation are not well understood. Here, we evaluated the hypothesis that reactive oxygen species (ROS) generated by unproductive activation of O2, the so-called O2 uncoupling, in the presence of the alternative substrate exert a selective pressure on the bacterium for increasing the oxygenation efficiency of Rieske oxygenases. To that end, we studied wild-type 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42 and five enzyme variants that have evolved from adaptive laboratory evolution experiments with 3- and 4-nitrotoluene as alternative growth substrates. The enzyme variants showed a substantially increased oxygenation efficiency toward the new target substrates concomitant with a reduction of ROS production, while mechanisms and kinetics of enzymatic O2 activation remained unchanged. Structural analyses and docking studies suggest that amino acid substitutions in enzyme variants occurred at residues lining both substrate and O2 transport tunnels, enabling tighter binding of the target substrates in the active site. Increased oxygenation efficiencies measured in vitro for the various enzyme (variant)-substrate combinations correlated linearly with in vivo changes in growth rates for evolved Acidovorax strains expressing the variants. Our data suggest that the selective pressure from oxidative stress toward more efficient oxygenation by Rieske oxygenases was most notable when O2 uncoupling exceeded 60%.

An outer membrane porin-lipoprotein complex modulates elongasome movement to establish cell curvature in Rhodospirillum rubrum.

Curved cell shapes are widespread among bacteria and important for cellular motility, virulence and fitness. However, the underlying morphogenetic mechanisms are still incompletely understood. Here, we identify an outer-membrane protein complex that promotes cell curvature in the photosynthetic species Rhodospirillum rubrum. We show that the R. rubrum porins Por39 and Por41 form a helical ribbon-like structure at the outer curve of the cell that recruits the peptidoglycan-binding lipoprotein PapS, with PapS inactivation, porin delocalization or disruption of the porin-PapS interface resulting in cell straightening. We further demonstrate that porin-PapS assemblies act as molecular cages that entrap the cell elongation machinery, thus biasing cell growth towards the outer curve. These findings reveal a mechanistically distinct morphogenetic module mediating bacterial cell shape. Moreover, they uncover an unprecedented role of outer-membrane protein patterning in the spatial control of intracellular processes, adding an important facet to the repertoire of regulatory mechanisms in bacterial cell biology.

Effects of benzothiazinone and ethambutol on the integrity of the corynebacterial cell envelope.

The mycomembrane (MM) is a mycolic acid layer covering the surface of Mycobacteria and related species. This group includes important pathogens such as Mycobacterium tuberculosis, Corynebacterium diphtheriae, but also the biotechnologically important strain Corynebacterium glutamicum. Biosynthesis of the MM is an attractive target for antibiotic intervention. The first line anti-tuberculosis drug ethambutol (EMB) and the new drug candidate, benzothiazinone 043 (BTZ) interfere with the synthesis of the arabinogalactan (AG), which is a structural scaffold for covalently attached mycolic acids that form the inner leaflet of the MM. We previously showed that C. glutamicum cells treated with a sublethal concentration of EMB lose the integrity of the MM. In this study we examined the effects of BTZ on the cell envelope. Our work shows that BTZ efficiently blocks the apical growth machinery, however effects in combinatorial treatment with ß-lactam antibiotics are only additive, not synergistic. Transmission electron microscopy (TEM) analysis revealed a distinct middle layer in the septum of control cells considered to be the inner leaflet of the MM covalently attached to the AG. This layer was not detectable in the septa of BTZ or EMB treated cells. In addition, we observed that EMB treated cells have a thicker and less electron dense peptidoglycan (PG). While EMB and BTZ both effectively block elongation growth, BTZ also strongly reduces septal cell wall synthesis, slowing down growth effectively. This renders BTZ treated cells likely more tolerant to antibiotics that act on growing bacteria.

Novel Pectin Binder for Satelliting Carbides to H13 Tool Steel for PBF-LB Processing.

In order to enhance the range of processable alloys of laser-based powder bed fusion, reinforced alloys have gained focus. Satelliting is a recently introduced method for adding fine additives to larger parent powder particles using a bonding agent. Satellited particles prevent a local demixing due to size and density effects of the powder. In this study, the satelliting method is used for the additivation of Cr3C2 to AISI H13 tool steel via a functional polymer binder (pectin). The investigation includes a detailed binder analysis and comparison to the previously used PVA binder as well as processability in PBF-LB and the microstructure of the alloy. The results reveal that pectin is a suitable binder for the satelliting process and the demixing behavior that appears when using a simple powder blend can be significantly reduced. However, the alloy is enriched with carbon, which results in austenite being retained. Thus, in future research, a reduced binder content will be investigated.

Subcellular Dynamics of a Conserved Bacterial Polar Scaffold Protein.

In order to survive, bacterial cells rely on precise spatiotemporal organization and coordination of essential processes such as cell growth, chromosome segregation, and cell division. Given the general lack of organelles, most bacteria are forced to depend on alternative localization mechanisms, such as, for example, geometrical cues. DivIVA proteins are widely distributed in mainly Gram-positive bacteria and were shown to bind the membrane, typically in regions of strong negative curvature, such as the cell poles and division septa. Here, they have been shown to be involved in a multitude of processes: from apical cell growth and chromosome segregation in actinobacteria to sporulation and inhibition of division re-initiation in firmicutes. Structural analyses revealed that DivIVA proteins can form oligomeric assemblies that constitute a scaffold for recruitment of other proteins. However, it remained unclear whether interaction with partner proteins influences DivIVA dynamics. Using structured illumination microscopy (SIM), single-particle tracking (SPT) microscopy, and fluorescent recovery after photobleaching (FRAP) experiments, we show that DivIVA from Corynebacterium glutamicum is mobilized by its binding partner ParB. In contrast, we show that the interaction between Bacillus subtilis DivIVA and its partner protein MinJ reduces DivIVA mobility. Furthermore, we show that the loss of the rod-shape leads to an increase in DivIVA dynamics in both organisms. Taken together, our study reveals the modulation of the polar scaffold protein by protein interactors and cell morphology. We reason that this leads to a very simple, yet robust way for actinobacteria to maintain polar growth and their rod-shape. In B. subtilis, however, the DivIVA protein is tailored towards a more dynamic function that allows quick relocalization from poles to septa upon division.

Single-cell growth inference of Corynebacterium glutamicum reveals asymptotically linear growth.

Regulation of growth and cell size is crucial for the optimization of bacterial cellular function. So far, single bacterial cells have been found to grow predominantly exponentially, which implies the need for tight regulation to maintain cell size homeostasis. Here, we characterize the growth behavior of the apically growing bacterium Corynebacterium glutamicum using a novel broadly applicable inference method for single-cell growth dynamics. Using this approach, we find that C. glutamicum exhibits asymptotically linear single-cell growth. To explain this growth mode, we model elongation as being rate-limited by the apical growth mechanism. Our model accurately reproduces the inferred cell growth dynamics and is validated with elongation measurements on a transglycosylase deficient ΔrodA mutant. Finally, with simulations we show that the distribution of cell lengths is narrower for linear than exponential growth, suggesting that this asymptotically linear growth mode can act as a substitute for tight division length and division symmetry regulation.

FtsZ induces membrane deformations via torsional stress upon GTP hydrolysis.

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.

Imaging and milling resolution of light ion beams from helium ion microscopy and FIBs driven by liquid metal alloy ion sources.

While the application of focused ion beam (FIB) techniques has become a well-established technique in research and development for patterning and prototyping on the nanometer scale, there is still a large underused potential with respect to the usage of ion species other than gallium. Light ions in the range of m = 1-28 u (hydrogen to silicon) are of increasing interest due to the available high beam resolution in the nanometer range and their special chemical and physical behavior in the substrate. In this work, helium and neon ion beams from a helium ion microscope are compared with ion beams such as lithium, beryllium, boron, and silicon, obtained from a mass-separated FIB using a liquid metal alloy ion source (LMAIS) with respect to the imaging and milling resolution, as well as the current stability. Simulations were carried out to investigate whether the experimentally smallest ion-milled trenches are limited by the size of the collision cascade. While He+ offers, experimentally and in simulations, the smallest minimum trench width, light ion species such as Li+ or Be+ from a LMAIS offer higher milling rates and ion currents while outperforming the milling resolution of Ne+ from a gas field ion source. The comparison allows one to select the best possible ion species for the specific demands in terms of resolution, beam current, and volume to be drilled.

Untargeted metabolomics links glutathione to bacterial cell cycle progression.

Cell cycle progression requires the coordination of cell growth, chromosome replication, and division. Consequently, a functional cell cycle must be coupled with metabolism. However, direct measurements of metabolome dynamics remained scarce, in particular in bacteria. Here, we describe an untargeted metabolomics approach with synchronized Caulobacter crescentus cells to monitor the relative abundance changes of ~400 putative metabolites as a function of the cell cycle. While the majority of metabolite pools remains homeostatic, ~14% respond to cell cycle progression. In particular, sulfur metabolism is redirected during the G1-S transition, and glutathione levels periodically change over the cell cycle with a peak in late S phase. A lack of glutathione perturbs cell size by uncoupling cell growth and division through dysregulation of KefB, a K+/H+ antiporter. Overall, we here describe the impact of the C. crescentus cell cycle progression on metabolism, and in turn relate glutathione and potassium homeostasis to timely cell division.

Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle.

Methanol is a biotechnologically promising substitute for food and feed substrates since it can be produced renewably from electricity, water and CO2. Although progress has been made towards establishing Escherichia coli as a platform organism for methanol conversion via the energy efficient ribulose monophosphate (RuMP) cycle, engineering strains that rely solely on methanol as a carbon source remains challenging. Here, we apply flux balance analysis to comprehensively identify methanol-dependent strains with high potential for adaptive laboratory evolution. We further investigate two out of 1200 candidate strains, one with a deletion of fructose-1,6-bisphosphatase (fbp) and another with triosephosphate isomerase (tpiA) deleted. In contrast to previous reported methanol-dependent strains, both feature a complete RuMP cycle and incorporate methanol to a high degree, with up to 31 and 99% fractional incorporation into RuMP cycle metabolites. These strains represent ideal starting points for evolution towards a fully methylotrophic lifestyle.

Propionate supplementation promotes the expansion of peripheral regulatory T-Cells in patients with end-stage renal disease.

Patients with end-stage renal disease (ESRD) suffer from a progressively increasing low-grade systemic inflammation, which is associated with higher morbidity and mortality. Regulatory T cells (Tregs) play an important role in regulation of the inflammatory process. Previously, it has been demonstrated that short-chain fatty acids reduce inflammation in the central nervous system in a murine model of multiple sclerosis through an increase in tissue infiltrating Tregs. Here, we evaluated the effect of the short-chain fatty acid propionate on the chronic inflammatory state and T-cell composition in ESRD patients. Analyzing ESRD patients and healthy blood donors before, during, and 60 days after the propionate supplementation by multiparametric flow cytometry we observed a gradual and significant expansion in the frequencies of CD25highCD127- Tregs in both groups. Phenotypic characterization suggests that polarization of naïve T cells towards Tregs is responsible for the observed expansion. In line with this, we observed a significant reduction of inflammatory marker CRP under propionate supplementation. Of interest, the observed anti-inflammatory surroundings did not affect the protective pathogen-specific immunity as demonstrated by the stable frequencies of effector/memory T cells specific for tetanus/diphtheria recall antigens. Collectively, our data suggest that dietary supplements with propionate have a beneficial effect on the elevated systemic inflammation of ESRD patients. The effect can be achieved through an expansion of circulating Tregs without affecting the protective pathogen-reactive immunity.