Identification and recombinant expression of a cutinase from Papiliotrema laurentii that hydrolyzes natural and synthetic polyesters.

Given the multitude of extracellular enzymes at their disposal, many of which are designed to degrade nature's polymers (lignin, cutin, cellulose, etc.), fungi are adept at targeting synthetic polyesters with similar chemical composition. Microbial-influenced deterioration of xenobiotic polymeric surfaces is an area of interest for material scientists as these are important for the conservation of the underlying structural materials. Here, we describe the isolation and characterization of the Papiliotrema laurentii 5307AH (P. laurentii) cutinase, Plcut1. P. laurentii is basidiomycete yeast with the ability to disperse Impranil-DLN (Impranil), a colloidal polyester polyurethane, in agar plates. To test whether the fungal factor involved in this clearing was a secreted enzyme, we screened the ability of P. laurentii culture supernatants to disperse Impranil. Using size exclusion chromatography (SEC), we isolated fractions that contained Impranil-clearing activity. These fractions harbored a single ~22 kD band, which was excised and subjected to peptide sequencing. Homology searches using the peptide sequences identified, revealed that the protein Papla1 543643 (Plcut1) displays similarities to serine esterase and cutinase family of proteins. Biochemical assays using recombinant Plcut1 confirmed that this enzyme has the capability to hydrolyze Impranil, soluble esterase substrates, and apple cutin. Finally, we confirmed the presence of the Plcut1 in culture supernatants using a custom antibody that specifically recognizes this protein. The work shown here supports a major role for the Plcut1 in the fungal degradation of natural polyesters and xenobiotic polymer surfaces.IMPORTANCEFungi play a vital role in the execution of a broad range of biological processes that drive ecosystem function through production of a diverse arsenal of enzymes. However, the universal reactivity of these enzymes is a current problem for the built environment and the undesired degradation of polymeric materials in protective coatings. Here, we report the identification and characterization of a hydrolase from Papiliotrema laurentii 5307AH, an aircraft-derived fungal isolate found colonizing a biodeteriorated polymer-coated surface. We show that P. laurentii secretes a cutinase capable of hydrolyzing soluble esters as well as ester-based compounds forming solid surface coatings. These findings indicate that this fungus plays a significant role in biodeterioration through the production of a cutinase adept at degrading ester-based polymers, some of which form the backbone of protective surface coatings. The work shown here provides insights into the mechanisms employed by fungi to degrade xenobiotic polymers.

The microbiota drives diurnal rhythms in tryptophan metabolism in the stressed gut.

Chronic stress disrupts microbiota-gut-brain axis function and is associated with altered tryptophan metabolism, impaired gut barrier function, and disrupted diurnal rhythms. However, little is known about the effects of acute stress on the gut and how it is influenced by diurnal physiology. Here, we used germ-free and antibiotic-depleted mice to understand how microbiota-dependent oscillations in tryptophan metabolism would alter gut barrier function at baseline and in response to an acute stressor. Cecal metabolomics identified tryptophan metabolism as most responsive to a 15-min acute stressor, while shotgun metagenomics revealed that most bacterial species exhibiting rhythmicity metabolize tryptophan. Our findings highlight that the gastrointestinal response to acute stress is dependent on the time of day and the microbiome, with a signature of stress-induced functional alterations in the ileum and altered tryptophan metabolism in the colon.

Draft genome sequence of potentially dikaryotic black fungus Aureobasidium melanogenum isolated from aircraft.

The Ascomycota yeast Aureobasidium melanogenum strain W12 was isolated from an aircraft polymer-coated surface. The genome size is 53,160,883 bp with a G + C content of 50.13%. The genome contains fatty acid transporters, cutinases, hydroxylases, and lipases potentially used for survival on polymer coatings on aircraft.

Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes.

Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house E. coli extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.

Silky Liquid Metal Electrodes for On-Skin Health Monitoring.

Next generation on-skin electrodes will require soft, flexible, and gentle materials to provide both high-fidelity sensing and wearer comfort. However, many commercially available on-skin electrodes lack these key properties due to their use of rigid hardware, harsh adhesives, uncomfortable support structures, and poor breathability. To address these challenges, this work presents a new device paradigm by joining biocompatible electrospun spider silk with printable liquid metal to yield an incredibly soft and scalable on-skin electrode that is strain-tolerant, conformable, and gentle on-skin. These electrodes, termed silky liquid metal (SLiM) electrodes, are found to be over five times more breathable than commercial wet electrodes, while the silk's intrinsic adhesion mechanism allows SLiM electrodes to avoid the use of harsh artificial adhesives, potentially decreasing skin irritation and inflammation over long-term use. Finally, the SLiM electrodes provide comparable impedances to traditional wet and other liquid metal electrodes, offering a high-fidelity sensing alternative with increased wearer comfort. Human subject testing confirmed the SLiM electrodes ability to sense electrophysiological signals with high fidelity and minimal irritation to the skin. The unique properties of the reported SLiM electrodes offer a comfortable electrophysiological sensing solution especially for patients with pre-existing skin conditions or surface wounds.

The impact of acute and chronic stress on gastrointestinal physiology and function: a microbiota-gut-brain axis perspective.

The physiological consequences of stress often manifest in the gastrointestinal tract. Traumatic or chronic stress is associated with widespread maladaptive changes throughout the gut, although comparatively little is known about the effects of acute stress. Furthermore, these stress-induced changes in the gut may increase susceptibility to gastrointestinal disorders and infection, and impact critical features of the neural and behavioural consequences of the stress response by impairing gut-brain axis communication. Understanding the mechanisms behind changes in enteric nervous system circuitry, visceral sensitivity, gut barrier function, permeability, and the gut microbiota following stress is an important research objective with pathophysiological implications in both neurogastroenterology and psychiatry. Moreover, the gut microbiota has emerged as a key aspect of physiology sensitive to the effects of stress. In this review, we focus on different aspects of the gastrointestinal tract including gut barrier function as well as the immune, humoral and neuronal elements involved in gut-brain communication. Furthermore, we discuss the evidence for a role of stress in gastrointestinal disorders. Existing gaps in the current literature are highlighted, and possible avenues for future research with an integrated physiological perspective have been suggested. A more complete understanding of the spatial and temporal dynamics of the integrated host and microbial response to different kinds of stressors in the gastrointestinal tract will enable full exploitation of the diagnostic and therapeutic potential in the fast-evolving field of host-microbiome interactions.

Iridescent biofilms of Cellulophaga lytica are tunable platforms for scalable, ordered materials.

Nature offers many examples of materials which exhibit exceptional properties due to hierarchical assembly of their constituents. In well-studied multi-cellular systems, such as the morpho butterfly, a visible indication of having ordered submicron features is given by the display of structural color. Detailed investigations of nature's designs have yielded mechanistic insights and led to the development of biomimetic materials at laboratory scales. However, the manufacturing of hierarchical assemblies at industrial scales remains difficult. Biomanufacturing aims to leverage the autonomy of biological systems to produce materials at lower cost and with fewer carbon emissions. Earlier reports documented that some bacteria, particularly those with gliding motility, self-assemble into biofilms with polycrystalline structures and exhibit glittery, iridescent colors. The current study demonstrates the potential of using one of these bacteria, Cellulophaga lytica, as a platform for the large scale biomanufacturing of ordered materials. Specific approaches for controlling C. lytica biofilm optical, spatial and temporal properties are reported. Complementary microscopy-based studies reveal that biofilm color variations are attributed to changes in morphology induced by cellular responses to the local environment. Incorporation of C. lytica biofilms into materials is also demonstrated, thereby facilitating their handling and downstream processing, as would be needed during manufacturing processes. Finally, the utility of C. lytica as a self-printing, photonic ink is established by this study. In summary, autonomous surface assembly of C. lytica under ambient conditions and across multiple length scales circumvent challenges that currently hinder production of ordered materials in industrial settings.

Screening Enzymatic Degradation of Polyester Polyurethane with Fluorescent Single-walled Carbon Nanotube and Polymer Nanoparticle Conjugates.

Enzymatic biodegradation is a promising method to reclaim plastic materials. However, to date, a high-throughput method for screening potential enzyme candidates for biodegradation is still lacking. Here, we propose a single-walled carbon nanotube (SWCNT) fluorescence sensor for screening the enzymatic degradation of polyester polyurethane nanoparticles. Through wrapping the SWCNT with cationic chitosan, an electrostatic bond is formed between the SWCNT and Impranil, a widely applied model substrate of polyester polyurethane. As Impranil is being degraded by the enzymes, a characteristic quenching at a short reaction time followed by a brightening at a longer reaction time in the fluorescence signal is observed. The time-dependent fluorescence response is compared with turbidity measurement, and we conclude that the brightening in fluorescence results from the binding of the degradation product with the SWCNT. The proposed SWCNT sensor design has the potential to screen enzyme candidates for selective degradation of other plastic particles.

Accurate prediction by AlphaFold2 for ligand binding in a reductive dehalogenase and implications for PFAS (per- and polyfluoroalkyl substance) biodegradation.

Despite the success of AlphaFold2 (AF2), it is unclear how AF2 models accommodate for ligand binding. Here, we start with a protein sequence from Acidimicrobiaceae TMED77 (T7RdhA) with potential for catalyzing the degradation of per- and polyfluoroalkyl substances (PFASs). AF2 models and experiments identified T7RdhA as a corrinoid iron-sulfur protein (CoFeSP) which uses a norpseudo-cobalamin (BVQ) cofactor and two Fe4S4 iron-sulfur clusters for catalysis. Docking and molecular dynamics simulations suggest that T7RdhA uses perfluorooctanoic acetate (PFOA) as a substrate, supporting the reported defluorination activity of its homolog, A6RdhA. We showed that AF2 provides processual (dynamic) predictions for the binding pockets of ligands (cofactors and/or substrates). Because the pLDDT scores provided by AF2 reflect the protein native states in complex with ligands as the evolutionary constraints, the Evoformer network of AF2 predicts protein structures and residue flexibility in complex with the ligands, i.e., in their native states. Therefore, an apo-protein predicted by AF2 is actually a holo-protein awaiting ligands.

Aerosol Jet Printed Surface-Enhanced Raman Substrates: Application for High-Sensitivity Detection of Perfluoroalkyl Substances.

Printing technologies offer an attractive means for producing low-cost surface-enhanced Raman spectroscopy (SERS) substrates with high-throughput methods. The development of these substrates is especially important for field-deployable detection of environmental contaminants. Toward this end, we demonstrate SERS-based substrates fabricated through aerosol jet printing of silver nanoparticles and graphene inks on Kapton films. Our printed arrays exhibited measurable intensities for fluorescein and rhodamine dyes down to concentrations of 10-7 M, with the highest SERS intensities obtained for four print passes of Ag nanoparticles. The substrates also exhibited an excellent shelf life, with little reduction in fluorescein intensities after 9 months of shelf storage. We also demonstrated the capability of our substrates to sense perfluoroalkyl substances (PFAS), the so-called forever chemicals that resist degradation due to their strong C-F bonds and persist in the environment. Interestingly, the addition of graphene to the Ag nanoparticles greatly enhanced the SERS intensity of the perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) molecules under basic conditions (pH ∼ 9) compared to that of fluorescein and rhodamine. We were able to successfully detect SERS spectra from nano- and picomolar (∼0.4 ppt) concentrations of PFOA and PFOS, respectively, demonstrating the viability of deploying our SERS sensors in the environment for the ultrasensitive detection of contaminants.

Draft Genome Sequence of the Nonmotile Tremellomycetes Yeast Naganishia albida, Isolated from Aircraft.

The Basidiomycota yeast Naganishia albida strain 5307AI was isolated from an aircraft polymer-coated surface. The genome size is 20,642,279 bp, with a G+C content of 53.99%. The genome contains fatty acid transporters, cutinases, hydroxylases, and lipases that are likely used for survival on polymer coatings on aircraft.

Molecular Dynamics Investigation into pH Dependent Metal Binding of the Intrinsically Disordered Worm Jaw Protein, Nvjp-1.

Sclerotization of the Nereis virens jaw is mediated by metal binding to the histidine-rich jaw protein, Nvjp-1. Previous studies showed that the mechanical properties of Nvjp-1 hydrogels could be modulated with zinc binding as well as the associated anion. Here, we show that the mechanical properties of Nvjp-1 hydrogels can be modulated by pH and that zinc binding to Nvjp-1 is stable at both acidic and alkaline pH conditions. To probe the mechanism of Zn2+ binding to Nvjp-1 at different pH conditions, we utilized all atom molecular dynamics simulations employing a polarizable force field. At low pH conditions, polar residues predominantly interacted with Zn2+, with at most two residues interacting with a given zinc ion. Surprisingly, little to no Zn2+ binding was observed with the abundant Nvjp-1 acidic residues, which form salt-bridges with the protonated histidines to effectively block their binding to Zn2+ ions. As the pH was shifted to alkaline conditions, Zn2+ binding residues reconfigured to form additional coordination bonds with histidine, resulting in a reduction in the radius of gyration that correlated with hydrogel sclerotization. Furthermore, acetate ions were shown to facilitate the capture of zinc ions through association with protonated histidines at low pH, freeing acidic residues to interact with Zn2+ ions and increasing the number of Zn2+ ions that diffuse into the Nvjp-1 interior. Thus, these studies provide valuable molecular insights into how amino acid residues in Nvjp-1 manage metal salt binding and coordination in hydrogels as a function of the pH and ionic environments.

Molecular dynamics simulations explore effects of electric field orientations on spike proteins of SARS-CoV-2 virions.

Emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its current worldwide spread have caused a pandemic of acute respiratory disease COVID-19. The virus can result in mild to severe, and even to fatal respiratory illness in humans, threatening human health and public safety. The spike (S) protein on the surface of viral membrane is responsible for viral entry into host cells. The discovery of methods to inactivate the entry of SARS-CoV-2 through disruption of the S protein binding to its cognate receptor on the host cell is an active research area. To explore other prevention strategies against the quick spread of the virus and its mutants, non-equilibrium molecular dynamics simulations have been employed to explore the possibility of manipulating the structure-activity of the SARS-CoV-2 spike glycoprotein by applying electric fields (EFs) in both the protein axial directions and in the direction perpendicular to the protein axis. We have found out the application of EFs perpendicular to the protein axis is most effective in denaturing the HR2 domain which plays critical role in viral-host membrane fusion. This finding suggests that varying irradiation angles may be an important consideration in developing EF based non-invasive technologies to inactivate the virus.

AlphaFold2 models indicate that protein sequence determines both structure and dynamics.

AlphaFold 2 (AF2) has placed Molecular Biology in a new era where we can visualize, analyze and interpret the structures and functions of all proteins solely from their primary sequences. We performed AF2 structure predictions for various protein systems, including globular proteins, a multi-domain protein, an intrinsically disordered protein (IDP), a randomized protein, two larger proteins (> 1000 AA), a heterodimer and a homodimer protein complex. Our results show that along with the three dimensional (3D) structures, AF2 also decodes protein sequences into residue flexibilities via both the predicted local distance difference test (pLDDT) scores of the models, and the predicted aligned error (PAE) maps. We show that PAE maps from AF2 are correlated with the distance variation (DV) matrices from molecular dynamics (MD) simulations, which reveals that the PAE maps can predict the dynamical nature of protein residues. Here, we introduce the AF2-scores, which are simply derived from pLDDT scores and are in the range of [0, 1]. We found that for most protein models, including large proteins and protein complexes, the AF2-scores are highly correlated with the root mean square fluctuations (RMSF) calculated from MD simulations. However, for an IDP and a randomized protein, the AF2-scores do not correlate with the RMSF from MD, especially for the IDP. Our results indicate that the protein structures predicted by AF2 also convey information of the residue flexibility, i.e., protein dynamics.

Draft Genome Sequence of the Bacterium Delftia acidovorans Strain D4B, Isolated from Soil.

Delftia acidovorans strain D4B is an aerobic bacterium within the Betaproteobacteria lineage that was isolated from soil. The genome size is 6.26 Mbp, with a G+C content of 67%. The genome encodes enzymes potentially involved in the degradation of fluorinated compounds.

Preparation of Multifunctional Silk-Based Microcapsules loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing ß-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

Meeting report of the fourth annual Tri-Service Microbiome Consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations. The annual TSMC symposium is designed to enable information sharing between DoD scientists and leaders in the field of microbiome science, thereby keeping DoD consortium members informed of the latest advances within the microbiome community and facilitating the development of new collaborative research opportunities. The 2020 annual symposium was held virtually on 24-25 September 2020. Presentations and discussions centered on microbiome-related topics within four broad thematic areas: (1) Enabling Technologies; (2) Microbiome for Health and Performance; (3) Environmental Microbiome; and (4) Microbiome Analysis and Discovery. This report summarizes the presentations and outcomes of the 4th annual TSMC symposium.

Characterization of synthetic riboswitch in cell-free protein expression systems.

Riboswitches are RNA-based regulatory elements that utilize ligand-induced structural changes in the 5'-untranslated region of mRNA to regulate the expression of associated genes. The majority of synthetic riboswitches have been selected and tested in cell-based systems. Cell-free protein expression systems (CFPS) have several advantages for the development and testing of synthetic riboswitches, including eliminating interactions with complex cellular networks, and the decoupling of transcription and translation processes. To gain a better understanding of the riboswitch regulatory mechanism, to allow for more efficient riboswitch optimization and use for biosensing applications, we studied the performance of a theophylline-responsive synthetic riboswitch coupled with the superfolder green fluorescent protein (sfGFP) reporter gene in E. coli cellular extract and PURE cell-free systems. To monitor the mRNA dynamics, a malachite green aptamer sequence was added to the 3'-untranslated region of sfGFP mRNA. Performance of the theophylline riboswitch was compared with a constitutively expressed sfGFP (control). Transcription dynamics of the riboswitch mRNA was very similar to the transcription of the control mRNA for all theophylline concentrations tested in both E. coli extract and PURE CFPS. However, sfGFP expression in the riboswitch construct was one order of magnitude lower, even at the highest concentration of theophylline. A mathematical model of riboswitch activation governed by the kinetic trapping mechanism was developed. Two factors - a reduced fraction of mRNA in the 'ON' state and a considerably lower translation initiation rate in the riboswitch - contribute to the much lower level of protein expression in the theophylline riboswitch compared to the control construct.