A Designed <i>A. vinelandii</i>–<i>S. elongatus</i> Coculture for Chemical Photoproduction from Air, Water, Phosphate, and Trace Metals

Microbial mutualisms play critical roles in a diverse number of ecosystems and have the potential to improve the efficiency of bioproduction for desirable chemicals. We investigate the growth of a photosynthetic cyanobacterium, <i>Synechococcus elongatus</i> PCC 7942, and a diazotroph, <i>Azotobacter vinelandii</i>, in coculture. From initial studies of the coculture grown in media with glutamate, we proposed a model of cross-feeding between these organisms. We then engineer a new microbial mutualism between <i>Azotobacter vinelandii</i> AV3 and <i>cscB Synechococcus elongatus</i> that grows in the absence of fixed carbon or nitrogen. The coculture cannot grow in the absence of a sucrose-exporting <i>S. elongatus,</i> and neither organism can grow alone without fixed carbon or nitrogen. This new system has the potential to produce industrially relevant products, such as polyhydroxybutyrate (PHB) and alginate, from air, water, phosphate, trace metals, and sunlight. We demonstrate the ability of the coculture to produce PHB in this work

Recyclable Thermoresponsive Polymer–Cellulase Bioconjugates for Biomass Depolymerization

Here we report the construction and characterization of a recoverable, thermoresponsive polymer–endoglucanase bioconjugate that matches the activity of unmodified enzymes on insoluble cellulose substrates. Two copolymers exhibiting a thermoresponsive lower critical solution temperature (LCST) were created through the copolymerization of an aminooxy-bearing methacrylamide with <i>N</i>-isopropylacrylamide (NIPAm) or <i>N</i>-isopropylmethacrylamide (NIPMa). The aminooxy group provided a handle through which the LCST was adjusted through small-molecule quenching. This allowed materials with LCSTs ranging from 20.9 to 60.5 °C to be readily obtained after polymerization. The thermostable endoglucanase EGPh from the hypothermophilic <i>Pyrococcus horikoshii</i> was transaminated with pyridoxal-5′-phosphate to produce a ketone-bearing protein, which was then site-selectively modified through oxime linkage with benzylalkoxyamine or 5 kDa-poly­(ethylene glycol)-alkoxyamine. These modified proteins showed activity comparable to the controls when assayed on an insoluble cellulosic substrate. Two polymer bioconjugates were then constructed using transaminated EGPh and the aminooxy-bearing copolymers. After 12 h, both bioconjugates produced an equivalent amount of free reducing sugars as the unmodified control using insoluble cellulose as a substrate. The recycling ability of the NIPAm copolymer–EGPh conjugate was determined through three rounds of activity, maintaining over 60% activity after two cycles of reuse and affording significantly more soluble carbohydrates than unmodified enzyme alone. When assayed on acid-pretreated Miscanthus, this bioconjugate increased the amount of reducing sugars by 2.8-fold over three rounds of activity. The synthetic strategy of this bioconjugate allows the LCST of the material to be changed readily from a common stock of copolymer and the method of attachment is applicable to a variety of proteins, enabling the same approach to be amenable to thermophile-derived cellulases or to the separation of multiple species using polymers with different recovery temperatures

Direct Electrochemical Bioconjugation on Metal Surfaces

DNA has unique capabilities for molecular recognition and self-assembly, which have fostered its widespread incorporation into devices that are useful in science and medicine. Many of these platforms rely on thiol groups to tether DNA to gold surfaces, but this method is hindered by a lack of control over monolayer density and by secondary interactions between the nucleotide bases and the metal. In this work, we report an electrochemically activated bioconjugation reaction as a mild, reagent-free strategy to attach oligonucleotides to gold surfaces. Aniline-modified DNA was coupled to catechol-coated electrodes that were oxidized to <i>o</i>-quinones using an applied potential. High levels of coupling could be achieved in minutes. By changing the reaction time and the underlying catechol content, the final DNA surface coverage could be specified. The advantages of this method were demonstrated through the electrochemical detection of the endocrine disruptor bisphenol A, as well as the capture of living nonadherent cells on electrode surfaces by DNA hybridization. This method not only improves the attachment of DNA to metal surfaces but also represents a new direction for the site-specific attachment of biomolecules to device platforms

N‑Terminal Modification of Proteins with <i>o</i>‑Aminophenols

The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses <i>o</i>-aminophenols or <i>o</i>-catechols that are oxidized to active coupling species <i>in situ</i> using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods

Near-Quantitative Aqueous Synthesis of Rotaxanes via Bioconjugation to Oligopeptides and Proteins

In spite of widespread interest in rotaxane-based molecular machines and materials, rotaxanes have not been attached covalently to proteins. We describe the near-quantitative aqueous synthesis of [2]­rotaxanes based on neutral and charged aqueous hostscucurbit[7]­uril (CB7) and cyclobis­(paraquat-<i>p</i>-phenylene) (CBPQT⁴⁺), respectivelyusing the thiol-ene addition of cysteine and maleimide as a stoppering protocol. After verifying the high efficiency of the reaction using glutathione (GSH) as an oligopeptide stopper, we have employed cytochrome C (CytC) as a protein stopper to produce the first well-characterized protein-rotaxane bioconjugates. We anticipate that this methodology will enable the preparation of novel materials that combine the unique properties of proteins and mechanical bonds

Quantifying Hormone Disruptors with an Engineered Bacterial Biosensor

Endocrine disrupting compounds are found in increasing amounts in our environment, originating from pesticides, plasticizers, and pharmaceuticals, among other sources. Although the full impact of these compounds is still under study, they have already been implicated in diseases such as obesity, diabetes, and cancer. The list of chemicals that disrupt normal hormone function is growing at an alarming rate, making it crucially important to find sources of contamination and identify new compounds that display this ability. However, there is currently no broad-spectrum, rapid test for these compounds, as they are difficult to monitor because of their high potency and chemical dissimilarity. To address this, we have developed a new detection strategy for endocrine disrupting compounds that is both fast and portable, and it requires no specialized skills to perform. This system is based on a native estrogen receptor construct expressed on the surface of <i>Escherichia coli</i>, which enables both the detection of many detrimental compounds and signal amplification from impedance measurements due to the binding of bacteria to a modified electrode. With this approach, sub-ppb levels of estradiol and ppm levels of bisphenol A are detected in complex solutions. Rather than responding to individual components, this system reports the total estrogenic activity of a sample using the most relevant biological receptor. As an applied example, estrogenic chemicals released from a plastic baby bottle following microwave heating were detectable with this technique. This approach should be broadly applicable to the detection of chemically diverse classes of compounds that bind to a single receptor

DNA Hybridization To Interface Current-Producing Cells with Electrode Surfaces

As fossil fuels are increasingly linked to environmental damage, the development of renewable, affordable biological alternative fuels is vital. <i>Shewanella oneidensis</i> is often suggested as a potential component of bioelectrochemical cells because of its ability to act as an electron donor to metal surfaces. These microbes remain challenging to implement, though, due to inconsistency in biofilm formation on electrodes and therefore current generation. We have applied DNA hybridization-based cell adhesion to immobilize <i>S. oneidensis</i> on electrodes. High levels of current are reproducibly generated from these cell layers following only 30 min of immobilization without the need for the formation of a biofilm. Upon incorporation of DNA mismatches in the microbe immobilization sequence, significant attenuation in current production is observed, suggesting that at least part of the electron transfer to the electrode is DNA-mediated. This method of microbe assembly is rapid, reproducible, and facile for the production of anodes for biofuel cells

Characterization of MS2 conjugates.

<p>(a) The periodate-mediated oxidative coupling reaction takes place between <i>o</i>-aminophenol peptides and aniline containing MS2 capsids. (b) Alexa Fluor 680 and peptide-MS2 conjugates were analyzed by SDS-PAGE, with visualization of fluorescent (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100678#pone.0100678.s005" target="_blank">Figure S5</a>) and Coomassie-stained bands (shown). Lanes 1–3 show disassembled MS2 monomers labeled with Alexa Fluor 680. In lane 2, the GPR peptide was added and in lane 3 the GPS peptide was added. The upper bands represent the fraction of the MS2 monomers conjugated to the peptides. (c) Transmission electron microscopy, (d) dynamic light scattering, and (e) size-exclusion chromatography (fluorescence: λ_ex = 280 nm, λ_em = 330 nm) of MS2, GPR-MS2, and GPS-MS2 confirmed that the capsids remained intact after modification. Wide-field TEM images appear in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100678#pone.0100678.s007" target="_blank">Figure S7</a>.</p

Multivalent Viral Capsids with Internal Cargo for Fibrin Imaging

<div><p>Thrombosis is the cause of many cardiovascular syndromes and is a significant contributor to life-threatening diseases, such as myocardial infarction and stroke. Thrombus targeted imaging agents have the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of thrombosis-related diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. In this work, we report the synthesis and use of chemically functionalized bacteriophage MS2 capsids as biomolecule-based nanoparticles for fibrin imaging. The capsids were modified using an oxidative coupling reaction, conjugating ∼90 copies of a fibrin targeting peptide to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents. The in vitro assessment of the capsids suggests that fibrin-targeted MS2 capsids could be used as delivery agents to thrombi for diagnostic or therapeutic applications.</p></div

Vascular Cell Adhesion Molecule-Targeted MS2 Viral Capsids for the Detection of Early-Stage Atherosclerotic Plaques

Atherosclerosis is a cardiovascular disease characterized by the formation of lipid-rich plaques within the walls of large arteries. Over time, a portion of these lesions can detach and lead to serious complications, such as strokes or heart attacks. Currently, there is no clinically effective way to detect the presence of atherosclerosis in patients until it has reached a relatively advanced stage. Furthermore, increasing evidence suggests that the pathobiological behavior of plaques is determined mainly by their composition, and not their size, which is the parameter usually monitored with current imaging techniques. In this work, we report protein-based agents that target the vascular cell adhesion molecule (VCAM1), a protein that plays a crucial role in atherosclerosis progression. <i>In vivo</i> experiments with murine atherosclerosis models indicated that the targeted protein nanoparticles were successful in detecting plaques of various sizes in the descending aorta and the aortic arch. This finding encourages the further development of these nanoscale agents for applications in the imaging, diagnosis, and treatment of cardiovascular diseases