Sustainable biopolymer soil stabilisation: the effect of microscale chemical characteristics on macroscale mechanical properties.

Sustainable biopolymer additives offer a promising soil stabilisation methodology, with a strong potential to be tuned to soil's specific nature, allowing the tailoring of mechanical properties for a range of geotechnical applications. However, the biopolymer chemical characteristics driving soil mechanical property modifications have yet to be fully established. Within this study we employ a cross-scale approach, utilising the differing galactose:mannose (G:M) ratios of various Galactomannan biopolymers (Guar Gum G:M 1:2, Locust Bean Gum G:M 1:4, Cassia Gum G:M 1:5) to investigate the effect of microscale chemical functionality upon macroscale soil mechanical properties. Molecular weight effects are also investigated, utilising Carboxy Methyl Cellulose (CMC). Soil systems comprising of SiO2 (100%) (SiO2) and a Mine Tailing (MT) exemplar composed of SiO2 (90%) + Fe2O3 (10%) (SiO2 + Fe) are investigated. The critical importance of biopolymer additive chemical functionality for the resultant soil mechanical properties, is demonstrated..For Galactomannan G:M 1:5 stabilised soils the 'high-affinity, high-strength', mannose-Fe interactions at the microscale (confirmed by mineral binding characterisation) are attributed to the 297% increase in the SiO2 + Fe systems Unconfined Compressive Strength (UCS), relative to SiO2 only. Conversely for SiO2 Galactomannan-stabilised soils, when increasing the G:M ratio from 1:2 to 1:5, a 85% reduction in UCS is observed, attributed to mannose's inability to interact with SiO2. UCS variations of up to a factor of 12 were observed across the biopolymer-soil mixes studied, in line with theoretically and experimentally expected values, due to the differences in the G:M ratios. The limited impact of molecular weight upon soil strength properties is also shown in CMC-stabilised soils. When considering a soil's stiffness and energy absorbance, the importance of biopolymer-biopolymer interaction strength and quantity is discussed, further deciphering biopolymer characteristics driving soil property modifications. This study highlights the importance of biopolymer chemistry for biopolymer stabilisation studies, illustrating the use of simple low-cost, accessible chemistry-based instrumental tools and outlining key design principles for the tailoring of biopolymer-soil composites for specific geotechnical applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s11440-022-01732-0.

Development of a Personalised Device for Systemic Magnetic Drug Targeting to Brain Tumours.

Delivering therapies to deeply seated brain tumours (BT) is a major clinical challenge. Magnetic drug targeting (MDT) could overcome this by rapidly transporting magnetised drugs directly into BT. We have developed a magnetic device for application in murine BT models using an array of neodymium magnets with a combined strength of 0.7T. In a closed fluidic system, the magnetic device trapped magnetic nanoparticles (MNP) up to distances of 0.8cm. In mice, the magnetic device guided intravenously administered MNP (<50nm) from the circulation into the brain where they localised within mouse BT. Furthermore, MDT of magnetised Temozolomide (TMZmag+) significantly reduced tumour growth and extended mouse survival to 48 days compared to the other treatment groups. Using the same principles, we built a proof of principle scalable magnetic device for human use with a strength of 1.1T. This magnetic device demonstrated trapping of MNP undergoing flow at distances up to 5cm. MDT using our magnetic device provides an opportunity for targeted delivery of magnetised drugs to human BT.

A versatile non-fouling multi-step flow reactor platform: demonstration for partial oxidation synthesis of iron oxide nanoparticles.

In the last decade flow reactors for material synthesis were firmly established, demonstrating advantageous operating conditions, reproducible and scalable production via continuous operation, as well as high-throughput screening of synthetic conditions. Reactor fouling, however, often restricts flow chemistry and the common fouling prevention via segmented flow comes at the cost of inflexibility. Often, the difficulty of feeding reagents into liquid segments (droplets or slugs) constrains flow syntheses using segmented flow to simple synthetic protocols with a single reagent addition step prior or during segmentation. Hence, the translation of fouling prone syntheses requiring multiple reagent addition steps into flow remains challenging. This work presents a modular flow reactor platform overcoming this bottleneck by fully exploiting the potential of three-phase (gas-liquid-liquid) segmented flow to supply reagents after segmentation, hence facilitating fouling free multi-step flow syntheses. The reactor design and materials selection address the operation challenges inherent to gas-liquid-liquid flow and reagent addition into segments allowing for a wide range of flow rates, flow ratios, temperatures, and use of continuous phases (no perfluorinated solvents needed). This "Lego®-like" reactor platform comprises elements for three-phase segmentation and sequential reagent addition into fluid segments, as well as temperature-controlled residence time modules that offer the flexibility required to translate even complex nanomaterial synthesis protocols to flow. To demonstrate the platform's versatility, we chose a fouling prone multi-step synthesis, i.e., a water-based partial oxidation synthesis of iron oxide nanoparticles. This synthesis required I) the precipitation of ferrous hydroxides, II) the addition of an oxidation agent, III) a temperature treatment to initiate magnetite/maghemite formation, and IV) the addition of citric acid to increase the colloidal stability. The platform facilitated the synthesis of colloidally stable magnetic nanoparticles reproducibly at well-controlled synthetic conditions and prevented fouling using heptane as continuous phase. The biocompatible particles showed excellent heating abilities in alternating magnetic fields (ILP values >3 nH m2 kgFe-1), hence, their potential for magnetic hyperthermia cancer treatment. The platform allowed for long term operation, as well as screening of synthetic conditions to tune particle properties. This was demonstrated via the addition of tetraethylenepentamine, confirming its potential to control particle morphology. Such a versatile reactor platform makes it possible to translate even complex syntheses into flow, opening up new opportunities for material synthesis.

Sustainable biopolymer soil stabilization in saline rich, arid conditions: a 'micro to macro' approach.

Water scarcity in semi-arid/arid regions is driving the use of salt water in mining operations. A consequence of this shift, is the potentially unheeded effect upon Mine Tailing (MT) management. With existing stabilization/solidification methodologies exhibiting vulnerability to MT toxicity and salinity effects, it is essential to explore the scope for more environmentally durable sustainable alternatives under these conditions. Within this study we investigate the effects of salinity (NaCl, 0-2.5 M) and temperatures associated with arid regions (25 °C, 40 °C), on Locust Bean Gum (LB) biopolymer stabilization of MT exemplar and sand (control) soil systems. A cross-disciplinary 'micro to macro' pipeline is employed, from a Membrane Enabled Bio-mineral Affinity Screen (MEBAS), to Mineral Binding Characterisation (MBC), leading finally to Geotechnical Verification (GV). As predicted by higher Fe2O3 LB binding affinity in saline in the MEBAS studies, LB with 1.25 M NaCl, results in the greatest soil strength in the MT exemplar after 7 days of curing at 40 °C. Under these most challenging conditions for other soil strengthening systems, an overall UCS peak of 5033 kPa is achieved. MBC shows the critical and direct relationship between Fe2O3-LB in saltwater to be 'high-affinity' at the molecular level and 'high-strength' achieved at the geotechnical level. This is attributed to biopolymer binding group's increased availability, with their 'salting-in' as NaCl concentrations rises to 1.25 M and then 'salting-out' at higher concentrations. This study highlights the potential of biopolymers as robust, sustainable, soil stabilization additives in challenging environments.

Nanobugs as Drugs: Bacterial Derived Nanomagnets Enhance Tumor Targeting and Oncolytic Activity of HSV-1 Virus.

The survival strategies of infectious organisms have inspired many therapeutics over the years. Indeed the advent of oncolytic viruses (OVs) exploits the uncontrolled replication of cancer cells for production of their progeny resulting in a cancer-targeting treatment that leaves healthy cells unharmed. Their success against inaccessible tumors however, is highly variable due to inadequate tumor targeting following systemic administration. Coassembling herpes simplex virus (HSV1716) with biocompatible magnetic nanoparticles derived from magnetotactic bacteria enables tumor targeting from circulation with magnetic guidance, protects the virus against neutralizing antibodies and thereby enhances viral replication within tumors. This approach additionally enhances the intratumoral recruitment of activated immune cells, promotes antitumor immunity and immune cell death, thereby inducing tumor shrinkage and increasing survival in a syngeneic mouse model of breast cancer by 50%. Exploiting the properties of such a nanocarrier, rather than tropism of the virus, for active tumor targeting offers an exciting, novel approach for enhancing the bioavailability and treatment efficacy of tumor immunotherapies for disseminated neoplasms.

Magnetosomes and Magnetosome Mimics: Preparation, Cancer Cell Uptake and Functionalization for Future Cancer Therapies.

Magnetic magnetite nanoparticles (MNP) are heralded as model vehicles for nanomedicine, particularly cancer therapeutics. However, there are many methods of synthesizing different sized and coated MNP, which may affect their performance as nanomedicines. Magnetosomes are naturally occurring, lipid-coated MNP that exhibit exceptional hyperthermic heating, but their properties, cancer cell uptake and toxicity have yet to be compared to other MNP. Magnetosomes can be mimicked by coating MNP in either amphiphilic oleic acid or silica. In this study, magnetosomes are directly compared to control MNP, biomimetic oleic acid and silica coated MNP of varying sizes. MNP are characterized and compared with respect to size, magnetism, and surface properties. Small (8 ± 1.6 nm) and larger (32 ± 9.9 nm) MNP are produced by two different methods and coated with either silica or oleic acid, increasing the size and the size dispersity of the MNP. The coated larger MNP are comparable in size (49 ± 12.5 nm and 61 ± 18.2 nm) to magnetosomes (46 ± 11.8 nm) making good magnetosome mimics. All MNP are assessed and compared for cancer cell uptake in MDA-MB-231 cells and importantly, all are readily taken up with minimal toxic effect. Silica coated MNP show the most uptake with greater than 60% cell uptake at the highest concentration, and magnetosomes showing the least with less than 40% at the highest concentration, while size does not have a significant effect on uptake. Finally, surface functionalization is demonstrated for magnetosomes and silica coated MNP using biotinylation and EDC-NHS, respectively, to conjugate fluorescent probes. The modified particles are visualized in MDA-MB-231 cells and demonstrate how both naturally biosynthesized magnetosomes and biomimetic silica coated MNP can be functionalized and readily up taken by cancer cells for realization as nanomedical vehicles.

Biopolymer Stabilization/Solidification of Soils: A Rapid, Micro-Macro, Cross-Disciplinary Approach.

In this study, we describe a novel high throughput, micro-macro approach for the identification and efficient design of biopolymer stabilized soil systems. At the "microscopic" scale, we propose a rapid Membrane Enabled Bio-Mineral Affinity Screening (MEBAS) approach supported by Mineral Binding Characterization (MBC) (TGA, ATR-FTIR and ζ Potential), while at the "macroscopic" scale, micro scale results are confirmed by Geotechnical Verification (GV) through unconfined compression testing. We illustrate the methodology using an exemplar mine tailings Fe2O3-SiO2 system. Five different biopolymers were tested against Fe2O3: locust bean gum, guar gum, gellan gum, xanthan gum, and sodium carboxymethyl cellulose. The screening revealed that locust bean gum and guar gum have the highest affinity for Fe2O3, which was confirmed by MBC and in agreement with GV. This affinity is attributed to the biopolymer's ability to form covalent C-O-Fe bonds through ß-(1,4)-d-mannan groups. Upon their 1% addition to a "macroscopic" Fe2O3 based exemplar MT system, unconfined compressive strengths of 5171 and 3848 kPa were obtained, significantly higher than those for the other biopolymers and non-Fe systems. In the current study, MEBAS gave an approximately 50-fold increase in rate of assessment compared to GV alone. Application of the proposed MEBAS-MBC-GV approach to a broad range of soil/earthwork components and additives is discussed.

Systematic Screening and Deep Analysis of CoPt Binding Peptides Leads to Enhanced CoPt Nanoparticles Using Designed Peptides.

Using protein and peptide additives to direct the crystallization of inorganic materials is a very attractive and environmentally friendly strategy to access complex and sometimes inaccessible mineral phases. CoPt is a very desirable high-magnetoanisotropic material in its L10 phase, but this is acquired by annealing at high temperatures which is incompatible with delicate nanomaterial assembly. Previous studies identified one peptide with high affinity to CoPt and four peptides with high affinity to FePt L10 phase nanoparticles (NPs) through phage display biopanning selection. While synthesis mediated by these peptides offered a small degree of L10 character to the NPs, they do not have the magnetoanistropy required for applications. In this study, we improve the activity of peptide directed crystallization by designing second generation peptides. We use the five literature sequences (LS) to probe the binding affinity deeper through dissection (alanine scanning), reduction (truncations), and substitution of the LS to find key amino acids and motifs. This is performed using a SPOT peptide array, importantly probing interactions at three stages of NP formation: with precursor, during synthesis, and with NPs. We found four universal features: 1) the importance of basic residues, particularly lysine flanking both ends of the sequence; 2) the importance of methionine; 3) shorter sequences show higher affinity than longer ones; and 4) acidic residues have a negative impact on binding with aspartic acid less favorable than glutamic acid. However, an acidic amino acid benefits, presumably to balance charge. The short motif KSLS had high affinity in all assays. Three sequences were selected from the screening, and three sequences were designed from the rules above. These were used to mediate a green synthesis of CoPt nanoparticles. The screened peptides mediated the formation of NPs with improved coercivity (90-110 Oe) compared to the LS (30-80 Oe), while the designed peptides facilitated formation of CoPt NPs with the highest coercivity (109 to 132 Oe), representing a massive improvement on L10 character. This result along with deeper insight this methodology brings offers vast potential for the future.

Rational Design and Self-Assembly of Coiled-Coil Linked SasG Protein Fibrils.

Protein engineering is an attractive approach for the self-assembly of nanometer-scale architectures for a range of potential nanotechnologies. Using the versatile chemistry provided by protein folding and assembly, coupled with amino acid side-chain functionality, allows for the construction of precise molecular "protein origami" hierarchical patterned structures for a range of nanoapplications such as stand-alone enzymatic pathways and molecular machines. The Staphyloccocus aureus surface protein SasG is a rigid, rod-like structure shown to have high mechanical strength due to "clamp-like" intradomain features and a stabilizing interface between the G5 and E domains, making it an excellent building block for molecular self-assembly. Here we characterize a new two subunit system composed of the SasG rod protein genetically conjugated with de novo designed coiled-coils, resulting in the self-assembly of fibrils. Circular dichroism (CD) and quartz-crystal microbalance with dissipation (QCM-D) are used to show the specific, alternating binding between the two subunits. Furthermore, we use atomic force microscopy (AFM) to study the extent of subunit polymerization in a liquid environment, demonstrating self-assembly culminating in the formation of linear macromolecular fibrils.

Investigating the ferric ion binding site of magnetite biomineralisation protein Mms6.

The biomineralization protein Mms6 has been shown to be a major player in the formation of magnetic nanoparticles both within the magnetosomes of magnetotactic bacteria and as an additive in synthetic magnetite precipitation assays. Previous studies have highlighted the ferric iron binding capability of the protein and this activity is thought to be crucial to its mineralizing properties. To understand how this protein binds ferric ions we have prepared a series of single amino acid substitutions within the C-terminal binding region of Mms6 and have used a ferric binding assay to probe the binding site at the level of individual residues which has pinpointed the key residues of E44, E50 and R55 involved in Mms6 ferric binding. No aspartic residues bound ferric ions. A nanoplasmonic sensing experiment was used to investigate the unstable EER44, 50,55AAA triple mutant in comparison to native Mms6. This suggests a difference in interaction with iron ions between the two and potential changes to the surface precipitation of iron oxide when the pH is increased. All-atom simulations suggest that disruptive mutations do not fundamentally alter the conformational preferences of the ferric binding region. Instead, disruption of these residues appears to impede a sequence-specific motif in the C-terminus critical to ferric ion binding.

Macrofluidic Coaxial Flow Platforms to Produce Tunable Magnetite Nanoparticles: A Study of the Effect of Reaction Conditions and Biomineralisation Protein Mms6.

Magnetite nanoparticles' applicability is growing extensively. However, simple, environmentally-friendly, tunable synthesis of monodispersed iron-oxide nanoparticles is challenging. Continuous flow microfluidic synthesis is promising; however, the microscale results in small yields and clogging. Here we present two simple macrofluidics devices (cast and machined) for precision magnetite nanoparticle synthesis utilizing formation at the interface by diffusion between two laminar flows, removing aforementioned issues. Ferric to total iron was varied between 0.2 (20:80 Fe3+:Fe2+) and 0.7 (70:30 Fe3+:Fe2+). X-ray diffraction shows magnetite in fractions from 0.2-0.6, with iron-oxide impurities in 0.7, 0.2 and 0.3 samples and magnetic susceptibility increases with increasing ferric content to 0.6, in agreement with each other and batch synthesis. Remarkably, size is tuned (between 20.5 nm to 6.5 nm) simply by increasing ferric ions ratio. Previous research shows biomineralisation protein Mms6 directs magnetite synthesis and controls size, but until now has not been attempted in flow. Here we report Mms6 increases magnetism, but no difference in particle size is seen, showing flow reduced the influence of Mms6. The study demonstrates a versatile yet simple platform for the synthesis of a vast range of tunable nanoparticles and ideal to study reaction intermediates and additive effects throughout synthesis.

Artificial coiled coil biomineralisation protein for the synthesis of magnetic nanoparticles.

Green synthesis of precise inorganic nanomaterials is a major challenge. Magnetotactic bacteria biomineralise magnetite nanoparticles (MNPs) within membrane vesicles (magnetosomes), which are embedded with dedicated proteins that control nanocrystal formation. Some such proteins are used in vitro to control MNP formation in green synthesis; however, these membrane proteins self-aggregate, making their production and use in vitro challenging and difficult to scale. Here, we provide an alternative solution by displaying active loops from biomineralisation proteins Mms13 and MmsF on stem-loop coiled-coil scaffold proteins (Mms13cc/MmsFcc). These artificial biomineralisation proteins form soluble, stable alpha-helical hairpin monomers, and MmsFcc successfully controls the formation of MNP when added to magnetite synthesis, regulating synthesis comparably to native MmsF. This study demonstrates how displaying active loops from membrane proteins on coiled-coil scaffolds removes membrane protein solubility issues, while retains activity, enabling a generic approach to readily-expressible, versatile, artificial membrane proteins for more accessible study and exploitation.

A biomimetic magnetosome: formation of iron oxide within carboxylic acid terminated polymersomes.

Bioinspired macromolecules can aid nucleation and crystallisation of minerals by mirroring processes observed in nature. Specifically, the iron oxide magnetite (Fe3O4) is produced in a dedicated liposome (called a magnetosome) within magnetic bacteria. This process is controlled by a suite of proteins embedded within the liposome membrane. In this study we look to synthetically mimic both the liposome and nucleation proteins embedded within it using preferential orientation polymer design. Amphiphilic block co-polymers self-assemble into vesicles (polymersomes) and have been used to successfully mimic liposomes. Carboxylic acid residue-rich motifs are common place in biomineralisation nucleating proteins and several magnetosome membrane specific (Mms) proteins (namely Mms6) have a specific carboxylic acid motifs that are found to bind both ferrous and ferric iron ions and nucleate the formation of magnetite. Here we use a combination of 2 diblock co-polymers: Both have the hydrophobic 2-hydroxypropyl methacrylate (PHPMA) block with either a poly(ethylene glycol) (PEG) block or a carboxylic acid terminated poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) block. These copolymers ((PEG113-PHPMA400) and (PMPC28-PHPMA400) respectively) self-assemble in situ to form polymersomes, with PEG113-PHPMA400 displaying favourably on the outer surface and PMPC28-PHPMA400 on the inner lumen, exposing numerous acidic iron binding carboxylates on the inner membrane. This is a polymersome mimic of a magnetosome (PMM28) containing interior nucleation sites. The resulting PMM28 were found to be 246 ± 137 nm in size. When the PMM28 were subjected to electroporation (5 pulses at 750 V) in an iron solution, iron ions were transported into the PMM28 polymersome core where magnetic iron-oxide was crystallised to fill the core; mimicking a magnetosome. Furthermore it has been shown that PMM28 magnetopolymersomes (PMM28Fe) exhibit a 6 °C temperature increase during in vitro magnetic hyperthermia yielding an intrinsic loss power (ILP) of 3.7 nHm2 kg-1. Such values are comparable to commercially available nanoparticles, but, offer the added potential for further tuning and functionalisation with respect to drug delivery.

Enhanced Tubulation of Liposome Containing Cardiolipin by MamY Protein from Magnetotactic Bacteria.

Lipid tubules are of particular interest for many potential applications in nanotechnology. Among various lipid tubule fabrication techniques, the morphological regulation of membrane structure by proteins mimicking biological processes may provide the chances to form lipid tubes with highly tuned structures. Magnetotactic bacteria synthesize magnetosomes (a unique prokaryotic organelle comprising a magnetite crystal within a lipid envelope). MamY protein is previously identified as the magnetosome protein responsible for magnetosome vesicle formation and stabilization. Furthermore, MamY is shown in vitro liposome tubulation activity. In this study, the interaction of MamY and phospholipids is investigated by using a lipids-immobilized membrane strip and a peptide array. Here, the binding of MamY to the anionic phospholipid, cardiolipin, is found and enhanced liposome tubulation efficiency. The authors propose the interaction is responsible for recruiting and locating cardiolipin to elongate liposome in vitro. The authors also suggest a similar mechanism for the invagination site in magnetosomes vesicle formation, where the lipid itself contributes further to increasing the curvature. These findings are highly important to develop an effective biomimetic synthesis technique of lipid tubules and to elucidate the unique prokaryotic organelle formation in magnetotactic bacteria.

Draft Genome Sequence of Magnetovibrio blakemorei Strain MV-1, a Marine Vibrioid Magnetotactic Bacterium.

We report here the genome sequence of Magnetovibrio blakemorei MV-1, a marine vibrioid magnetotactic bacterium with a single polar flagellum. The current assembly consists of 91 contigs with a combined size of 3,638,804 bp (54.3% G+C content). This genome allows for further investigations of the molecular biomineralization mechanisms of magnetosome formation.

Manufacturing Man-Made Magnetosomes: High-Throughput In Situ Synthesis of Biomimetic Magnetite Loaded Nanovesicles.

A new synthetic method for the production of artificial magnetosomes, i.e., lipid-coated vesicles containing magnetic nanoparticles, is demonstrated. Magnetosomes have considerable potential in biomedical and other nanotechnological applications but current production methods rely upon magnetotactic bacteria which limits the range of sizes and shapes that can be generated as well as the obtainable yield. Here, electrohydrodynamic atomization is utilized to form nanoscale liposomes of tunable size followed by electroporation to transport iron into the nanoliposome core resulting in magnetite crystallization. Using a combination of electron and fluorescence microscopy, dynamic light scattering, Raman spectroscopy, and magnetic susceptibility measurements, it is shown that single crystals of single-phase magnetite can be precipitated within each liposome, forming a near-monodisperse population of magnetic nanoparticles. For the specific conditions used in this study the mean particle size is 58 nm (±8 nm) but the system offers a high degree of flexibility in terms of both the size and composition of the final product.

Crystallizing the function of the magnetosome membrane mineralization protein Mms6.

The literature on the magnetosome membrane (MM) protein, magnetosome membrane specific6 (Mms6), is reviewed. Mms6 is native to magnetotactic bacteria (MTB). These bacteria take up iron from solution and biomineralize magnetite nanoparticles within organelles called magnetosomes. Mms6 is a small protein embedded on the interior of the MM and was discovered tightly associated with the formed mineral. It has been the subject of intensive research as it is seen to control the formation of particles both in vivo and in vitro Here, we compile, review and discuss the research detailing Mms6's activity within the cell and in a range of chemical in vitro methods where Mms6 has a marked effect on the composition, size and distribution of synthetic particles, with approximately 21 nm in size for solution precipitations and approximately 90 nm for those formed on surfaces. Furthermore, we review and discuss recent work detailing the structure and function of Mms6. From the evidence, we propose a mechanism for its function as a specific magnetite nucleation protein and summaries the key features for this action: namely, self-assembly to display a charged surface for specific iron binding, with the curvature of the surfaces determining the particle size. We suggest these may aid design of biomimetic additives for future green nanoparticle production.

Ferrous Iron Binding Key to Mms6 Magnetite Biomineralisation: A Mechanistic Study to Understand Magnetite Formation Using pH Titration and NMR Spectroscopy.

Formation of magnetite nanocrystals by magnetotactic bacteria is controlled by specific proteins which regulate the particles' nucleation and growth. One such protein is Mms6. This small, amphiphilic protein can self-assemble and bind ferric ions to aid in magnetite formation. To understand the role of Mms6 during in vitro iron oxide precipitation we have performed in situ pH titrations. We find Mms6 has little effect during ferric salt precipitation, but exerts greatest influence during the incorporation of ferrous ions and conversion of this salt to mixed-valence iron minerals, suggesting Mms6 has a hitherto unrecorded ferrous iron interacting property which promotes the formation of magnetite in ferrous-rich solutions. We show ferrous binding to the DEEVE motif within the C-terminal region of Mms6 by NMR spectroscopy, and model these binding events using molecular simulations. We conclude that Mms6 functions as a magnetite nucleating protein under conditions where ferrous ions predominate.

Biomagnetic Recovery and Bioaccumulation of Selenium Granules in Magnetotactic Bacteria.

UNLABELLED: Using microorganisms to remove waste and/or neutralize pollutants from contaminated water is attracting much attention due to the environmentally friendly nature of this methodology. However, cell recovery remains a bottleneck and a considerable challenge for the development of this process. Magnetotactic bacteria are a unique group of organisms that can be manipulated by an external magnetic field due to the presence of biogenic magnetite crystals formed within their cells. In this study, we demonstrated an account of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria alongside and independent of magnetite crystal biomineralization when grown in a medium containing selenium oxyanion (SeO3 (2-)). Quantitative analysis shows that magnetotactic bacteria accumulate the largest amount of target molecules (Se) per cell compared with any other previously reported nonferrous metal/metalloid. For example, 2.4 and 174 times more Se is accumulated than Te taken up into cells and Cd(2+) adsorbed onto the cell surface, respectively. Crucially, the bacteria with high levels of Se accumulation were successfully recovered with an external magnetic field. The biomagnetic recovery and the effective accumulation of target elements demonstrate the potential for application in bioremediation of polluted water. IMPORTANCE: The development of a technique for effective environmental water remediation is urgently required across the globe. A biological remediation process of waste removal and/or neutralization of pollutant from contaminated water using microorganisms has great potential, but cell recovery remains a bottleneck. Magnetotactic bacteria synthesize magnetic particles within their cells, which can be recovered by a magnetic field. Herein, we report an example of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria independent of magnetic particle synthesis. The cells were able to accumulate the largest amount of Se compared to other foreign elements. More importantly, the Se-accumulating bacteria were successfully recovered with an external magnetic field. We believe magnetotactic bacteria confer unique advantages of biomagnetic cell recovery and of Se accumulation, providing a new and effective methodology for bioremediation of polluted water.

Using a biomimetic membrane surface experiment to investigate the activity of the magnetite biomineralisation protein Mms6.

Magnetotactic bacteria are able to synthesise precise nanoparticles of the iron oxide magnetite within their cells. These particles are formed in dedicated organelles termed magnetosomes. These lipid membrane compartments use a range of biomineralisation proteins to nucleate and regulate the magnetite crystallisation process. A key component is the membrane protein Mms6, which binds to iron ions and helps to control the formation of the inorganic core. We have previously used Mms6 on gold surfaces patterned with a self-assembled monolayer to successfully produce arrays of magnetic nanoparticles. Here we use this surface system as a mimic of the interior face of the magnetosome membrane to study differences between intact Mms6 and the acid-rich C-terminal peptide subregion of the Mms6 protein. When immobilised on surfaces, the peptide is unable to reproduce the particle size or homogeneity control exhibited by the full Mms6 protein in our experimental setup. Moreover, the peptide is unable to support anchoring of a dense array of nanoparticles to the surface. This system also allows us to deconvolute particle binding from particle nucleation, and shows that Mms6 particle binding is less efficient when supplied with preformed magnetite nanoparticles when compared to particles precipitated from solution in the presence of the surface immobilised Mms6. This suggests that Mms6 binds to iron ions rather than to magnetite surfaces in our system, and is perhaps a nucleating agent rather than a controller of magnetite crystal growth. The comparison between the peptide and the protein under identical experimental conditions indicates that the full length sequence is required to support the full function of Mms6 on surfaces.