Éva
Bereczk-Tompa
a,
Ferenc
Vonderviszt
bc,
Barnabás
Horváth
d,
István
Szalai
d and
Mihály
Pósfai
*a
aDepartment of Earth and Environmental Sciences, University of Pannonia, Egyetem u. 10, 8200 Veszprém, Hungary. E-mail: eva.tompa88@gmail.com; mihaly.posfai@gmail.com
bBio-Nanosystems Laboratory, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Egyetem u. 10, 8200 Veszprém, Hungary. E-mail: von007@almos.uni-pannon.hu
cInstitute of Technical Physics and Materials Science, Centre for Energy Research, Konkoly-Thege u. 29-33, 1121 Budapest, Hungary
dInstitute of Physics and Mechatronics, University of Pannonia, Egyetem u. 10, 8200 Veszprém, Hungary. E-mail: bhorvath@almos.uni-pannon.hu; szalai@almos.uni-pannon.hu
First published on 26th September 2017
With the aim of creating one-dimensional magnetic nanostructures, we genetically engineered flagellar filaments produced by Salmonella bacteria to display iron- or magnetite-binding sites, and used the mutant filaments as templates for both nucleation and attachment of the magnetic iron oxide magnetite. Although nucleation from solution and attachment of nanoparticles to a pre-existing surface are two different processes, non-classical crystal nucleation pathways have been increasingly recognized in biological systems, and in many cases nucleation and particle attachment cannot be clearly distinguished. In this study we tested the magnetite-nucleating ability of four types of mutant flagella previously shown to be efficient binders of magnetite nanoparticles, and we used two other mutant flagella that were engineered to periodically display known iron-binding oligopeptides on their surfaces. All mutant filaments were demonstrated to be efficient as templates for the synthesis of one-dimensional magnetic nanostructures under ambient conditions. Both approaches resulted in similar final products, with randomly oriented magnetite nanoparticles partially covering the filamentous biological templates. In an external magnetic field, the viscosity of a suspension of the produced magnetic filaments showed a twofold increase relative to the control sample. The results of magnetic susceptibility measurements were also consistent with the magnetic nanoparticles occurring in linear structures. Our study demonstrates that biological templating can be used to produce one-dimensional magnetic nanostructures under benign conditions, and that modified flagellar filaments can be used for creating model systems in which crystal nucleation from solution can be experimentally studied.
The use of bacterial filaments in nanofabrication is attractive because their main component – the flagellin (FliC) protein – can be produced in large quantities, exported out of the cell and can self-assemble into long, homogeneous fibers,3 which can be easily purified.4 The length of the filaments can be controlled during in vitro polymerization by changing the conditions, i.e. the applied precipitant or flagellin monomer concentration.5 Using flagellin in nanofabrication offers one more benefit: it can be easily tailored by genetic manipulation. The middle hypervariable region of the FliC gene forms the surface-exposed D3 domain in the filament structure, which can be removed, modified or replaced by foreign proteins without adversely affecting the polymerization ability of flagellin.6 Mutant flagellin variants obtained by genetic engineering can be used as building blocks to add various functionalities to the filament.1,7,8
In our recent work9 flagellar filaments of Salmonella were engineered to facilitate the formation of 1D magnetic nanostructures under ambient conditions. We constructed four different flagellin mutants displaying magnetite-binding motifs, two of which contained fragments of magnetosome-associated proteins from magnetotactic bacteria (MamI and Mms6), and another two contained synthetic sequences. The self-assembled mutant filaments were used as scaffolds to which separately synthesized magnetite nanoparticles could attach. Even though MamI was previously reported to play a crucial role in magnetite nucleation,10 the mutant filaments containing the fragment of the MamI protein proved to be efficient templates for magnetite nanoparticle attachment. Considering that nucleation and nanoparticle binding are closely related phenomena, in this paper we explore whether the nucleation of magnetic particles could be induced by the modified filaments. Therefore, magnetite was synthesized in the presence of mutant Salmonella bacteria. In addition to the above mentioned sequences (Table 1), we created two additional mutants which were engineered to display known iron-binding motifs. Our goal was to use these motifs to bind iron ions from solution and initiate the nucleation of magnetite particles on the surfaces of the filaments. We compared the magnetite templating and magnetite binding activities of the six different motifs, as well as the products resulting from the two different (iron- and magnetite-binding) processes (Fig. 1).
Peptide name | Abbreviation | Sequence | Isoelectric point |
---|---|---|---|
Loop region of MamI9 | MamI_L | WWWSVTEFLRG | 6.00 |
C-terminal region of Mms611 | Mms6_C | YAYMKSRDIESAQSDEEVELRDALA | 4.19 |
Synthetic magnetite-binding oligopeptide112 | SP1 | SGVYKVAYDWQH | 6.74 |
Synthetic magnetite-binding oligopeptide213 | SP2 | TLNKPNRALHFN | 11.00 |
Iron-binding oligopeptide114 | IB1 | DLGEQYFKG | 4.37 |
Iron-binding oligopeptide215 | IB2 | HREERHKEEKR | 8.60 |
Synthesis of magnetite onto surface-modified flagellar filaments was performed by the co-precipitation method: 60 μL of iron chloride solution (66 mM; FeII:FeIII = 1:2) was added to 500 μL cell suspension and was gently mixed. The pH of the reaction mixture was ∼2.5, and then a base (0.1 M NaOH) was gradually added dropwise to increase the pH until a black precipitate (magnetite) formed. Although reconstructed filaments are stable between pH 4 and 10,16 the distal ends of filaments formed in vivo on bacteria are covered by the pentameric HAP2 cap17 which may help to preserve their stability even at extreme values of pH.
In order to avoid oxidation, all solutions were degassed before use, and the system was kept under argon during the synthesis.
The experiments were performed with three controls that were not supposed to contain iron- or magnetite-binding sites on their flagella: (1) wild-type bacteria, (2) a mutant with its D3 domain removed, and the two ends of D2 connected with a small, uncharged linker (ΔD3_FliC_GNLSA),6 and (3) another mutant in which the D3 domain was replaced by a longer linker (LETGPGEL),8 encoded by a gene cassette containing the recognition sites of the restriction enzymes used in the genetic engineering experiments (ΔD3_FliC_LETGPGEL).
The rheological behavior of magnetic nanofibers in an external magnetic field was determined by an Anton Paar MCR 301 rotational rheometer equipped with an MRD 70/1 T magnetorheological accessory. The measurements were conducted at a constant shear rate of 5 s−1 with parallel plate geometry (at T = 20.5 °C). During constant shear the samples were subjected to an external homogeneous magnetic field (20 s long rectangular pulse, B = 800 mT) perpendicular to the shear flow.
The dynamic magnetic response of the nanofibers was determined by a special magnetic susceptibility measurement method, which is the magnetic counterpart of the dielectric response-time measurement technique.18 The sample was placed inside a solenoid, which is the frequency-determining element of an LC oscillator. The frequency was measured by an HP 53310A modulation domain analyzer. A Helmholtz coil pair was used to generate a 50 ms long ramp pulse of a homogeneous magnetic field (25 ms rise time, Bmax = 4.5 mT). The Helmholtz coil was driven by a Labworks PA-138 power amplifier, and the input signal was supplied by a data acquisition card (National Instruments PCI-6052E).
The controlled precipitation led to the formation of networks of magnetite chains in all six iron-binding mutants and in one of the controls (ΔD3_FliC_LETGPGEL) (Fig. 4A–F). In contrast, cells in two control samples (WT and ΔD3_FliC_GLNSA) were tightly embedded in magnetite aggregates, but networks of magnetite chains were absent around the cells (Fig. 4G and H). In the case of the mutants producing networks of magnetite-covered filaments, the question arises whether (1) iron from solution was bound by the modified filaments, initiating magnetite crystal formation directly on the surfaces of filaments, or (2) magnetite nanoparticles formed by homogeneous nucleation in the solution and then attached to the filaments. The resulting particle size distribution suggests that process (1) was primarily responsible for the formation of magnetite. We observed particle sizes in the range of 1.5–15 nm showing a bimodal distribution centered around 1.5–3 nm and 10–15 nm (Fig. 5). Magnetite nanoparticles <5 nm (Fig. 6) were observed neither in inorganic co-precipitation experiments in which the same reagents were used (and that yielded 13.5(±6) nm diameter nanoparticles),9 nor in the experiments in which pre-made magnetite particles were attached to mutant filaments.9 Although the homogeneous nucleation of magnetite in the solution and its later attachment to the flagella cannot be entirely excluded, we found in a previous study that the FliC-Mms6_C mutant could not interact with magnetite nanoparticles.9 We also checked the magnetite-binding ability of the IB1 and IB2 variants but failed to observe any binding. The fact that Mms6_C, IB1 and IB2 flagella were not able to interact with pre-existing magnetite surfaces but were efficient in the nucleation experiments (Table 2) also suggests that crystals nucleated directly on these filaments (Fig. 7).
Fig. 7 Bright-field TEM image of a web of magnetic nanofibers synthesized by co-precipitation of Fe(II) and Fe(III) onto mutant FliC-Mms6_C flagella. Since Mms6_C flagella were not able to interact with pre-existing magnetite surfaces,8 the presence of magnetite on these filaments indicates that the crystals nucleated directly on the filaments (the large crystal with dark contrast on the top is NaCl). |
Filament sample | Results of the magnetite-binding experiment | Results of the nucleation experiment |
---|---|---|
MamI_L | + | + |
Mms6_C | − | + |
SP1 | + | + |
SP2 | + | + |
IB1 | − | + |
IB2 | − | + |
ΔD3_FliC_LETGPGEL (control) | − | + |
ΔD3_FliC_GLNSA (control) | − | − |
Wild-type (control) | − | − |
HRTEM images of the nm-sized crystallites and their Fourier transforms indicate that the particles are magnetite. In many systems the formation of amorphous precursors was observed before crystallization of the final product.19 For magnetite, a ferrihydrite precursor was found by cryo-TEM to form in the solution first and then to convert to magnetite.20 Here, we observed cyrstalline, nm-sized magnetite without any evidence for the presence of a precursor. Interestingly, the nm-sized crystallites did not arrange themselves along the protein templates by oriented attachment, and did not fuse into larger single crystals, but were present in random crystallographic orientations along the filaments.
In the present study we tested the magnetite-nucleating ability of six types of mutant flagella, some of which had been previously shown to be efficient binders of magnetite nanoparticles.9 The results of both magnetite nucleation and magnetite attachment experiments are outlined in Table 2. The loop segment of MamI can perform a double role: it may both nucleate and bind magnetite nanoparticles. Previous studies suggested that MamI may be involved in magnetite nucleation in magnetotactic bacteria;21 our experiments provide the first direct evidence for this role. For the C-terminal part of Mms6, both iron- and magnetite-binding capacities have been reported.22,23 However, a recent study found that the 20 amino acid C-terminal residue of Mms6 binds iron ions (especially Fe(II)) rather than magnetite, suggesting that Mms6 is a magnetite-nucleating protein.24 This is confirmed by our experiments in which the C-terminal segment of Mms6 performed as a nucleating agent but was unable to bind pre-made magnetite particles. The apparent contradiction between reports about the magnetite-binding ability of Mms6_C probably results from the same protein adopting different conformations in the various fusion constructs used in the above studies.
It is not straightforward to interpret the interactions between the mutant flagellar filaments and iron ions, as well as between filaments and the magnetite nanoparticles. Based on both experimental studies of the interface between magnetite and magnetosome proteins25 and molecular dynamics simulations of interactions between the magnetite surface and acidic amino acids,26 electrostatic forces are thought to dominate. In our experiments, the attachment of magnetite nanoparticles to the mutant filaments was performed at pH 7, where magnetite has a positive surface charge.27 Simple coulombic interactions between iron ions or the magnetite surface and the functional olygopeptides built into the flagellar filaments are insufficient to explain our observations. For example, both SP2 and MamI_L bound pre-made magnetite nanoparticles, even though the isoelectric point of SP2 is at pH 11 (Tables 1 and 2) and MamI_L has a highly hydrophobic character. We have no clear explanation for the observed binding behavior of the applied segments. The process of magnetite nucleation appears to be less specific than magnetite binding, since in addition to the six mutants designed to bind iron, even the ΔD3_FliC_LETGPGEL mutant (intended as a control) was able to nucleate magnetite. In contrast, only three of the mutant filaments could attach to magnetite nanoparticles. Although the ΔD3_FliC_LETGPGEL mutant did not contain the D3 domain of flagellin, two glutamic acid residues were present in the construct. We believe that negatively charged amino acids probably play an important role in binding iron irons from solution and thereby initiating magnetite nucleation.
Even though the atomic scale interactions between the mutant filaments and magnetite remain obscure, the efficiency of the inserted oligopeptide sequences in either nucleating and/or binding magnetite has been clearly established by our experiments.
To test whether the magnetic nanofibers could be aligned by exposure to a static magnetic field, a drop of suspension containing magnetite-covered MamI_L filaments was placed on a TEM grid and then introduced into a strong magnetic field (up to 800 mT) until the sample dried. TEM images revealed that the magnetic filaments aligned in roughly parallel ropes of several μm long (Fig. 8A), and each rope was composed of bundles of smaller chains of magnetite-covered filaments.
The effect of a magnetic field on the viscosity of a suspension containing magnetite-covered filaments was investigated experimentally. Fig. 8B shows the viscosity versus time of the MamI_L sample and two controls (one containing unmodified filaments with magnetite and the other only magnetite particles), with and without application of an external magnetic field. The viscosity of the sample containing the magnetite-covered filaments increased about twofold upon application of an external magnetic field, and returned to its original value when the magnetic field was switched off. The change of viscosity is caused by the alignment of the magnetic filaments parallel to the applied field (that is perpendicular to the shear exerted by the flow). No viscosity change could be observed in the controls. Apparently, the chains formed from the free magnetite particles under the magnetic field were much less stable than the ordered, linear arrangement of magnetite along the filament templates.
The dynamics of structure formation (alignment of the magnetic filaments) in an external magnetic field was characterized by response time measurements. The structure formation causes a change in the magnetic susceptibility of the nanofiber samples. The response time was extracted from the dynamic magnetic response (change of magnetic susceptibility versus time) as a characteristic time constant. We found that the magnetic nanofiber samples have a response time of τ = 10.2 ms, which is considerably higher than the τ = 6.6 ms response time of the reference sample (magnetite nanoparticles without filaments). The slower response of the magnetic nanofibers is consistent with the magnetite particles being bound to the filaments.
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