biomineralization and biomimetics articles spatial...

J. Mater. Res., Vol. 31, No. 5, Mar 14, 2016

BIOMINERALIZATION AND BIOMIMETICS ARTICLES

Spatial localization of Mms6 during biomineralization of Fe3O4

nanocrystals in Magnetospirillum magneticum AMB-1

Zachery OestreicherSchool of Natural System, College of Science and Engineering, Kanazawa University, Kakuma machi, Kanazawa 9201192, Japan; and School of Environment and Natural Resources, The Ohio State University, Columbus, Ohio 43210, USA

Eric MumperSchool of Earth Sciences, The Ohio State University, Columbus, Ohio 43210, USA

Carol GassmanDepartment of Chemistry, Columbia Basin College, Pasco, Washington 99301, USA

Dennis A. BazylinskiSchool of Life Sciences, University of Nevada, Las Vegas, Las Vegas, Nevada 89154, USA

Steven K. LowerSchool of Earth Sciences, The Ohio State University, Columbus, Ohio 43210, USA; and School of Environment andNatural Resources, The Ohio State University, Columbus, Ohio 43210, USA

Brian H. Lowera)

School of Environment and Natural Resources, The Ohio State University, Columbus, Ohio 43210, USA

(Received 26 June 2015; accepted 25 January 2016)

Magnetotactic bacteria mineralize nanometer-size crystals of magnetite (Fe3O4) through a seriesof protein-mediated reactions that occur inside of organelles called magnetosomes. Mms6 isa transmembrane protein thought to play a key role in magnetite mineralization. We used bothelectron and fluorescent microscopy to examine the subcellular location of Mms6 protein withinsingle cells of Magnetospirillum magneticum AMB-1 using Mms6-specific antibodies. We alsopurified magnetosomes from M. magneticum to determine if Mms6 was physically attached tomagnetite crystals. Our results show that Mms6 proteins are present during crystal growth, andMms6 is found in direct contact with the magnetite crystals or within the lipid/protein membranesurrounding the magnetite crystals. Mms6 was not detected at other subcellular locations withinthe bacteria or isolated fractions. Because Mms6 was found to completely surround themagnetosomes rather than being localized to one specific area of the magnetosome, it appears thatthis protein could act on the entire magnetite crystal during the biomineralization process. Thissupports a model in which Mms6 functions to regulate Fe3O4 crystal morphology. Thisknowledge is important for future in vitro experiments utilizing Mms6 to synthesize tailorednanomagnets with specific physical or magnetic properties.

I. INTRODUCTION

Magnetic nanoparticles hold great promise for manyapplications including, magnetic separations in biotech-nology, delivery of cancer treatments, magnetic reso-nance imaging, data storage, and clean up ofenvironmental contaminants.1–10 Humans have thereforespent much of the past decade trying to synthesizemagnetic nanoparticles with controllable size and mor-phology (e.g., Ref. 11). There is another organism thathas spent considerably more time perfecting the art offabricating nanometer scale magnets. A group of prokar-yotes called magnetotactic bacteria (MTB) have evolved

a unique set of genes to direct the biological synthesis ofnanometer size, monodisperse magnets. The human questto synthesize the ideal nanomagnet may benefit from ourability to mimic the biomineralization reaction within thissingle-celled organism.MTBs use a specialized set of proteins to mineralize

chemically pure, single domain, nanometer-size crystalsof magnetite (Fe3O4) and or greigite (Fe3S4) underphysiological conditions.12–14 The morphology of thecrystals can be elongate hexagonal, tooth/bullet-shaped,cuboctahedral, or octahedral, depending on the species ofMTB. Crystal length ranges from 35 to 120 nm, whichputs the magnets in the single domain size range.15 Themagnetic crystals are formed and contained within a in-tracellular, organelle known as the magnetosome.16 MostMTB cells contain between 20 and 50 of thesemagnetosomes.17

Contributing Editor: Laurie Gowera)Address all correspondence to this author.e mail: [email protected]

DOI: 10.1557/jmr.2016.41

�Materials Research Society 2016 527


A major challenge to replicating the bacterial productionof nano-magnetic crystals is determining which proteins arespecific to MTB biomineralization and how those proteinsparticipate in the process. Biomagnetite proteins exclusivelyassociated with the magnetosome are named membranespecific proteins (Mam) and magnetic particle membranespecific proteins (Mms).18–20 These proteins are encoded asa cluster of 48 genes on the highly conserved magnetosomeisland.19,20 However, little is know about how these proteinsget emplaced in the magnetosome membrane and thetemporal arrangement of particular proteins in relation tothe appearance of magnetite.21,22

Mms6 has been shown to be a key protein involved inthe mineralization of magnetite in vivo.23–27 Mms6contains 59 amino acids and is 6.3 kDa in its maturestate (12.5 kDa in its premature state).23 This protein hasa hydrophilic C-terminal which is tightly bound to themagnetite crystal and a membrane-bound, hydrophobic,leucine–glycine rich N-terminus.23,24,28 The goal of thisstudy is to determine the location of Mms6 proteinsinside of individual cells of M. magneticum AMB-1. Thishas been accomplished by using two different techniques:gold immunolabeling with transmission electron micros-copy (TEM) and fluorescent labeling with confocal laserscanning microscopy (CLSM).

II. MATERIALS AND METHODS

A. Purification of the protein

Mms6 was purified using the technique described byProzorov et al.24 Plasmids (pTrcHis-TOPO) containing thegene sequence for the mature Mms6 protein fromM. magneticum AMB-1 and a poly His-tag on theN-terminus were used to transform XL-1 Blue CompetentCells (Stratagene, La Jolla, California). The proteins wereover-expressed in the cells using IPTG induction and thenpurified using TALON metal affinity resin (Clontech,Mountain View, California). Most of the proteins wereinsoluble, so 8 M guanidine was used to solubilize theinclusion bodies. The recombinant protein was dialyzed toallow Mms6 to refold into its native state. Purifiedrecombinant Mms6 was resolved by SDS-PAGE, stainedwith Coomassie Simply Blue Safe Stain (Invitrogen,Carlsbad, California) and sequenced using mass spectrom-etry to confirm that the correct protein was purified.

B. Mass spectrometry of recombinant protein

Mms6 protein was sequenced at The Ohio StateUniversity Mass Spectrometry and Proteomics Facility.The protein band was cut from the gel and then digestedand sequenced using capillary-liquid chromatographytandem mass spectrometry (Cap-LC/MS/MS) usinga Thermo Finnigan LTQ mass spectrometer equippedwith a CaptiveSpray source (Bruker Michrom, Billerica,

MA) operated in positive ion mode. Sequence informa-tion from the MS/MS data was processed by convertingthe raw data files into a merged file (.mgf) using anin-house program, RAW2MZXML n MGF batch(merge.pl, a Perl script). The resulting mgf files weresearched using Mascot Daemon by Matrix Scienceversion 2.3.2 (Boston, MA) and the database searchedagainst the full SwissProt database version 2012 06(536,489 sequences; 190,389,898 residues) or NCBIdatabase version 20120515 (18,099,548 sequences;6,208,559,787 residues). Protein identifications werechecked manually and proteins with a Mascot score of50 or higher with a minimum of two unique peptidesfrom one protein having a -b or -y ion sequence tag offive residues or better were accepted.

C. Production of the antibody

Purified recombinant Mms6 was sent to ProSci In-corporated (Poway, CA) to produce polyclonal antibodiesin rabbits. After eight weeks, the serum was removedfrom the rabbit and the Mms6 antibodies were purifiedfrom rabbit serum using affinity purification by attachingrecombinant Mms6 to a resin and running the serumthrough a column containing the Mms6 resin. The finalconcentration of the antibody was 1.42 mg/mL as de-termined by direct ELISA. Pre-immune serum was re-moved from the rabbit before injecting the antigen(recombinant Mms6) into the rabbit which was later usedas a control for immunoblotting.

D. Immunoblotting analysis

Two different amounts of recombinant Mms6 protein(0.5 and 0.05 lg) and three different cell fractions ofM. magneticum AMB-1 were resolved by SDS using10 well 16% Tris–glycine gels (Invitrogen). The threecell fractions and the total amount used in each lanewere: (a) magnetosome membrane (10 lg), (b) cellularsoluble protein (10 lg), and (c) cell membrane (10 lg).Two identical gels were run simultaneously; one gel wasused for blotting, the other was imaged after stainingwith Simply Blue Safe Stain (Invitrogen). BenchMarkprestained protein ladder (Invitrogen) was used on bothgels. The proteins were blotted onto a PVDF membrane(Invitrogen), blocked with 5% BSA, labeled with theanti-Mms6 antibody at a concentration of 1:50,000 andlabeled with a secondary goat anti-rabbit HRP (horse-radish peroxidase) antibody at a concentration of 1:200using the Clean-Blot IP Detection Kit HRP (Thermo-Pierce, Rockford, Illinois). The membrane was analyzedon a Gel Logic 1500 using Kodak software (Rochester,New York). As a control to test whether or not theantibody binds to Mms6, the procedure above wasrepeated, but 300 lg of recombinant Mms6 was addedto the antibody solution.

Z. Oestreicher et al.: Spatial localization of Mms6 during biomineralization of Fe3O4 nanocrystals in Magnetospirillum magneticum AMB-1

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by another protein that associates with Mms6, or theresult of actual uneven distribution of Mms6 epitopes onthe magnetosomes. If the latter is the case, then labelingis occurring because the protein is present in higherconcentrations on some magnetosomes. This suggeststhat the Mms6 is actively controlling the morphology ofgrowing magnetite crystals. Conversely, the absence oflabel suggests that the crystal has finished growing.

Some of the first reports of Mms6 described the proteinas being involved in crystal nucleation.23,24,32,33 How-ever, these examinations have all been done in vitro usingisolated proteins.23,24 Indeed, the ability of isolatedMms6 proteins to nucleate magnetite crystals has beenconvincingly demonstrated.27,31 Still, the in vivo functionof Mms6 within a bacterium may be to control the growthand mineralization process inside the magnetosome. Theresults of our localization experiments support this idea.Further, our results match the conceptual model proposedby Lohße et al., 2014.37 Their work illustrates a temporalmodel of magnetosome biosynthesis in which Mms6 isonly present during the “crystal maturation” stage. Weobserved Mms labeling only when a mature Fe3O4 crystalwas present. Furthermore, in their model,37 Mms6 is notpresent during the late “crystal maturation” stage, nor isthe protein present during the final stage of “magneto-some chain assembly and positioning”, both consistentwith the work presented herein.

The significance of understanding magnetite biominer-alization in MTB is critical if one wants to mimic MTB’scapacity to biomineralize single domain magnets withuniform morphology. Previous studies have shown thatMms6 plays a strong role in shaping the magnetitecrystals, but the location and timing of the activity ofMms6 during the biomineralization has not been dis-cussed. Our results suggest that Mms6 is present duringthe process of mineral growth, which helps to understandthe timing of the proteins’ activity within MTB. Thisneeds to be confirmed with additional time-point experi-ments (e.g., determine if gold nanoparticles are largelyassociated with mature magnetosomes as opposed toimmature or empty magnetosomes from iron-limitedcells). This knowledge is important for future studies,which would use Mms6 to synthesize customized mag-nets having a specific morphology and size. Individuallytailored magnets could have many uses ranging fromdrug delivery in the human body to technologicalapplications involving magnetic data storage and evenstrategies for environmental remediation.

ACKNOWLEDGMENTS

This research was supported by the U.S. NationalScience Foundation grants EAR1424138 andEAR1423939. We would like to thank Marit Nilsen-Hamilton and Pierre Palo for providing us with the

plasmid used in this study and their assistance withprotein purification; Sara Cole and Richard Montione atThe Ohio State University Campus Microscopy andImaging Facility for their assistance with the microscopy;and Todd Matulnik for assisting us with the batchculturing of M. magneticum AMB-1.

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