The ISME Journal (2015) 9, 1399–1409 & 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE Sudden motility reversal indicates sensing of magnetic field gradients in Magnetospirillum magneticum AMB-1 strain Lina M Gonza´lez1, Warren C Ruder2, Aaron P Mitchell3, William C Messner1,4 and Philip R LeDuc1 1Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA; 2Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA, USA; 3Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA, USA and 4Department of Mechanical Engineering, Tufts University, Medford, MA, USA Many motile unicellular organisms have evolved specialized behaviors for detecting and responding to environmental cues such as chemical gradients (chemotaxis) and oxygen gradients (aerotaxis). Magnetotaxis is found in magnetotactic bacteria and it is defined as the passive alignment of these cells to the geomagnetic field along with active swimming. Herein we show that Magnetospirillum magneticum (AMB-1) show a unique set of responses that indicates they sense and respond not only to the direction of magnetic fields by aligning and swimming, but also to changes in the magnetic field or magnetic field gradients. We present data showing that AMB-1 cells exhibit sudden motility reversals when we impose them to local magnetic field gradients. Our system employs permalloy (Ni80Fe20) islands to curve and diverge the magnetic field lines emanating from our custom-designed Helmholtz coils in the vicinity of the islands (creating a drop in the field across the islands). The three distinct movements we have observed as they approach the permalloy islands are: unidirectional, single reverse and double reverse. Our findings indicate that these reverse movements occur in response to magnetic field gradients. In addition, using a permanent magnet we found further evidence that supports this claim. Motile AMB-1 cells swim away from the north and south poles of a permanent magnet when the magnet is positioned less than B30 mm from the droplet of cells. All together, these results indicate previously unknown response capabilities arising from the magnetic sensing systems of AMB-1 cells. These responses could enable them to cope with magnetic disturbances that could in turn potentially inhibit their efficient search for nutrients. The ISME Journal (2015) 9, 1399–1409; doi:10.1038/ismej.2014.224; published online 5 December 2014 Introduction pigeons. Among other eukaryotic organisms in which magnetite has been experimentally found Diverse organisms can sense and respond to the are fish (Mann et al., 1988; Diebel et al., 2000) and Earth’s magnetic field. This ability is dependent termites (Maher 1998). All of these organisms upon the intracellular biomineralization of presumably use this mineral as a ‘compass’ to sense magnetic particles and the formation of organic magnetic fields when navigating and homing. compartments or vesicles; this magnetic field sen- Certain unicellular microorganisms such as sing behavior can be found in a diversity of magnetotactic bacteria (MTB) have the ability to organisms (Lowenstam, 1981). For example, Gould synthesize chains of mineral crystals (Ruder et al., et al. (1978) discovered that honeybees contain 2012), each of which is enveloped by individual magnetite localized in their abdomen, and Walcott lipid bilayers in organelles known as magnetosomes and colleagues (Walcott, 1977; Walcott et al., 1979) (Frankel et al., 1979). These magnetosome inclu- discovered magnetite in the head of homing sions render these creatures a model organism to study compartmentalization in prokaryotes Correspondence: P LeDuc, Department of Mechanical Engineering, (Komeili, 2012), and the formation mechanisms of Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA these structurally organized magnetosomes under 15213, USA or W Messner, Department of Mechanical Engineering, genetic control have been elucidated (Komeili et al., Tufts University, 204 Anderson Hall, Medford, MA 02155, USA. E-mail: [email protected] or [email protected] 2004, 2006; Scheffel et al., 2006). According to Received 6 May 2014; revised 14 September 2014; accepted 23 previous studies the proposed mechanisms of the October 2014; published online 5 December 2014 assembly of magnetosomes are: (1) the magnetosome Reversal indicates sensing of magnetic gradients LM Gonza´lez et al 1400 compartment invaginates from the inner cell mem- In this paper, we have found sudden motility brane (Komeili et al., 2004); (2) individual magneto- reversals (Figure 1a) exhibited by the Magneto- somes are recruited to the actin-like protein, MamK spirillum magneticum (AMB-1) that are related to (Komeili et al., 2006; Katzmann et al., 2010) magnetic field gradients. We proposed that these (providing a structural scaffold), where an acidic organisms may utilize these reversals to respond to protein, MamJ (Scheffel et al., 2006), (running along magnetic disturbances. the cell, from pole to pole), attaches them to this cytoskeleton filaments in a beads-on-a-string man- ner; (3) ferric ions are chelated by siderophores and Materials and methods transported into these cells; and (4) magnetite crystallization ensues within the nucleated vesicles. Helmholtz coils There is a plethora of information about magneto- In order to examine the interaction of AMB-1 cells, somes and the proteins involved in their formation we custom built a pair of Helmholtz coils that processes. However, there is no study showing produced an area with a uniform magnetic field evidence of the ability of magnetotactic bacteria to (Gonza´lez et al., 2014) . This system consisted of two cope with magnetic disturbances or fluctuations identical coils separated by the radius of the coils. (that is, gradients) in their surrounding environ- The radius of the coil was 4.5 inch and the gauge of ments that can misguide them, pull them and the magnet wire used was 22 AWG. For this pair of inhibit their efficient search for nutrients. Investiga- Helmholtz coils, there were 400 turns in each coil, tion of the swimming behavior in the presence of and the windings were stacked in 7 layers. For the magnetic field gradients could shed light on a U-turn experiments, we used another larger pair of possible defense mechanism that these organisms Helmholtz coils with a radius of 13 inch with 150 could have adopted under physiological stress in turns in each coil and the magnet wire gage used their natural environment or in the laboratory was 18 AWG. An Arduino Uno microcontroller was setting to impart a protection against self-magnetics. used to rapidly alternate the stimulation between Figure 1 Observed reversals in magnetotactic bacteria in the vicinity of magnetic islands indicate magnetic field gradient sensing. (a) Schematic of the three observed modes of motility, UM, SRM and DRM, for AMB-1 approaching NiFe islands. Bright-field microscopy images of a time sequence of the motility path of representative AMB-1 approaching the magnetic islands and exhibiting (b) single- reverse and (c) double-reverse responses (scale bar, 5 mm). The ISME Journal Reversal indicates sensing of magnetic gradients LM Gonza´lez et al 1401 the two coils to do the U-turn experiments. A circuit field lines, but the ability of these bacteria to switch with a transistor (TIP102) and diode (1N4004) was directions in response to local magnetic field built to protect the electrical circuitry of the gradients has not been explored. Sudden motility Arduino microcontroller as the maximum current reversals in axial magnetotactic bacteria have been the Arduino can take is B30 mA and we needed previously observed in response to oxygen gradients over 0.1 A˚ to drive the coils. In the circuitry, in a mechanism known as magneto-aerotaxis, we included light-emitting diodes (LEDs) to because it is a ‘magnetically assisted aerotaxis’ indicate which coils were operating at each (Frankel et al., 1997). In this study, we imposed time (Supplementary Figure S1 shows the coil sharp concentrated magnetic field gradients using B arrangement and the circuit used to control thin film permalloy (Ni80Fe20) island ( 30 nm in both coils). height) to concentrate magnetic fields generated through our custom-designed Helmholtz coils that produced a uniform magnetic field (Gonza´lez et al., Permalloy island fabrication 2014). We exposed AMB-1 cells to magnetic field Permalloy islands (Figures 1b and c) were fabricated gradients as they approached and crossed the star- and then magnetized using the previously described shaped islands. They experienced a change in the Helmholtz coils. To visualize the interaction of the magnetic field from high to low and back to high AMB-1 cells with these islands, we used a  63 again. The bacteria were observed to exhibit three objective on a modified inverted microscope (Zeiss distinct behavior modes, unidirectional movement Anxiovert 200, Go¨ttingen, Germany). The permalloy (UM), single-reverse movement (SRM) and double- islands and the microscope slide thickness was reverse movement (DRM), as shown in Figure 1a. o170 mm for visualization. To make the permalloy The UM mode was characterized by continued, island master mold, we used an image reversal straight-line movement with only very slight devia- fabrication process (Long and Newman, 1984). The tions from the straight path in one direction as the ensuing master was used as the mold to