Magnetotactic Protists at the OxicâAnoxic Transition Zones of Coastal ...

Biodata of Dennis A. Bazylinski, author of “Magnetotactic Protists at the Oxic–Anoxic Transition Zones of Coastal Aquatic Environments,” with coauthors Christopher T. Lefèvre and Richard B. Frankel. Dennis A. Bazylinski received his Ph.D. in Microbiology from the University of New Hampshire in 1984. He is currently Director of and a Professor in the School of Life Sciences at the University of Nevada at Las Vegas. He joined this Department after spending ten years in the Department of Microbiology at Iowa State University. His main research interests are in Microbial Geochemistry and Microbial Ecophysiology with a focus on Biomineralization. His organisms of study are the magnetotactic bacteria, prokaryotes that biomineralize intracellular magnetic crystals, which he has been working on for over 25 years after being introduced to them during his Ph.D. work. E-mail: [email protected] Christopher T. Lefèvre received his Ph.D. in Marine Biology from the Centre d’Océanologie de Marseille, Université Aix-Marseille II, France in 2008. He joined Professor Bazylinski’s lab as a Postdoctoral Associate in 2008. He recently started a second Postdoctoral at the Comissariat à l’Energie Atomique of Cadarache in France. His research interest is the ecophysiology and evolution of the magnetotactic bacteria. E-mail: [email protected]

Christopher T. Lefèvre

Dennis A. Bazylinski

131 A.V. Altenbach et al. (eds.), Anoxia: Evidence for Eukaryote Survival and Paleontological Strategies, Cellular Origin, Life in Extreme Habitats and Astrobiology 21, 131–143 DOI 10.1007/978-94-007-1896-8_7, © Springer Science+Business Media B.V. 2012

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DENNIS A. BAZYLINSKI ET AL.

Richard B. Frankel received his Ph.D. from the University of California, Berkeley in 1965. He is currently Emeritus Professor of Physics at the California Polytechnic State University, San Luis Obispo. He came to Cal Poly in 1988 after 23 years at the F. Bitter National Magnet Laboratory, MIT. His main research interests are in biophysics of magnetotaxis and biomineralization of magnetic iron minerals. He started working on magnetotactic bacteria in 1978. E-mail: [email protected]

MAGNETOTACTIC PROTISTS AT THE OXIC–ANOXIC TRANSITION ZONES OF COASTAL AQUATIC ENVIRONMENTS

DENNIS A. BAZYLINSKI1, CHRISTOPHER T. LEFÈVRE1, AND RICHARD B. FRANKEL2 1 School of Life Sciences, University of Nevada at Las Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154-4004, USA 2 Department of Physics, California Polytechnic State University, San Luis Obispo, CA 93407, USA

1. Introduction Magnetotactic bacteria are a diverse group of motile prokaryotes that biomineralize intracellular, membrane-bounded, tens-of-nanometer-sized crystals of a magnetic mineral, either magnetite (Fe3O4) or greigite (Fe3S4) (Bazylinski and Frankel 2004). These structures, called magnetosomes, cause cells to align along the Earth’s geomagnetic field lines as they swim, a trait called magnetotaxis (Frankel et al. 1997). Magnetotactic bacteria are known to mainly inhabit the oxic–anoxic transition zone (OATZ) of aquatic habitats (Bazylinski and Frankel 2004), and it is currently thought that the magnetosomes function as a means of making chemotaxis more efficient in locating and maintaining an optimal position for growth and survival at the OATZ (Frankel et al. 1997). In addition to magnetotactic bacteria, there have been a few reports of the presence of magnetotactic protists in similar environments (Bazylinski et al. 2000). Unfortunately, this discovery raised more questions than it answered! For example, what is the origin of the magnetic crystals in these organisms and do they consist of the same magnetic minerals as the magnetotactic bacteria? Do these organisms contribute to iron cycling in their respective habitats? etc. While the magnetotactic bacteria have been relatively well-studied as to their phylogeny, ecology, physiology, and genetics (Bazylinski and Frankel 2004), little to nothing is known regarding magnetotactic protists. The purpose of this chapter is to present what is known about magnetotactic protists and to discuss some of the questions and problems regarding these organisms that remain to be addressed in future studies. 2. Discovery of Magnetotactic Protists The first magnetotactic protist, a euglenoid alga, was discovered in brackish mud and water samples collected from a coastal mangrove swamp near Fortaleza, Brazil (Fig. 1) (Torres de Araujo et al. 1985). This organism was tentatively 133

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Figure 1. Transmission electron micrograph (TEM) image of a cell of the magnetotactic euglenoid alga described by Torres de Araujo et al. (1985). The parallel “lines” that traverse the cell along its long axis represent chains of bullet-shaped magnetite magnetosomes. Upper inset shows higher magnification electron micrograph image of the magnetosomes.

identified as Anisonema platysomum and contained numerous chains of bulletshaped magnetite crystals ranging from 80 to 180 nm long by 40 to 50 nm wide. Chemical concentrations of the environment where this organism was located were not determined, but it seems likely from the presence of magnetotactic coccoid and spirillar bacteria that conditions were microaerobic/microoxic. While studying magnetotactic bacteria populations related to the chemistry and magnetic properties of the water column of a chemically stratified coastal salt pond, Bazylinski et al. (2000) and later Simmons and Edwards (2007) reported the presence of numerous, different magnetotactic protists whose presence in the water column appeared to be correlated with depth and specific chemical parameters.

MAGNETOTACTIC PROTISTS AT THE OXIC–ANOXIC TRANSITION ZONES

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3. Magnetotactic Protists at Salt Pond Salt Pond is a semi-anoxic eutrophic marine basin in Woods Hole (MA, USA) that becomes chemically stratified during the summer and fall (Wakeham et al. 1984, 1987). It has a maximum depth of about 5.5 m and receives significant freshwater input resulting in a well-defined pycnocline (density gradient), which helps to stabilize the chemical gradients (Fig. 2). Hydrogen sulfide, resulting from the activity of sulfate-reducing bacteria in the anoxic hypolimnion, diffuses toward the surface causing the formation of steep opposing gradients of hydrogen sulfide and oxygen diffusing from the surface. The OATZ rises to within 2–3.5 m of the surface in summer. Dense populations of prokaryotes exist in the OATZ in Salt Pond, including magnetotactic and purple photosynthetic bacteria, the latter of which cause the water to appear pink when collected.

3.1. TYPES OF MAGNETOTACTIC PROTISTS Magnetotactic protistan types observed in Salt Pond included dinoflagellates, biflagellates, and one ciliate (Fig. 3). The sizes of these organisms were typical of eukaryotes rather than prokaryotes and ranged from 3.8–9.3 to 15–28 mm. Fe(μM) 0

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Figure 2. Chemical concentration profiles versus depth in Salt Pond determined during July 1995 in the summer when this semi-anaerobic basin becomes chemically stratified. The OATZ occurred from about 3.3 to 3.7 m. Magnetite-producing magnetotactic bacteria are generally found at the OATZ proper, while greigite-producing types are found below the OATZ in the anaerobic sulfidic zone. The distribution of magnetotactic protists is shown in Table 1 with depths relating to this figure.

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Table 1. Magnetotactic protists present in Salt Pond water column samples collected in July 1995a.

Protist

Type

Tentative identification

Depth distribution (m)

Zone

Ab B C D

Dinoflagellate Biflagellate Biflagellate Ciliate

Dinoflagellate Cryptomonad Cryptomonad Cyclidium species

3.3–3.6 3.5–3.7 3.7–4.1 3.8–3.9

OATZ to anoxic Anoxic Anoxic Anoxic

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Data from Bazylinski et al. (2000) Letter denotes protists shown in Fig. 3

b

All types had clearly identifiable eukaryotic cell structures, including nuclei, flagella, and/or cilia. Magnetotactic protists were associated with specific depths in Salt Pond, as shown in Table 1, and, in general, most types were more abundant in the anoxic zone. It is presently unclear whether they follow magnetotactic or other bacteria to graze on or whether they require specific chemical and/or anoxic conditions for growth and survival. Apparently, the types of magnetotactic protists present in Salt Pond change, as Simmons and Edwards (2007) show images of three types, two of which are clearly different in morphology than those described here. What controls the types of magnetotactic protists present in Salt Pond is not currently known but may be related to changes or disruptions in water column chemical concentration profiles which have been shown to occur (Moskowitz et al. 2008; Bazylinski et al. unpublished data).

3.2. BEHAVIOR OF MAGNETOTACTIC PROTISTS Several different types of magnetotactic protists were found in Salt Pond, all of which displayed magnetotaxis but differed in how they swam. Like magnetotactic bacteria, they migrated and accumulated at the edge of a hanging water droplet in a magnetic field (Fig. 4). A dinoflagellate (Fig. 3a) and a specific biflagellate (Fig. 3c) migrated more or less directly along magnetic field lines and responded to a reversal of the magnetic field by rotating 180° and continued to swim in the same direction relative to the field direction. In contrast, another biflagellate (Fig. 3b) did not migrate directly along the magnetic field lines; instead rather it swam with frequent, spontaneous changes in direction, only gradually migrating along magnetic field lines. The ciliate shown in Fig. 3d exhibited the most unusual behavior. It frequently attached to the microscope slide and/or cover slip, making rapid excursions before attaching again. Cells that apparently died and became free-floating responded to a reversal of the magnetic field, as described for the dinoflagellate. Unlike magnetotactic bacteria, it took the protists a significantly


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Figure 3. Light micrographs of four types of magnetotactic protozoa. (a) Differential interference contrast (DIC) micrograph of a dinoflagellate. Arrows denote the girdle or annulus surrounding the test typical of the dinoflagellate group. One flagellum has separated from the cell. (b) Phase-contrast (PC) micrograph of a biflagellate. Neither flagellum is visible. Arrows denote structures that are sometimes extruded from the cell and that orient in a magnetic field. (c) PC micrograph of another biflagellate showing the two flagella typical of the group. (d) DIC micrograph of a ciliate probably belonging to the genus Cyclidium (Figure adapted from Bazylinski et al. 2000).

longer period of time to accumulate near the edge of the drop and, in addition, they never actually reached the very edge of the drop (Fig. 4). In the original report on these organisms (Bazylinski et al. 2000), they were described as magnetic (meaning that they are magnetically responsive) rather than magnetotactic. “Magnetotactic” may be more appropriate as all those described from natural environments showed a polar preference in their swimming direction like polar magnetotactic bacteria (Frankel et al. 1997; Simmons and Edwards 2007).

3.3. “MAGNETOSOMES” IN MAGNETOTACTIC PROTISTS? Electron microscopy in conjunction with selected area electron diffraction and energy dispersive x-ray analyses was consistent with the presence of magnetite crystals in cells of the dinoflagellate and the biflagellate shown in Fig. 3a–c (Bazylinski et al. 2000) (Fig. 5). Although crystals were similar in size to (about 55–75 nm in diameter) and morphologically resembled those of magnetotactic bacteria, precise crystal morphologies could not be determined due to the thickness of the cells. Interestingly, despite the fact that greigite is a magnetic component in a number of magnetotactic bacteria in Salt Pond, it was never identified in any protistan cell. It is possible that this mineral dissolves rapidly in protistan cells, as discussed in Sect. 4. Whether the magnetite crystals in these magnetotactic protists can be considered as “magnetosomes” depends on whether they are enclosed in a lipid bilayer membrane as they are in their bacterial counterparts. This is presently

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Figure 4. Effect of a magnetic field on the behavior of two protists collected from Salt Pond, the dinoflagellate shown in Fig. 3a and the biflagellate shown in Fig. 3b. The microscope was focused on a point at the edge of the water drop closest to the south pole of a bar magnet. (a) shows the organisms in that region before the magnet was placed on the microscope stage. (b) shows the effect of the magnet with the south pole closest to the drop, producing a local field direction indicated by the arrow in the upper left corner. (c) shows the effect of reversing the bar magnet so that the north magnetic pole is closest to the edge of the drop. (d) shows the reversal of the magnet again, with the same orientation as in (b). (e) and (f ) are a repeat of (c) and (d). Images were taken approximately 45–60 s after the field was reversed. Bar in panel A represents 20 mm.


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Figure 5. TEM image of the biflagellate shown in Fig. 3c. Dark, electron-dense structures in chains are the mineral crystals identified as magnetite. Inset depicts a high-magnification TEM of the magnetite crystals that are bracketed in the image of the whole cell. Note that the magnetite crystals are not organized as they are in the organism shown in Fig. 1.

unknown. Nonetheless, the presence of these crystals explains the magnetotactic behavior of these organisms.

4. Origin of Magnetite in Magnetotactic Protists An important question that needs to be answered deals with the origin of the putative “magnetosomes” in magnetotactic protists. Two possibilities have been raised: (1) Do the protists biomineralize the magnetite crystals themselves? and (2) Do the protists ingest magnetotactic bacteria and/or bacterial magnetosomes from lysed cells and incorporate them either temporarily or permanently in the cell? Most researchers feel that both possibilities occur in nature and that what occurs is species dependent despite the fact that there is only direct evidence of the second possibility. Because the arrangement of magnetosomes appears to be so precisely structured in the euglenoid alga described by Torres de Araujo et al. (1985) (Fig. 1), it seems unlikely that this arrangement could occur after the ingestion of what would have to be significant numbers of magnetotactic bacteria. Instead, it seems more likely that this organism biomineralizes and arranges endogenous magnetite crystals in a highly controlled fashion within the cell where intracellular structural

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elements play a significant role in magnetosome, as has been shown for magnetotactic prokaryotes (Komeili et al. 2006; Scheffel et al. 2006). On the other hand, other protists seem to have magnetite crystals that might be in partial chains but not very organized within the cell (Fig. 5). One biflagellate was observed to extrude magnetic, dark-orange, roughly spherical inclusions (Fig. 3b) after which the cell was no longer magnetically responsive. It is possible that these inclusions represent indigestible remains of ingested magnetotactic bacteria in vacuoles. In the study of Bazylinski et al. (2000), none of the magnetic protistan cells were observed to be engulfing significant amounts of magnetotactic bacteria although the bacteria were abundant at the same depths in the water column and present in the water droplets examined. However, Simmons and Edwards (2007) observed direct feeding of protists on magnetotactic bacteria and subsequent egestion of magnetosomes. Unfortunately, to our knowledge, there is only one published laboratory study on the ingestion of magnetotactic bacteria by a protozoan (Martins et al. 2007). In this study, the filter-feeding ciliate Euplotes vannus (E. vannus) was fed units of the multicellular magnetotactic prokaryote Candidatus Magnetoglobus multicellularis (Ca. M. multicellularis). However, cells of E. vannus did not respond to a magnetic field after confirmed ingestion of Ca. M. multicellularis. The reason for this is unclear although this organism biomineralizes greigite rather than magnetite in its magnetosomes, and it was shown that most of the greigite crystals within ingested cells were dissolved within 30–120 min of when the bacteria were present in vacuoles. 5. Role of Magnetotactic Protists in Iron Cycling Simmons and Edwards (2007) observed up to 2.9 ± 0.6 × 103 magnetotactic protists per ml in the OATZ of Salt Pond that, as shown here and previously (Bazylinski et al. 2000), can contain a large number of magnetosomes. Thus, these organisms clearly have a great potential for iron cycling in aquatic environments like Salt Pond. Iron is well recognized as a limiting factor in primary production in some oceanic environments and is often present in seawater in particulate and colloidal forms (Barbeau et al. 1996). Barbeau et al. (1996) and later Pernthaler (2005) showed that digestion of colloidal iron in the food vacuoles of protozoans during grazing of particulate and colloidal matter might generate more bioavailable iron for other species, such as phytoplankton. The observations of Simmons and Edwards (2007) together with the work of Martins et al. (2007) suggest that protists that ingest magnetotactic bacteria could play an important role in iron cycling by solubilizing iron in magnetosomes. If this is true of those that ingest magnetite-producing magnetotactic bacteria in habitats like Salt Pond, this would contribute to the high ferrous iron concentration at the OATZ and the high microbial concentrations present there. A key point here is whether magnetite is dissolved either partially or completely in the acidic


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environment of the digestive vacuoles (Ramoino et al. 1996) of the protists, as has been shown for greigite (Martins et al. 2007). Alternatively, magnetotactic protists that take up soluble forms of iron and biomineralize magnetic mineral crystals might do the opposite, making potentially significant amounts of iron unavailable to other organisms. 6. Future Research Directions As stated early in this brief chapter, the interesting discovery of magnetotactic protists raises many important questions that should be addressed in future work. These questions range from the simple to the complex. The protists obviously need to be precisely identified. Are magnetotactic protists distributed widely in aquatic environments? It is sometimes difficult to determine the presence of magnetotactic protists because they are overlooked when samples contain large numbers of magnetotactic bacteria. In addition, it takes much more time for them to swim and accumulate at the edge of a water drop than magnetotactic bacteria. However, we have observed: magnetotactic protists in samples collected from the chemically stratified Pettaquamscutt Estuary (Donaghay et al. 1992); magnetotactic Gymnodinioid dinoflagellates in samples collected from salt marsh pools at the Ebro Delta, Spain; and magnetotactic biflagellates similar to that shown in Fig. 3b in samples collected from the Mediterranean Sea in Marseille, France [Lefèvre and Wu unpublished data]. Do the magnetite crystals in protists have a magnetosome membrane? Do any of the magnetotactic protists biomineralize their own magnetosomes? Axenic cultures of these organisms would certainly help in this regard. Clearly, more magnetotactic bacteria grazing experiments involving different types of protists are necessary to understand how these organisms behave after ingestion of the bacteria. These and more environmental studies would help to ascertain the role and estimate the impact of magnetotactic protists in iron cycling in natural habitats. Regardless of the origin of the magnetite crystals, the protists described here exhibit a magnetotactic response albeit a seemingly weak one. Does their magnetic dipole moment help them in any way as it is thought to do for the magnetotactic bacteria in locating and maintaining position at the OATZ? Many types of protists appear to prefer microoxic conditions and are known to be distributed in the OATZ (Fenchel 1969; Fenchel and Finlay 1984; Fenchel et al. 1989). Some appear to use geotactic mechanisms that involve mineral mechanoreceptors containing barium (Finlay et al. 1983) and strontium (Rieder et al. 1982), and aerotaxis to locate and maintain an optimal position in vertical oxygen concentration gradients (Fenchel and Finlay 1984). What is the link between a specific protist and the chemistry in stratified aquatic environments? Many were found in the anoxic zone below the OATZ. Are these organisms following specific types of magnetotactic bacteria for grazing or are the chemical conditions necessary for their growth and survival? Do the

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protists possess any type of anaerobic metabolism? We hope this chapter and the studies cited herein stimulate the research studies necessary to answer these intriguing questions. 7. Acknowledgments We are grateful to K.J. Edwards, S.S. Epstein, M. Pósfai, and S.L. Simmons for their collaboration in this work. DAB and CTL are supported by U.S. National Science Foundation grant EAR-0920718. 8. References Barbeau K, Moffett JW, Caron DA, Croot PL, Erdner DL (1996) Role of protozoan grazing in relieving iron limitation of phytoplankton. Nature 380:61–64 Bazylinski DA, Frankel RB (2004) Magnetosome formation in prokaryotes. Nat Rev Microbiol 2:217–230 Bazylinski DA, Schlezinger DR, Howes BL, Frankel RB, Epstein SS (2000) Occurrence and distribution of diverse populations of magnetic protists in a chemically stratified coastal salt pond. Chem Geol 169:319–328 Donaghay PL, Rines HM, Sieburth JM (1992) Simultaneous sampling of fine scale biological, chemical and physical structure in stratified waters. Arch Hydrobiol Beih Ergeb Limnol 36:97–108 Fenchel T (1969) The ecology of marine microbenthos: IV. Structure and function of the benthic ecosystem, its chemical and physical factors and the microfauna communities with special reference to the ciliated protozoa. Ophelia 6:1–182 Fenchel T, Finlay BJ (1984) Geotaxis in the ciliated protozoan, Loxodes. J Exp Biol 110:17–33 Fenchel T, Finlay BJ, Gianni A (1989) Microaerophily in ciliates: responses of an Euplotes species (Hypotrichida) to oxygen tension. Arch Protistenkd 137:317–330 Finlay BJ, Hetherington NB, Davison W (1983) Active biological participation in lacustrine barium chemistry. Geochim Cosmochim Acta 47:1325–1329 Frankel RB, Bazylinski DA, Johnson MS, Taylor BL (1997) Magneto-aerotaxis in marine coccoid bacteria. Biophys J 73:994–1000 Komeili A, Li Z, Newman DK, Jensen GJ (2006) Magnetosomes are cell membrane invaginations organized by the actin-like protein MamK. Science 311:242–245 Martins JL, Silveira TS, Abreu F, Silva KT, da Silva-Neto ID, Lins U (2007) Grazing protozoa and magnetosome dissolution in magnetotactic bacteria. Environ Microbiol 9:2775–2781 Moskowitz BM, Bazylinski DA, Egli R, Frankel RB, Edwards KJ (2008) Magnetic properties of marine magnetotactic bacteria in a seasonally stratified coastal salt pond (Salt Pond, MA, USA). Geophys J Int 174:75–92 Pernthaler J (2005) Predation on prokaryotes in the water column and its ecological implications. Nat Rev Microbiol 3:537–546 Ramoino P, Beltrame F, Diaspro A, Fato M (1996) Time-variant analysis of organelle and vesicle movement during phagocytosis in Paramecium primaurelia by means of fluorescence confocal laser scanning microscopy. Microsc Res Tech 35:377–384 Rieder N, Ott HA, Pfundstein P, Schoch R (1982) X-ray microanalysis of the mineral contents of some protozoa. J Protozool 29:15–18 Scheffel A, Gruska M, Faivre D, Linaroudis A, Plitzko JM, Schüler D (2006) An acidic protein aligns magnetosomes along a filamentous structure in magnetotactic bacteria. Nature 440:110–114


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Simmons SL, Edwards KJ (2007) Geobiology of magnetotactic bacteria. In: Schüler D (ed) Magnetoreception and magnetosomes in bacteria. Springer, Heidelberg, pp 77–102 Torres de Araujo FF, Pires MA, Frankel RB, Bicudo CEM (1985) Magnetite and magnetotaxis in algae. Biophys J 50:375–378 Wakeham SG, Howes BL, Dacey JWH (1984) Dimethyl sulphide in a stratified coastal salt pond. Nature 310:770–772 Wakeham SG, Howes BL, Dacey JWH, Schwarzenbach RP, Zeyer J (1987) Biogeochemistry of dimethylsulfide in a seasonally stratified coastal salt pond. Geochim Cosmochim Acta 51:1675–1684

Magnetotactic Protists at the OxicâAnoxic Transition Zones of Coastal ...

Magnetotactic Protists at the OxicâAnoxic Transition Zones of Coastal ...

Magnetotactic Protists at the OxicâAnoxic Transition Zones of Coastal ...

Magnetotactic Protists at the OxicâAnoxic Transition Zones of Coastal ...