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MAGNETOSPIRILLUM GRYPHISWALDENSE: FUNDAMENTALS AND APPLICATIONS I. Ardelean1, 4, C. Moisescu1, M. Ignat2, M. Constantin3 and M. Virgolici3 1 Bucharest Institute of Biology, Department of Microbiology, Bucharest, Romania 2 National Institute of Research for Electrical Engineering ICPE-CA, Bucharest, Romania 3 Institute IFIN-HH, IRASM Center, Magurele, Romania 4 “Ovidius” University, Faculty of Natural Sciences, Constanta, Romania Correspondence to: Ioan Ardelean E-mail: [email protected]

ABSTRACT In this contribution we show our results on the magnetotactic bacterium Magnetospirillum gryphiswaldense concerning the growth of this bacterium in batch cultures, biochemical analysis (proteins and fatty acids profile), the (respiratory) oxygen consumption in iron -rich and iron-limited cultures, magneto-aerotactic behaviour of the cells, magnetic characterization of intact cells and isolated magnetosomes, and magnetosome isolation. We propose a magneto-mechanic model of magnetosome chain with special aim to further develop nanoactuators and micromanipulators and claim that magnetotactic bacteria could be used for in vivo synthesis of gold nanoparticles and for heavy metals biosorbtion. Keywords: biotechnology, growth, magnetotactic bacteria, magnetotaxis

Introduction Magnetotactic bacteria (MTB) are prokaryotes belonging to the Domain Bacteria whose specific functional characteristic is magnetotaxis, the ability to orient and migrate along Earth’s geomagnetic field lines (4; 5; 13; 20). Magnetotaxis is determined both by the presence of magnetosomes and the ability to perform active movements. Magnetosomes are intracellular bodies which consist of magnetic iron mineral particles, either magnetite (Fe3O4) or greigite (Fe3S4) enclosed within a lipid bilayer containing different types of proteins, some of them being involved in the transformation of soluble iron in magnetic nanoparticles. In Magnetospirillum gryphiswaldense as well as in other MTB, magnetosomes are only produced at low atmospheric oxygen tensions, these microorganisms showing a microaerophilic behavior in which magnetosomes play an important role. It was proposed that in natural environments magnetotaxis enables the cells to locate and maintain an optimal position in water columns or in sediments, with respect to their main metabolically needs: molecular oxygen and other nutrients (7), all in all allowing them to keep their headings as they swim in the face of the disorienting Brownian buffering by BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE

the medium (3). Furthermore, in the last years the evaluation of the potential of magnetosomes in various biomedical and nanotechnological applications has received special attention (10; 11, 17; 19). The aim of this paper is to summarize the main results obtained so far by our group and to stress on the perspectives of our research.

Materials and methods Material and methods Bacterial strains. A wild-type M. gryphiswaldense strain, was used in this study. The stock culture of M. gryphiswaldense was provided by Prof. Dr. Dirk Schüler (Ludwig Maximilians University of Munich). Flask cultivation. M. gryphiswaldense was cultured at 28°C in flask standard medium (FSM) at two different iron concentration 100 µM and 0.7 μM as described previously (16). Cell growth was determined by measuring the optical density at 565 nm. Optical and transmission electron microscopy were performed as described previously (14; 15). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of total and periplasmic proteins of M. gryphiswaldense were performed as described previously (14). Magnetosomes isolation. Intact cells of M. gryphiswaldense were treated with 50 mM phosphate buffer, pH 7.0, followed by lysozyme

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treatment resulting in spheroplasts isolation. The intact magnetosomes (magnetite crystals surrounded by the magnetosome membrane) were extracted by an osmotic shock of the spheroplasts and magnetically separated from the cellular debris. O2 consumption rates were monitored with an oxygen meter (Rank Brothers, LTD) and titrated with different concentrations of KCN. Fatty acids profiling was as done according to Sasser M. (18) as shown (16). Quality control of FAME extraction procedure was made using reference strains Stenotrophomonas maltophilia ATCC 13637 and Bacillus subtilis as positive controls (18). Magnetic characterization of intact cells and isolated magnetsomes was done using Vibrating Sample Probe 7300 magnetometer (9).

gryphiswaldense cells, based on the capacity of magnetotactic bacteria to orient along magnetic field lines (Fig. 1a), even when dead, thus behaving like tiny, selfpropelled magnetic compass needles. The non-magnetic mutant NM failed to orient in the presence of magnetic field, fact demonstrated by the random orientation of the cells (Fig. 1b), confirming the absence of magnetite synthesis in these cells.

Results and Discussion Cellular growth and magnetosomes synthesis in M. gryphiswaldense. During this study, the growth conditions were optimized for the microaerobic growth of wild-type M. gryphiswaldense strain and a non-magnetic mutant (NM) in flasks in both iron-rich and iron-limited conditions (Table 1). These new methods allowed us to overcome one of the major problems associated with production of magnetosomes: the oxygen concentration. Considering the fact that oxygen seems to have an inhibitory effect on magnetite formation, the reduction of the oxygen amount introduced during inoculation, by injecting the inoculum through the stopper resulted in significantly increased cell and magnetosome yields. During this study we observed that the generation time and the lag period are strongly influenced by the history of the culture. TABLE 1 The generation time of M. gryphiswaldense grown in ironrich or iron-limited growth media. + + iron is present in both inoculum and culture media; + – iron is present only in the inoculum; – – iron is absent from both inoculum and culture media; – + iron is present only in the culture media. Culture type ++ +– –– –+

Lag period (h) 0 22 78 78

Generation time (h) 26 24 7 6

Microscopy methods for testing the magnetic properties of the culture. The optical microscopy method allowed us to demonstrate the existence of magnetic properties of M.

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Fig. 1. Optical microscopy images of (a) oriented magnetic cells of wild-type M. gryphiswaldense and (b) random arrangement of nonmagnetic cells of NM mutant. Electron micrographs of unstained cells of (c) wild-type M. gryphiwsaldense cell, showing the characteristic magnetosome chain (M) and (d) the non-magnetic mutant NM which exhibits the characteristic morphology of spirilla but lacks electron-dense magnetite particles. PHA, polyhydroxyalkanoate; PPH, polyphosphate. Bar represents 400 nm.

The absence of magnetosome chains in NM mutant was also verified by transmission electron microscopy (TEM) micrographs (Fig. 1c, d). The morphology of NM cells appeared to be very similar to that of the wild-type cells but we were unable to detect any particles resembling magnetosome crystals in electron micrographs. Loss of magnetism seemed to be permanent, and no reversions to the wild-type phenotype were observed. Analysis of protein profile. The comparison of protein spectra of the wild-type and the mutant NM revealed no notable differences between the two strains. These findings confirm that the mutant NM is a M. gryphiswaldense strain lacking magnetic properties. The lost of magnetic properties is probably due to a deleted gene, as a result of oxygen exposure.

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in Table 2. The saturation magnetization (or saturation induction) of the isolated magnetosomes are; M s = ± 0 , 009 [ emu ] to H = ± 1150 [ Oe ] , are assimilated with an ellipsoidal model (9).

Fig. 2. SDS-PAGE of (a) periplasmic proteins and (b) total protein extracts from wild-type (W-T) M. gryphiswaldense and non-magnetic mutant NM.

Magnetosomes. Magnetite particles extracted from the magnetotactic bacteria M. gryphiswaldense, exhibit a speciesspecific cuboctahedral crystal morphologies, with an average diameter of 46 nm (15). Magnetic characterization The magnetization characteristics (without saturation behaviour) of Magnetospirillum gryphiswaldense domain are presented

Fatty acids profiles. A significant variation of main fatty acids (coeluted “16:1 w7c/16:1 w6c” and “18:1 w7c”) was observed between the magnetic type and non-magnetic type of M. gryphiswaldense (Table 3). The fresh extracted magnetosomes seem to have similar fatty acid composition with the whole cell for magnetic type. Another variation was observed for octadecanoic acid (stearic) (18:0) thus if the magnetic type and non-magnetic type show a similar fatty acids composition, the magnetosomes membranes extracted from the magnetic type contain an increased percent compared to magnetic type and non-magnetic type. TABLE 2

The main point of the magnetization characteristics without saturation. Domain

H < 0, M < 0

H > 0, M > 0

Minimum point a) b) M ≈ −0,0003[emu ] M ≈ −0,00175[emu ] H ≈ −1000[Oe] H ≈ −980[Oe] _

Maximum point _

a)

M ≈ +0,0003[emu ] H ≈ 1000[Oe]

b)

M ≈ +0,00175[emu ] H ≈ +1200[Oe]

TABLE 3 The percent of fatty acids profiles present in the M. gryphiswaldense magnetic type membrane, non-magnetic type membrane and the fresh magnetosomes membranes obtain after GS-MS analysis. Fresh extracted Non-magnetic Magnetic type Fatty acid name magnetosomes type Percent of total extracted fatty acids dodecanoic acid (lauric) [12:0] 0.0 0.3 0.0 tetradecanoic acid (myristic) [14:0] 4.2 4.0 3.8 3-hydroxy-13-methyl tetradecanoic acid [15:0 iso 3OH] 0.0 0.4 0.0 hexadecanoic acid (palmitic) [16:0] 12.5 13.1 13.6 3-hydroxy hexadecanoic acid [16:0 3OH] 2.4 2.3 1.6 octadecanoic acid (stearic) [18:0] 2.5 3.5 2.1 3-hydroxy octadecanoic acid [18:0 3OH] 1.3 1.4 1.2 2-hydroxy 1-octadecenoic acid [18:1 2OH] 0.7 0.8 1.4 11-methyl cis-7-octadecenoic acid [18:1 w7c 11-methyl] 0.5 0.5 0.0 cis-8, 9-methylene-octadecanoic acid [19:0 cyclo w8c] 2.8 2.8 2.1 coeluted 3-hydroxy tetradecanoic acid / 14-methyl 5.7 5.5 6.3 pentadecenoic acid [14:0 3OH/16:1 iso I] coeluted cis-7-hexadecenoic acid / cis-6-hexadecenoic acid 13.5 12.5 18.4 [16:1 w7c/16:1 w6c] coeluted cis-7-octadecenoic acid [18:1 w7c] 53.3 52.7 50.3 Respiration. Typical results in M. gryphiswaldense show an O2 consumption rate of 35 nmoles O2/min/O.D for ironrich (100 µM) cultures and 23 nmoles O2/min/O.D for ironBIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE

limited (0.7 µM) cultures, suggesting that high O2 consumption rate could be correlated with the presence of magnetosomes. Furthermore, in iron-rich (100 µM) culture

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media, 20 µM KCN inhibits O2 consumption by 30% and 1,4 mM KCN by 90%, suggesting that cytochrome c oxidase and another terminal oxidase should be involved in respiratory O2 consumption. In M. gryphiswaldense cultures grown in ironlimited (0.7 µM) culture media, 20 µM KCN inhibits O2 consumption by 45% and 1,4 mM KCN by 90%, suggesting that this so far unknown terminal oxidase is used preferentially in iron-limited conditions compared with ironrich conditions. Magneto-Aerotactic behavior. Aerotaxis, the migratory response towards or away from O2, is a universal property of motile bacteria that enables the bacteria to move to a concentration of dissolved O2 that is optimal for their preferred metabolism. As it was expected, the magnetotactic M. gryphiswaldense cells showed a negative horizontal swimming response to 21% O2, the cells accumulating at the opposite end of the capillary tube, next to the bar magnet and faraway from the starting point, swimming a total distance of 3.5 cm in 110 min with a speed of 18.6 µm/s. These results correlate with the literature data (6; 21).

Conclusions As perspectives in our research we focus on the following: the potential of MTB for terraformation (2), elaboration of a magnetic model of magnetotactic bacteria (1; 8; 12; 21) heavy metal biosorbtion and gold nanoparticles synthesis.

3. Bazylinski et al. (2004) Nature Reviews, 2, 217-230. 4. Bellini S. (1963) Su di un particolare comportamento di batteri d'acqua dolce (About a peculiar behavior of freshwater bacteria). Instituto di Microbiologia dell'Universita di Pavia. 5. Blakemore R.P. (1975) Science, 190, 377-379. 6. Flies et al. (2005) FEMS Microbiol Ecology, 52, 185195. 7. Frankel et al. (1997) Biophys J., 73, 994-1000. 8. Ignat et al. (2004) Romanian J Phys., 49, 835-848. 9. Ignat et al. (2005) In Printech (ed.), Experimental aspects and magnetic characterization of Magnetospirillum gryphiswaldense. Proceedings of the 4th National Conference New Research Trends in Material Science, Constanta, pp. 433-442. 10. Ignat et al. (2007) J Optoelectronics and Advanced Materials, 4, 1169-1171. 11. Lang et al. (2006) J Phys Condens Matter., 18, S2815S2828. 12. Logofatu et al. (2008) J Appl Phys., 103, 094911094916. 13. Mann et al. (1990) Adv Microbiol Physiol., 31, 125-181. 14. Moisescu et al. (2007) Annals of West University of Timisoara, Series Chemistry, 16, 217-226. 15. Moisescu et al. (2008) Mineralogical Magazine, 72, 333336.

Acknowledgment Thanks are due to Dr. Cornel C. Ponta, Head of IRASM Center, Institute IFIN-Horia Hulubei, Magurele, Romania for scientific support.

(2008) Magnetospirillum 16. Moisescu et al. gryphiswaldense as a source of magnetite nanoparticles: biological and bionanotechnological significance. In Proceeding International Symposium on New research In Biotechnology, Serie F (Special volume), Cluj-Napoca, pp. 594-603.

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1. Ardelean et al. (2007) In M. A. Gutierrez-Naranjo, G. Paun, A. Romero-Jimenez, and A. Riscos-Nunez (eds.), Magnetotactic Bacteria and Their Significance for P Systems and Nanoactuators. Proceedings of the 5th Brainstorming Week on Membrane Computing, Seville, pp. 21-32.

18. Sasser M. (1990) Bacterial Identification by Gas Chromatographic Analysis of Fatty Acids Methyl Esters (GC-FAME) MIDI, Inc. Technical Note #101.

2. Ardelean I., Moisescu C., Popoviciu D.R. (2009) Magnetotactic bacteria and their potential for terraformation. In:. Seckbach J and Walsh M M (eds.) From Fossils to Astrobiology, 1st ed. Springer, pp. 335353.

21. Smith et al. (2006) Biophys J., 91, 1098-1107.

XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY

19. Schuler et al. (1999) Appl Microbiol Biotechnol., 52, 464-473. 20. Schuler D. (2004) Arch Microbiol., 181, 1-7.

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