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Magnetotactic bacteria from Lonar lake

Mahesh S. Chavadar* and Shyam S. Bajekal
Department of Microbiology, Yashwantrao Chavan College of Science, Karad 415 124, India

Magnetotactic bacteria (MTB) are motile, aquatic prokaryotes that swim along geomagnetic field lines. These bacteria display a myriad of cellular morphologies, including coccoid, rod, vibrioid, spirilloid etc. with their unique magnetosomes within the cells. The Lonar lake in Maharashtra, formed due to a meteorite impact crater, is a closed basin lake characterized by high alkalinity and salinity. The MTB isolated by the magnetic collection and capillary racetrack methods showed a typical response in the form of movement towards the magnet and precise alignment at the edge of the hanging drop. Intracellular iron accumulation studies on these bacteria showed up to 11.5 times more iron than non-magnetic bacteria. Keywords: Capillary racetrack, iron accumulation, magnetic collection, magnetosomes, magnetotactic bacteria. MAGNETOTACTIC bacteria (MTB), originally discovered by Blakemore in 1975, are to be found in diverse aquatic habitats. They include coccoid to ovoid cells, rods, vibrios and spirilla from different water bodies (freshwater, sea water), sediment and soil. These aquatic microorganisms have the ability to orient and migrate or swim along geomagnetic field lines, a behaviour referred to as magnetotaxis1. This property is based on specific intracellular structures, the magnetosomes, which in most MTB are nanometer-sized, enveloped, membrane-bound, single magnetic domain crystal particles composed of the iron mineral magnetite (Fe3O4) and/or greigite (Fe3S4)1,2. These magnetosomes are organized in one or more straight chains parallel to the long axis of the cells. Such an arrangement confers a magnetic dipole to the cell, which is sufficiently large so that it will orient the entire bacterium along the geomagnetic field lines at ambient temperature3. Today, MTB are the hotspot of research in microbiology with tremendous biotechnological and nanotechnological applications3, thereby attracting the interest of researchers from multiple fields. As of now, only few pure cultures of MTB are available, as cultivation of these bacteria is difficult due to their microaerophilic to anaerobic lifestyle. However, some workers have also reported the isolation of aerobic magnetic cultures4. The Lonar lake, situated in a hypervelocity meteorite impact crater5, formed some 52,000 years ago, is situated
*For correspondence. (e-mail: maheshchavadar@yahoo.co.in) CURRENT SCIENCE, VOL. 96, NO. 7, 10 APRIL 2009

in the Buldhana District of Maharashtra, India (lat. 1958 N, long. 7634E)5. This is the only crater in the world to be formed in basaltic rock6 and the lake water is saline and alkaline (pH 9.510.0, CaCO3 alkalinity 3.6 g/l, NaCl (as chloride) 3.0 g/l)6. As the formation of magnetosomes in MTB is carried out by a process of biomineralization7, these microorganisms have great importance in biogeochemical cycles1. We also noted evidences of magnetic impulses in rock samples surrounding the lake, which encouraged us to look for MTB in the lake environment. The littoral zone surrounding the Lonar lake was sampled at eight different sites for the upper sediment layer and surface water. About 1 kg soil from each location was collected using plastic spatulas into new polyethylene bags that were closed and tied tightly with string. Water samples were collected directly into previously disinfected (with isopropyl alcohol) plastic bottles of 1.01.5 l capacity and closed tightly with their lids. All samples were transported to the laboratory and stored under ambient temperature conditions till use. During transportation and storage the lids of the water bottles were kept slightly open to allow the gases formed under anaerobic conditions to escape. Collection of MTB was done by the magnetic collection method7. This technique is based on the swimming response of a cell to a magnetic field. The south pole of a permanent magnet was attached outside a jar containing water and sediment samples, 1 cm above the sediment surface. After 12 h, 0.10.2 ml of water in the bottle near the wall adjoining the magnet was collected with a sterile pipette and transferred to sterile tubes to be used for further studies. The modified capillary racetrack (CRT)8 method was used to purify and enrich the MTB obtained by magnetic collection. A capillary tube (length 69 cm) sealed at one end using a gas flame was filled with the chemically defined medium (pH 9.5) of Flies et al.7, by means of a long hypodermic syringe and was fitted to the narrow end of a Pasteur pipette. The sample material (magnetically collected cells) was placed on top of a sterile, wetted cotton plug in the wide-mouthed end of the pipette that served as a reservoir. The capillary was exposed to a magnetic field produced along it with a permanent magnet for 30 min to 3 h. The MTB migrated through the cotton plug towards the closed end of the capillary. The tip containing the accumulated MTB was then broken-off and using a sterile hypodermic needle the organisms were transferred to the sterile enrichment medium in test tubes that were incubated at 3035C for about 2 weeks. This method was repeated twice to purify the MTB. The purified magnetic cells were then isolated by the streak plate method and preserved (at 4C) on slants of the same medium. The isolates were tested for their magnetotactic response using the hanging drop technique under an optical microscope, with the south pole of a bar magnet
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Table 1. Isolate A B C D Control (Escherichia coli) Response of magnetic and non-magnetic bacteria to magnetic field, and their cellular iron content Morphology Gram-positive rod Gram-negative slender rod Gram-positive rod Gram-positive coccus Gram-negative short rods Magnetic response Moderate south-seeking Strong south-seeking Weak south-seeking Strong south-seeking No migration towards any pole Iron content (mg/g dry cell mass) 5.900 11.600 5.020 8.620 1.007

Figure 1. Magnetotactic bacteria (MTB) inside (a) and outside (b) the magnetic field.

Figure 2. Response of MTB (b) and non-magnetic bacteria (a) to the magnetic field.

being placed about 10 cm from the slide. Their magnetic response was also tested in terms of spreading of their growth on the surface of a semi-solid medium (0.8% agar). The isolates were inoculated in a straight line at the centre of the medium in petri dishes that were incubated in a magnetic field created by placing the opposite poles of two different bar magnets on either side perpendicular to the line of streaking. The growth pattern after incubation was observed for any spreading towards the magnet poles. The isolated magnetic cultures and a known nonmagnetic bacterial culture (Escherichia coli) as control
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were grown in the medium that contained ferric citrate as a source of iron. After obtaining sufficient growth (approximately 3040 mg dry weight), the cell mass was separated by centrifugation at 10,000 g in a REMI R24 research centrifuge. The cell mass thus obtained was dried to constant weight at 105C in a LabHosp hot-air oven. The iron content of the cell mass was determined by atomic absorption spectroscopy using Chemito Model 201 AAS, and the tri-acid digestion method of iron extraction10 from the cells. MTB were successfully enriched from the sediment samples of Lonar lake by the magnetic collection method and purified by the CRT method. The mixed bacterial culture obtained was observed under a microscope and was found to include more than one morphological type. It was subjected to the streak plate method of isolation and four different morphological forms of bacteria were obtained as pure cultures. They included two Grampositive, rod-shaped bacteria; one Gram-negative, slender rod and one Gram-positive coccus (Table 1). The mixture before isolation and the individual isolated cultures were tested for their magnetic properties by the hanging drop technique and the semi-solid medium method. Both the mixed culture and the isolated organisms showed a magnetotactic response as expected. In the hanging drop method, the cultures aligned parallel to each other along the magnetic field lines and showed a migration towards the magnet. In the semi-solid medium also, the MTB showed pronounced migration towards the magnetic pole (Figure 1); this is referred to as magnetotaxis. The non-magnetic, motile, control bacterial culture (E. coli) meanwhile did not show any migration towards any pole and grew as a straight line as inoculated (Figure 2). The magnetic culture kept outside the magnetic field also grew in a straight line as inoculated (Figure 1). Results of both the observations and the morphological characters of the isolates are shown in Table 1, along with data on iron content of each isolated bacterial strain. The unambiguous correlation between the magnetic response and the iron content of the cells is clearly evident. It can also be seen that the magnetic bacteria accumulated up to 11.5 times more iron within their cells (Table 1 and Figure 3) compared to the non-magnetic cells, an observation first reported by Blakemore et al.10.
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12. Sukumaran, P. V., Magnetotactic bacteria, magnetofossils and antiquity of life. Curr. Sci., 2005, 88, 879885.

ACKNOWLEDGEMENTS. We thank the Management and the Department of Microbiology, Yashwantrao Chavan College of Science, Karad for help and providing facilities during this study. We also thank M/s Nikhil Analytical Laboratory, Sangli for iron analysis.

Received 15 July 2008; revised accepted 20 February 2009

Figure 3.

Intracellular iron content of the isolates.

Karyotype analysis of Persian stone lapper, Garra persica Berg, 1913 (Actinopterygii: Cyprinidae) from Iran
Hamid Reza Esmaeili*, Mehrgan Ebrahimi, Talat H. Ansari, Azad Teimory and Ghorbanali Gholamhosseini
Department of Biology, College of Sciences, Shiraz University, Shiraz 71454, Iran

In light of the ecological importance of MTB in biogeochemical cycles, the study of such bacteria in hitherto unexplored environments can be significant. Fossil magnetosomes are considered to contribute significantly to magnetic properties of sediments and soils7,11. The magnetic nano-crystals in the 4.5-Ga-old Martian meteorite ALH84001 are found to be similar to the bacterial magnetosomes12. This seems to hint at a certain correlation between the MTB and the meteorites that could have tremendous implications on the search for extraterrestrial life.
1. Bazylinski, D. A. and Frankel, R. B., Magnetosome formation in prokaryotes, Nature Rev., 2004, 2, 217230. 2. Bazylinski, D. A., Synthesis of the bacterial magnetosome: the making of a magnetic personality. Int. Microbiol., 1999, 2, 7180. 3. Schuler, D., Formation of magnetosomes in magnetotactic bacteria, J. Mol. Microbiol. Biotechnol., 1999, 1, 7986. 4. Gao Jun et al., Isolation and biological characterization of aerobic marine magnetotactic bacterium YSC-1. Chin. J. Oceanol. Limnol., 2006, 24, 358363. 5. Rajasekhar, R. P. and Mishra, D. C., Analysis of gravity and magnetic anomalies over Lonar lake, India: An impact crater in a basalt province. Curr. Sci., 2005, 88, 18361840. 6. Thakker, C. D. and Ranade, D. R., An alkalophilic Methanosarcina isolated from Lonar crater. Curr. Sci., 2002, 82, 455458. 7. Schuler, D. and Frankel, R. B., Bacterial magnetosomes: microbiology, biomineralization and biotechnological applications. Appl. Microbiol. Biotechnol., 1999, 52, 464473. 8. Moench, T. T. and Konetzka, W. A., A novel method for the isolation and study of a magnetotactic bacterium. Arch. Microbiol., 1978, 119, 203212. 9. Flies, C. B., Peplies, J. and Schuler, D., Combined approach for characterization of uncultivated magnetotactic bacteria from various aquatic environments. Appl. Environ. Microbiol., 2005, 71, 27232731. 10. Blakemore, R. P., Maratia, D. and Wolfe, R. S., Isolation and pure culture of fresh water magnetic spirillum in chemically defined medium. J. Bacteriol., 1979, 140, 720729. 11. Pan, Y. et al., The detection of bacterial magnetite in recent sediments of Lake Chiemsee (Southern Germany). Earth Planet. Sci. Lett., 2005, 232, 109123. CURRENT SCIENCE, VOL. 96, NO. 7, 10 APRIL 2009

The karyotypic and cytological characteristics of an endemic cyprinid fish Garra persica Berg, 1913 have been investigated by examining metaphase chromosome spreads obtained from gill epithelial and kidney. The diploid chromosome number of this species was 2n = 48. The karyotype consisted of 15 pairs of metacentric, eight pairs of submetacentric and one pair of subtelocentric chromosomes (15m, 8Sm, 1St). The arm number was 94. No heteromorphic sex chromosomes were cytologically detected. Chromosome number, karyotype formula and arm number of G. persica differentiate it from G. rufa, a closely related species. Keywords: Chromosome, Garra persica, idiogram, karyotype analysis. CYPRINIFORMES or carps are a group of freshwater fishes with six families, 321 genera and about 3268 species found throughout the world, except Australia and South America1. The Cyprinidae or minnows are found in North America, Africa and Eurasia. The Cyprinidae with 220 genera and about 2420 species is the largest family of freshwater fishes and with possible exception of Gobiidae, the largest family of vertebrates1. They are mostly small (<5 cm) although some are very large (3 m). The genus Garra is one among this diverse group found throughout Southwest Asia and from Africa to Southeast Asia. There are about 73 species and four are recognized from` Iran, including Garra rufa (Heckel, 1843), G. rossica (Nikolskii, 1900), G. variabilis and G. persica (Heckel, 1843)2. G. persica (Figure 1) is recognized only as a sub*For correspondence. (e-mail: esmaeili@susc.ac.ir) 959