key: cord-0034170-t301bt08 authors: Xiao, Zhijie; Lian, Bin; Chen, Jun; Teng, H. Henry title: Design and application of the method for isolating magnetotactic bacteria date: 2007 journal: Chin J Geochem DOI: 10.1007/s11631-007-0252-y sha: 5e4904987c4a7f4f46238451b063574436cdfd86 doc_id: 34170 cord_uid: t301bt08 A simple apparatus was designed to effectively isolate magnetotactic bacteria from soils or sediments based on their magnetotaxis. Through a series of processes including sample incubation, MTB harvesting, isolation, purification and identification, several strains of bacteria were isolated from the samples successfully. By Transmission Electron Microscopy (TEM) and Energy-Dispersive X-ray Analysis (EDXA), these bacteria were certificated to be magnetotactic bacteria. The phylogenetic relationship between the isolated magnetic strains and some known magnetotactic bacteria was inferred by the construction of phylogenetic tree based on 16SrDNA sequences. This apparatus has been proven to have the advantages of being inexpensive, simple to assemble, easy to perform and highly efficient to isolate novel magnetotactic bacteria. The research indicated that the combined approach of harvesting MTB by home-made apparatus and the method of plate colony isolation could purify and isolate magnetotactic bacteria effectively. Magnetotactic bacteria are a general designation to a group of microorganisms which orient and migrate along geomagnetic field lines. Magnetotactic bacteria are of morphological and habitat diversity. The cells of various morphological types including cocci, short or long rods, vibrios, spirilla and multicellular aggregates (Bazylinski et al., 1994; Rodgers et al., 1990) , have been found from world-wide diverse aquatic environments (including marine, river, pond, etc.), surface of marine sediments, and soil. The magnetotaxis of magnetotactic bacteria is based on specific intracellular magnetic structures, magnetosomes, which comprise nano-sized, membrane-bound crystals of magnetic iron minerals. Magnetosomes consist mainly of magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ) (Bazylinski et al., 1988; Bazylinski and Garratt-Reed, 1993) . Additionally, FeS 2 and Fe 7 S 8 have also been reported recently (Mann et al., 1990; Farina et al., 1990) . Research on magnetotactic bacteria is of great significance, for example: as a model organism of biomagnetism to investigate the complex biomineralization in advanced organisms (Dunn, 1995) , as circumstantial evidence for ancient extraterrestrial life from a Martian meteorite (Kathie et al., 2002) . Research on magnetotactic bacteria is of significance for environmental markers (Lida and Akai, 1996) , for example, it has been widely concerned in geology, i.e., the contributions of magnetotactic bacteria to the stable remanence of sediments and to the magnetic susceptibility of Chinese loess-paleosol sequences (Kirschvink and Chang, 1984; Peng Xianzhi et al., 2002) . Additionally, research on the correlation between magnetotactic bacteria and iron in the environment is an issue involved in biogeochemistry, which is helpful to shed light on the mechanism of tolerance of some microbes and metal-tolerant plants to heavy-metal pollution (Yang Yuangen et al., 2004; Sadaf Naseem et al., 2005) . Besides the significant application of magnetotactic bacteria, magnetosomes also could widely be applied in many fields such as: material science, biology, pharmacy, electronics, optics, magnetism, and electrochemistry (Bazylinski, 2004) . Therefore, to establish an effective method to isolate magnetotactic bacteria is the crucial precondition for research on and application of them. Since the discovery of magnetotactic bacteria in 1975, magnetotactic bacteria differing in morphology have been isolated by the adscititious magnetic field based on their magnetotaxis from natural environment or www.gyig.ac.cn www.springerlink.com enriched samples. The methods of isolating magnetotactic bacteria could be roughly divided into two groups: (1) to isolate magnetotactic bacteria with motility and intracellular magnetosomes by the adscititious magnetic field, for example: magnetic collection and "race track" purification of magnetotactic bacteria (Moench and Konetzka, 1978; Wolfe et al., 1987) , or to design an apparatus to harvest magnetotactic bacteria (Ulysses et al., 2003; Wei Yangbao et al., 1994) ; and (2) to isolate non-motile and non-or weekly-magnetotactic bacteria as a result of environmental factors from enriched samples by colony formation, and optimization of conditions for growth and synthesis of magnetosomes (Sakaguchi et al., 1996) . A sulphate-reducing magnetic anaerobe, RS-1, which is weakly magnetotactic, was isolated by this method. The present paper reports an apparatus for harvesting magnetotactic bacteria and the method of isolating them, which have apparent advantages such as: being easy to perform, inexpensive, and effective to isolate a large number of novel magnetotactic bacteria. Several novel magnetotactic bacteria have been successfully isolated and purified by this apparatus and method. This apparatus is used to enrich and isolate motile-magnetotactic bacteria. It is made up of two parts ( Fig. 1 ), one part is the separating chamber, which is a flask used for enriching sample before the harvest procedure. The other is a sampling chamber which is refitted from tundish. At the top of the sampling chamber is a sampling hole, and there is a control piston at the joint of the two parts. The principle of the apparatus is: the arrangement of single-magnetic-domain magnetosomes in chains maximizes the dipole moment of the cell, when exposed to the magnetic field (approximately 4 Gauss) which is produced by placing the south pole of a circular magnet near the sampling hole, the motile-magnetotactic bacteria with intracellular magnetosomes would swim to the sampling chamber and could be isolated after a few hours. Laterite or the soil with a relatively dark color is usually selected as terrene samples. 20 g of soil were measured and put into a 300-mL flask filled with 200 mL of sterilized liquid-enriched medium (Blakemore et al., 1979) , and the flask was covered by pledget and left undisturbed in dim light at room temperature (22-28 ). ℃ In this paper, we sampled a paleosol layer with high magnetic susceptibility from a Chinese loess section at Qingyang, Gansu Province. As to aquatic samples, we usually select water and sediment samples on the surface of the boundary between water and sediment at about 1-m depth. In this paper, one liter of sample was collected at 1-m depth from a freshwater pond in Nanjing. Sediment and water in a proportion of approximately 1:2 were stored in loosely capped bottles, and left to be undisturbed for 1 month under dim light at room temperature (22-28 ). ℃ About 80 mL of sterilized enriched medium were taken to sterilize the sampling chamber of the home-made apparatus (Fig. 1 ), a sterilized filter paper was put at the joint of the sampling chamber and the separating chamber, the two chambers were joined by a rubber plug, and the piston was opened up to connect the water column in the two chambers of the apparatus. A magnet was put at the top of the apparatus with its south pole pointing downwards. Meanwhile, two control experiments were made with the north pole pointing downwards with no magnet. Under the effect of the magnetic field, magnetotactic bacteria swam to the south pole of the magnet and penetrated the filter paper to enter the sampling chamber. The piston was closed after 3 hours, and then the harvested sample containing magnetotactic bacteria was extracted by means of a sterilized injector from the sampling hole. The harvested sample was transferred to a 150-mL sterilized flask and piston; 5. rubber plug; 6. isolating chamber. cultured at 100 r/min at 30℃ to enrich magnetotactic bacteria secondarily. This paper aimed at isolating oxygen-tolerant magnetotactic bacteria. The enriched solid medium was used and the magnetotactic bacteria were isolated and purified in Petri dishes by the method of agar plate colony isolation from the collected liquor at 28℃ (Blakemore et al., 1979) . electron microscope and by energy-dispersive X-ray analysis (EDXA) One drop of the harvested sample was deposited on a grid. The drop was placed partially covering the grid surface. A magnet was positioned on the opposite side of the drop with an alignment of the magnetic field in such a way that the bacteria would swim to the side close to the middle of the grid (Ulysses et al., 2003) . In about 6 minutes, the bacteria were accumulated on the drop edge. One drop of distilled water was deposited by the side of the drop that did not cover the grid, and then the grid was air-dried. Usually through these procedures, the bacteria could spread evenly in some grid fields. The specimens were viewed under a HITACHI H600A-2 transmission electron microscope (TEM) operating at voltages of 75 kV. Purified cells of the isolated magnetotactic bacteria were prepared by centrifugation their liquid culture or scraping their colony formed on agar plate, and then the pure cells were diluted by sterilized phosphate buffer solution (pH=6.7，10 mM). The specimen was dropped directly to a grid without immobility or negative stain, the grid was air-dried, and then viewed under TEM operating at voltages of 75 kV. Microscope (SEM) and Energy-Dispersive X-ray Analysis (EDXA) of isolated bacteria Samples prepared by the method described in 2.2.4.2 were placed onto the surface of double-faced tape attached to a copper stub and then the specimens were air-dried. A coating of gold was vacuum-deposited on the specimens, which were then viewed under a JSM-5610LV SEM operating at voltages of 15 kV. Qualitative elemental composition of the magnetosome region and magnetosome-free region in isolated magnetotactic bacterial cells was determined by using VANTAGE energy-dispersive X-ray analysis system coupled to the SEM. The two presently isolated magnetotactic bacteria MG-1 and MG-2 were cultured in a liquid growth medium to exponential phase (Blakemore et al., 1979) . Bacterial genomic DNA was extracted and purified from each strain by Genomic DNA isolation Kit K713 (Shenergy Biocolor). 16SrDNA of MG-1 and MG-2 were amplified in a reaction containing 1.5 U of Thermus aquaticum DNA polymerase (Promega, Madison, Wis.), 10 nmol of each deoxynucleoside triphosphate, 5 µL of 10×PCR buffer (Promega), 75 nmol of MgCl 2 , and 50 pmol of each universal primer (Ksiazek et al., 2003; Peiris et al., 2003) . For the PCR template, 4 µL of extracted DNA was added. The reaction volume was made up to 50 µL with sterile distilled water. A thermal cycler (PTC-100) was used, with 5 min of initial denaturation at 94 , 35 cycles of ℃ 1 min at 94 , 1 min at 47 , and 1.5 min at 72 , ℃ ℃ ℃ and a 10 min extension to the last cycle at 72 . PCR ℃ products were electrophoresed at 10 Vcm -1 in 1% agarose gel in Tris-borate-EDTA buffer, stained in ethidium bromide solution, and photographed under UV illumination. PCR products were sent for sequencing if they are single bands confirmed by agarose gel electrophoresis. Six published 16SrDNA sequences of magnetotactic bacteria were selected from GenBank: Magnetospirillum magneticum MGT-1, M.magneticum AMB-1, M.magnetotacticum MS-1, Desulfovibrio magneticus RS-1, Magnetobacterium bavaricum and iron sulfide containing magnetotactic bacterium MMP1991. In addition, eight other bacterial 16SrDNA sequences were selected from GenBank in order to infer the phylogenetic position of the two new magnetotactic bacteria in relation to other Proteobacteria, such as: Rhodospirillum molischianum, Agrobacterium tumefaciens, Rhodobacter sphaeroides, Pseudomonas testosterone, Uncultured gamma proteobacterium SP_A2，Desulfovibrio desulfuricans, Leptospirillum ferrooxidans CF12 and Anacystis nidulans. The 16SrDNA sequences obtained were aligned with other 16SrDNA sequences on the basis of secondary structure and conserved sequence. Multiple alignments of the sixteen 16SrDNA sequences were carried out on a computer using the ClustalX (1.8) algorithm and fine-tuned manually. Phylogenetic trees were constructed from evolutionary distance data by the neighbor joining method. After one month of enrichment, brown coating and deposits appeared on the surface of sample and sediment. This is inferred to as the influence from a mass of enriched magnetotactic bacteria and dissociative magnetosomes after autolysis of cells, consistent with previous report (Wei Yangbao et al., 1994) . The bacteria in harvested samples are mostly bacillus and cocci under TEM. Most bacteria contain 3-8 of intracellular electron dense particles (Fig. 2) . The arrangement of these particles is in a single chain along the long-axis of the most cells (Fig. 2a, b, c) , as well as in a cluster in minor cells (Fig. 2d) . The dense particles are about 80 nm in diameter when measured along their long axes, which is consistent with the earlier report . In harvesting procedure, we set two control experiments: with a magnet and without a magnet with N pole downwards the apparatus. The result of control experiment showed that there are no bacteria or electron dense particles as shown above. This suggests that some bacteria with magnetotaxis swam to the S pole of the adscititious magnetic field and reached the sampling chamber. Through isolating single colony on agar plate aerobically from the harvested samples, three novel oxygen tolerant magnetotactic bacteria were purified successfully, named MG-1, MG-2, and MG-3, respectively (Fig. 3) . After cultivation for 4 days, TEM results showed that all cells of the three strains contain 3-8 of electron dense particles with a section of being circular-like and a diameter of about 80 nm (Fig. 3) . MG-2; c. MG-3. Strain MG-1 was selected for further research. MG-1 is rod-shaped, with a size of (0.4-0.7) μm×(1.1-1.5) μm as observed under SEM (Fig. 4) . Figure 5 shows the elemental composition of the magnetosome region and magnetosome-free region in a cell of MG-1 by EDXA. In magnetosome region of the cell (Fig. 5a) , except for C, O, P, Ca, the main metallic element is Fe, and its content accounts for 10.23% of the total elemental composition of the cell, in addition, the content of Co is 1.54% and Ni is 0.53%. However, the content of Fe is merely 0.43% while Co is 0.21% and no Ni is present in Fig. 4 . SEM image of MG-1. magnetosome-free region of the cell (Fig. 5b) . This indicates that the main elemental composition of the magnetosomes of MG-1 is Fe, while a small quantity of Co and Ni exists. All these three elements are significant constituents of magnetic materials. Amplified and sequenced 16S rDNA of the two new strains of magnetotactic bacteria MG-1 and MG-2, as well as the 1417bp and 1414bp sequences were obtained, respectively. The two sequences were aligned with six published sequences of magnetotactic bacteria and eight other bacterial 16S rDNA sequences which represent members of each subclass of the Proteobacteria. Phylogenetic analysis was carried out after 16 bacterial sequences were aligned, and the phylogenetic tree is constructed by the neighbor joining method (Fig. 6) . According to literature (Spring and Scheifer, 1995) , we designate magnetotactic bacteria into α-subclass, β-subclass, γ-subclass, and δ-subclass of the Proteobacteria, Cyanobacteria phylum and Nitrospira phylum. Since the majority of the present magnetotactic bacteria are proven to be affiliated with α-subclass of Proteobacteria (Bazylinski and Frankel, 2000; DeLong et al., 1993) , we divide α-subclass into α-1 subgroup, α-2 subgroup and α-3 subgroup according to literature (Burgess et al., 1993) , so as to further position magnetotactic bacteria within the α-subclass. As is shown in Fig. 6 , among the four strains of magnetotactic bacteria with magnetosomes of Fe 3 O 4 , three (AMB-1, MGT-1, MS-1) are affiliated with the α-1 subgroup of the α-subclass of Proteobacteria while a magnetic sulfate-reducing bacterium RS-1 belonged to the δ-subclass. A magnetotactic, many-celled procaryote MMP1991 with magnetosomes of Fe 3 S 4 and a magnetic sulfate-reducing bacterium RS-1 with magnetosomes of Fe 3 O 4 both belong to the δ-subclass of Proteobacteria. A new magnetotactic bacterium MG-1 is closely related to an uncultured gamma proteobacterium SP_A2, and both of them are affiliated with the γ-subclass of Proteobacteria. The other new magnetotactic bacterium MG-2 and a morphologically distinct large magnetic rod Magnetobacterium bavaricum were assigned to the Cyanobacteria phylum and Nitrospira phylum, respectively. Previously DeLong considered that magnetotactic bacteria with magnetosomes of Fe 3 O 4 are related to the α-subclass of Proteobacteria (DeLong et al., 1993) , while magnetotactic bacteria with magnetosomes of Fe 3 S 4 are related to sulfate-reducing bacteria of the δ-subclass of Proteobacteria, and the ability to form magnetosomes has been evolved from separate evolutionary origins. However, from the phylogenetic tree in Fig. 6 , we could see that not all the magnetotactic bacteria with magnetosomes of Fe 3 O 4 belong to the α-subclass of Proteobacteria. For example, a magnetic sulfate-reducing bacterium RS-1 with magnetosomes of Fe 3 O 4 is affiliated with the δ-subclass of Proteobacteria, and Magnetobacterium bavaricum with magnetosomes of Fe 3 O 4 is assigned to the Nitrospira phylum. This means that the phylogenetic origin of magnetotactic bacteria is of magnetotaxis which as a characteristic feature may has been multi-evolved. There is no genus particularity of magnetotaxis to bacteria. It is proven through the novel magnetotactic bacteria with phylogenetic diversity that the home-made apparatus could harvest and isolate novel magnetotactic bacteria efficiently. Several strains of magnetotactic bacteria were harvested successfully by the home-made apparatus. Through aerobical plate colony isolation, we obtained three strains of pure oxygen tolerant magnetotactic bacteria MG-1, MG-2, and MG-3. This apparatus has many unique advantages compared with the similar domestic and foreign apparatuses reported so far, for example: (1) low-cost, simple to assemble, and easy to implement; (2) the flask for enrichment directly links with the separating chamber of the apparatus, which significantly simplifies the working procedures; (3) the sampling chamber is filled with enrichment medium to balance the concentrations of solution in two chambers of the apparatus, so as to reduce the influence on the swimming of bacteria caused by molecule diffusion; (4) the enrichment medium in the sampling chamber could enrich a few harvested bacteria secondarily; (5) high efficiency as evidenced by the fact that we isolated three strains successfully after merely 3 hours of harvesting in this paper; (6) the isolated magnetotactic bacteria are of novelty on morphology and phylogenesis through comparing their phylogenetic relationship with other known magnetic strains. However, our apparatus has the deficiency that depends on the magnetotaxisto to retrieve a large number of magnetic bacteria. There are non-motile and non-or weekly-magnetotactic bacteria as a result of environmental factors in natural environments (Sakaguchi et al., 1996; Schuler and Frankel, 1999) . So it relies on the method that does not use magnetotaxis to retrieve magnetic bacteria (Sakaguchi et al., 1996) , which consists of several steps that include incubation of sediments, enrichment of bacteria in the medium and isolation by colony formation. Although our approach may not retrieve all possible forms of magnetotactic microorganisms, we believe that both methods are complementary and can be used in different situations or for different purposes, and it is proven to be easy to use and retrieve a large number of novel magnetotactic bacteria efficiently by our apparatus and method. 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