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Correspondence: Yongxin Pan, Biogeomagnetism Group, Paleomagnetism and Geochronology Laboratory, State Key Laboratory of Lithospheric Evolution, Institute of Geology and Geophysics, Chinese Academy of Sciences, 19 Bei Tu Cheng Xi Road, Beijing 100029, China. Tel.: +86 0 10 8299 8406; fax: +86 0 10 6237 2053; e-mail: firstname.lastname@example.org
The racetrack-based PCR approach is widely used in phylogenetic analysis of magnetotactic bacteria (MTB), which are isolated from environmental samples using the capillary racetrack method. To evaluate whether the capillary racetrack-based enrichment can truly reflect the diversity of MTB in the targeted environmental sample, phylogenetic diversity studies of MTB enriched from the Miyun lake near Beijing were carried out, using both the capillary racetrack-based PCR and a modified metagenome-based PCR approach. Magnetotactic cocci were identified in the studied sample using both approaches. Comparative studies showed that three clusters of magnetotactic cocci were revealed by the modified metagenome-based PCR approach, while only one of them (e.g. MYG-22 sequence) was detected by the racetrack-based PCR approach from the studied sample. This suggests that the result of capillary racetrack-based enrichment might have been biased by the magnetotaxis of magnetotactic bacteria. It appears that the metagenome-based PCR approach better reflects the original diversity of MTB in the environmental sample.
Magnetotactic bacteria (MTB) are a group of mostly aquatic prokaryotes possessing the trait of magnetotaxis, i.e., they can orientate and swim along geomagnetic field lines. This feature is related to intracellular membrane-bound magnetic crystals (magnetite or greigite), named magnetosomes, arranged in one or more chains (Balkwill et al., 1980; Bazylinski et al., 1993). The function of magnetotaxis is assumed to allow MTB to find a favorable position in vertical chemical gradients in stratified environments (Frankel et al., 1997). Despite their ubiquity in aquatic environments (Fassbinder et al., 1990; Bazylinski et al., 1995; Bazylinski & Frankel, 2004), only a few species of MTB are currently available as laboratory pure cultures because most of them are extremely difficult to cultivate. However, MTB can be enriched, relative to other uncultivated bacteria, by taking advantage of their magnetotactic behavior along magnetic field lines, e.g. by the capillary racetrack method (Wolfe et al., 1987). Morphological and phylogenetic diversities of uncultivated MTB have been uncovered using this approach, as shown in several studies (Meldrum et al., 1993a, b; Schüler et al., 1999; Flies et al., 2005). However, little is known about the effectiveness of this magnetic approach.
The recent development of the ‘metagenome’ of the environmental sample (Handelsman et al., 1998), which enables the exploration of the phylogenetic and metabolic diversity of microorganisms without cultivation, provides a promising opportunity to investigate MTB from the environmental samples. Thornhill et al. (1995) designed a set of PCR primers that are specific to the 16S rRNA genes of magnetotactic cocci and used them to detect cocci in metagenome extracted from aquatic sediment by direct lysis. Recently, another study has examined the diversity of MTB in different microcosms by denaturing gradient gel electrophoresis (DGGE) analysis with metagenomic DNA from a sediment sample (Flies et al., 2005). The data on the phylogenetic diversity of MTB revealed by the metagenome-based approach appear more valuable when considering the occurrence of mutants that might lose their magnetotactic ability in the environment.
In this study, the 16S rRNA gene sequences of magnetotactic cocci were compared, which were acquired by the capillary racetrack-based PCR and metagenome-based PCR approaches. In order to evaluate whether the capillary racetrack-based enrichment can truly reflect the diversity of MTB in the studied environmental sample, the studied sample was collected from aquatic sediments of the Miyun lake near Beijing.
Materials and methods
Natural environmental samples
A sample of surface sediment with water was obtained from the Miyun water reservoir in northeast Beijing, China, in 2005, at a water depth of c. 1–2 m. The sample was stored in a plastic bottle and incubated under ambient temperature and low-light conditions in the laboratory. Distilled water was periodically added during the storage. Bacteriodrome (Pan et al., 2005) examinations showed that the fresh sediment sample hosted plenty of live MTB, mainly cocci.
Extraction and purification of metagenomic DNA from sediments
The metagenomic DNA was extracted from the fresh sediment sample. The procedure was based on the method of Zhou et al. (1996) with the following two modifications. First, sediments of 5 g (wet weight) were frozen in liquid nitrogen and ground with a mortar and pestle until thawed. This process was repeated three times until the color of the sample turned white. Second, the resulting crude nucleic acids pellet was resuspended in 100 μL double-distilled water instead of 500 μL. The quality of nucleic acid was evaluated on a 1% (w/v) agarose gel. The extracted DNA was purified using a Sepharose 4B spin column to minimize humic contamination based on the method of Jackson et al. (1997).
Capillary racetrack-based approach for isolation of MTB
Enrichment of MTB from the same sediment sample was obtained using the capillary racetrack method (Wolfe et al., 1987) modified by Schüler et al. (1999), using capillaries of 9 cm length. The wide-mouth end of the capillaries was not plugged. The tip of the capillaries containing MTB was broken off after 30 min collection with the south pole of a permanent magnet next to it. Collections of MTB from a total of 42 capillaries were pooled to contain sufficient cells for the following PCR amplification.
PCR amplification, cloning and sequencing
For metagenomic DNA, c. 270 bp fragments of 16S rRNA gene were amplified from the total DNA using the oligonucleotide primers MCF and MCR (Thornhill et al., 1995). The PCR reaction buffer was set in 50 μL volume with the concentration of reagents as follows: 5 μL 10 × Taq Reaction Buffer (Mg2+ free, TIANGEN, China), 3 μL MgCl2 (TIANGEN), 2 μL of each primer, 1 μL dNTPs (TIANGEN), 0.5 μL Taq DNA polymerase (50 U μL−1, TIANGEN), 1 μL template DNA and 35.5 μL sterile, double-distilled H2O. The amplification was carried out for 32 cycles using a Biometra thermal controller (TGRADIENT). The PCR reaction conditions were 3 min of initial denaturation at 95 °C and 30 cycles of 1 min at 95 °C, 2 min at 55 °C, and 2 min at 72 °C. Final extension was carried out for 5 min at 72 °C.
For the MTB enriched by the capillary racetrack method, nearly complete 16S rRNA gene was directly amplified in vitro (Saris et al., 1990) with the universal bacterial primers GM3F and GM4R described previously (Muyzer et al., 1995). The PCR reaction buffer was set in 50 μL volume with the following concentration of reagents: 5 μL 10 × Taq Reaction Buffer (Mg2+ free, TIANGEN), 3 μL MgCl2 (TIANGEN), 2 μL of each primer, 1 μL dNTPs (TIANGEN), 0.5 μL Taq DNA polymerase (50 U μL−1, TIANGEN) and 36.5 μL MTB cell collection enriched by the capillary racetrack method. The amplification was carried out for 35 cycles using the Biometra thermal controller (TGRADIENT). The PCR reaction conditions were as follows: 5 min of initial denaturation at 95 °C and 33 cycles of 1 min at 95 °C, 1 min at 42 °C and 2 min at 72 °C. Final extension was carried out for 10 min at 72 °C.
The PCR products were run on 1% (w/v) agarose gel with 0.5 × TBE buffer and stained with ethidium bromide. The resulting DNA bands were located under UV illumination, excised from the gel, and purified with the BioSpin Gel Extraction Kit (BioFlux, China). The purified PCR products were then ligated into a pMD18-T TA cloning vector (TaKaRa, Japan) and transformed into Escherichia coli DH5α-competent cells. Esherichia coli were cultured overnight and the plasmids were extracted from the cloned cells and purified with the Mini-Plasmid Rapid Isolation Kit (BioDev, China). DNA sequencing was performed with an ABI3730 automatic sequencer using the dideoxy chain-termination method (Sanger et al., 1977).
The partial and nearly complete 16S rRNA gene sequences retrieved in this study were compared with sequences stored in GenBank database using the blast algorithm. The tool clustal w version 1.8 (Thompson et al., 1994) was used for automatic sequence alignment. The alignment was further visually inspected and corrected manually. Phylogenetic analyses were conducted using mega version 3.1 (Kumar et al., 2004) based on the neighbor-joining analysis of selected sequences affiliated with known MTB and the sequences retrieved in this study. Bootstrap analysis of 100 replicates was performed to validate the reproducibility of the branching pattern of the tree. The sequences retrieved in this study were deposited in GenBank database under accession numbers EF370484–EF370499.
16S rRNA gene of magnetotactic cocci from metagenome-based PCR approaches
267-bp-long DNA fragments were amplified from metagenomic DNA in this study using the primers MCF, corresponding to nucleotides 385–404 of 16S rRNA gene in strain CS310 and MCR, corresponding to the complement of nucleotides 635–651 of the CS310 sequence, which were completely conserved with the freshwater magnetotactic cocci of strains CS103, CS308 and CS310 (Thornhill et al., 1995). A partial 16S rRNA gene clone library was then constructed. Twelve sequences were acquired and submitted to the GenBank database. All of them were associated with the freshwater magnetotactic cocci. The similarities among them were relatively low, ranging from 88.8% to 99.6% (Table 1).
Table 1. Similarity values of 16S rRNA gene sequences of magnetotactic cocci acquired from this study
According to the alignment results, only 267 bp were chosen from 1462 nucleotides for this similarity analysis.
16S rRNA gene of magnetotactic cocci from racetrack-based PCR approaches
Almost complete 16S rRNA genes were directly amplified by PCR using universal bacterial primers GM3F and GM4R from the racetrack-enriched MTB cells without a prior DNA extraction. Twelve clones were randomly selected and sequenced. In contrast to the results from the metagenome-based approach, only four sequences were found to be similar to magnetotactic cocci; all of these four sequences have very high similarity (>99.8%) and are thus believed to belong to the same species. For detailed analysis, MYG-22 was selected as the representative. According to the comparison of MYG-22 and the sequences obtained using the metagenome-based PCR approach, it was MY-3 obtained using the metagenome-based PCR approach was identical with corresponding fragments of MYG-22 (Table 1).
Phylogenetic analysis and comparison
According to the phylogenetic tree (Fig. 1), all sequences retrieved in this study were closely related to freshwater magnetotactic cocci affiliated with the Alphaproteobacteria, which could be further grouped into three clusters (A–C). Clusters A and B were comprised exclusively of sequences obtained using the metagenome-based PCR approach. In contrast, only one sequence (MYG-22) in cluster C was obtained by both approaches. Clusters A and C comprised very closely related sequences with similarities from 93.6% to 97% and 97.4% to 99.6%, respectively. In contrast, the degree of relatedness in cluster B was low, ranging from 88.4% to 94% sequence identity.
Both the racetrack-based PCR approach and the metagenome-based PCR approach revealed magnetotactic cocci in the studied Miyuan lake sample. Bacteriodrome and light microscopy confirmed the dominance of magnetotactic cocci. The capillary racetrack enrichment is mainly based on the magnetically directed swimming behaviour of the magnetotactic bacteria. An obvious advantage of the capillary racetrack-based method is that almost complete 16S rRNA gene can be directly amplified from the enriched pure cells by PCR using universal bacterial primers (e.g. GM3F and GM4R). For comparison, the metagenome-based PCR approach (Thornhill et al., 1995; Zhou et al., 1996; Handelsman et al., 1998) was used to study the phylogenetic diversity of cocci in the same sample.
Sequences belonging to magnetotactic cocci acquired from the metagenome-based approach of this study are distributed in three clusters (Fig. 1). As limited by the shortness of amplified DNA fragments, some loss of resolution is to be expected and deeper phylogenetic relationships are not well resolved (e.g. note the low bootstrap value between MY-15 and CS103). Identical sequences (e.g. MY-3 and MYG-22) are revealed from the same sediment sample using both the metagenome-based PCR and racetrack-based PCR approaches, demonstrating the effectiveness of the two different methods. Although the same resulting sequences were found by both methods, the 16S rRNA gene retrieved from the racetrack-based PCR approach had a lower diversity than that obtained using the metagenome-based PCR approach (Table 1).
It is not surprising that the racetrack-based PCR approach revealed fewer cocci species than that of the metagenome-based PCR approach, because the racetrack enrichment sample may have been biased due to the following reasons. The species with lower swimming ability may have been missed in the racetrack enrichment process. The intensity of the magnetic field, the swimming distance and medium, and chemotaxis may have also negatively affected the efficiency of the racetrack enrichment. In other words, the racetrack enrichment heavily relies on the magnetotaxis of MTB and thus the magnetically extracted species may be dominated by ‘good swimmers’. In contrast, the metagenome-based approach does not need the magnetic extraction process. Moreover, it should be noted that possible artifacts could arise from the different quality of templates and the different length of amplified 16S rRNA gene for the two approaches. Almost full-length 16S rRNA gene was directly amplified from cells, which could decrease the yield of the cloned genes in the racetrack-based PCR approach. Thus, it would be worthwhile to compare the two approaches using exactly the same experimental parameters in future investigations.
Based on transmission electron microscopy observations on magnetic extraction samples, one kind of magnetotactic coccus was noted to be dominant in the studied sample, which had a diameter of 1–2 μm and contained one straight magnetosome chain. Therefore, it is tempting to speculate that these sequences (e.g. MY-3 and MYG-22) may come from the observed most abundant magnetotactic coccus. It is also unclear whether magnetotactic cocci in clusters A and B (Fig. 1), which occurred with the metagenome-based PCR approach but not with the racetrack-based PCR approach, are nonmagnetosome mutants or have, for some reason, lost their ability to swim. Additional examination by FISH could help resolve these questions. Studies such as these will further advance one's understanding of the true diversity of magnetotactic cocci in the Miyun lake sediments. In brief, these findings suggest that the capillary racetrack-based PCR approach may reflect a biased composition of MTB mainly due to the limitation of the enrichment process. To fully understand the diversity of MTB in environmental samples (e.g. sediments), it is thus important to combine metagenome-based and racetrack-based PCR approaches with FISH and transmission electron microscope analyses.
The authors thank the microbiology group at Shandong University for their kind help in the 16S rRNA gene analyses, C. Jogler and C. Lang at Ludwig-Maximilians-University for constructive suggestions and A.J. Biggin at the University of Utrecht for improving the English on an earlier version of manuscript. The authors also thank two anonymous reviewers for their constructive comments. This work was supported by NSFC grants (40325011 and 40221402) and CAS project (KZCX-3-SW-150).