A biogeographic distribution of magnetotactic bacteria influenced by salinity


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NameA biogeographic distribution of magnetotactic bacteria influenced by salinity
A typeDocumentation
A biogeographic distribution of magnetotactic bacteria influenced by salinity

Wei Lin*, Yinzhao Wang, Bi Li and Yongxin Pan*

E-mail: weilin0408@gmail.com; yxpan@mail.iggcas.ac.cn
Supplemental information
Supplementary Table 1. Site and clone library information for nine sediment samples from which MTB 16S rRNA genes were retrieved. (.xls)
Supplementary Table 2. Information on the previously reported three sites and publicly available 16S rRNA gene sequences which are included in this analysis. (.xls)
Supplementary Table 3. Phylogenetic distribution of potential non-MTB sequences associated with magnetic enrichments retrieved in this study. (.xls)
Materials and methods

Site description and sample collection

Sediment samples were collected from nine sites for comparison of the diversity and distribution of MTB communities in a different range of ecosystem types, including freshwater lakes, mangrove swamp, estuary, and intertidal zone across northern and southern China (Supplementary Table 1).

Four sites are in and around Beijing City. The climate in Beijing is characteristically temperate, with an average annual temperature of 10-12°C and annual rainfall values of 600 mm. Lake Kunming (YHY) is located in the Summer Palace Park and has a water surface area ca. 2.2 km2, with an average water depth about 1.5 m. At the sampling time, the bottom water oxygen content was 0.16 mg L-1, pH of water 8.15. Lake Beihai (BH) is situated in Beihai Park in the city center. The lake has a water surface area of about 0.39 km2 with a water depth of 1-3 m. Temperature and pH during the sampling time was 29°C and 7.66, respectively; whereas the dissolved concentration of oxygen in surface sediment is 0.12 mg L-1. The Lake Yuyuantan (YY) is in the Yuyuantan Park, which has water surface area about 0.61 km2. The temperature, pH, and oxygen concentration are determined to be 28°C, 8.08, and 0.10 mg L-1, respectively.

Lake Miyun (MY) is located in front of the Yanshan Mountain, about 80 km northeast of Beijing. Its physical characteristics were described previously . Briefly, MY has a water body exceeding 1 billion m3 and a maximum water depth of 40 m. The pH of water is determined to be 7.50 in this study. The salinities of above-mentioned 4 lakes are all below 0.5 ppt, indicating that they are freshwater lakes.

Two sediment samples (HSL2 and HSL4) were collected from mangrove swamp in Wenchang County in Hainan Province, China. Hainan Island is characterized by a tropical climate with an average annual rainfall of 1600 mm and an average temperature of 23-25°C. The pH and salinity of samples ranged from 7.92 to 8.07, and 13.8 to 20.0 ppt, respectively (Table 1). Another two estuarine sediment samples (BMW1 and BMW2) were collected from Bamen Bay, near Wenchang. Two watersheds drain into the bay: Wenchang River and Wenjiao River (http://www.marsh.csdb.cn/survey/hainan.htm). The area of water surface and tidal flat is about 11.85 km2. The water depth of each sampling station was approximately 3-4 m. Salinities of BMW1 and BMW2 were determined to be 15.4 and 18.8 ppt, respectively.

The last sampling site (WH) was located in the intertidal zone of the Yellow Sea near Weihai City, Shandong Province. The average temperaure of Weihai is 26°C in August. Annual rainfall is about 665 mm. The pH and salinity of sea water were 7.71 and 28.6 ppt, respectively.

At each sampling site, sediments from the top 5-20 cm were collected and then were aliquoted into 4-14 plastic bottles (600 ml) covered with about 100 ml of in situ water. The oxygen concentrations of surface sediment were determined using an HQ40d Oxygen Meter (HACH, Loveland, Colorado, USA). The existence of MTB in all sediment samples was checked through the “hanging-drop” method . MTB in the sediment were magnetically enriched using a double-ended open magnetic separation apparatus (MTB trap) as previously described . Briefly, 200 mL of surface sediments from each site were scratched and directly transferred to the “MTB trap”. A homogeneous magnetic field, about seven times that of the Earth’s magnetic field, was applied for cell enrichment for 6 h. The enriched north- and south-seeking MTB for each location were then pooled for further TEM observations and phylogenetic analyses. After enrichment, the pore water was separated from the sediments by centrifugation at 1000 g for 20 min and was further filtered through 0.45-μm membrane filters. The pH and salinity of pore water were measured using a Mettler Toledo Delta 320 pH meter (Mettler-Toledo, Greifensee, Switzerland) and a Salinity Meter AZ-8371 (Instrument Corp., China), respectively.
TEM observation

Twenty microlitres of MTB enrichments were deposited on Formvar-carbon-coated copper grids. After waiting for 1 h, the remaining solution was wicked away using a piece of filter paper. The samples were then rinsed twice with sterile distilled water. Specimens were imaged using a JEM-2010 microscope operating at 200 kV (JEOL Corporation, Japan).
PCR amplification of 16S rRNA genes and construction of clone libraries

16S rRNA genes were directly amplified from the magnetically enriched MTB using the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as previously described . Each 20-μl PCR mixture contained 1 μl of template, 10 μl of DreamTaq PCR Master Mix (MBI Fermentas), and 8 pmol of each primer. The PCR conditions were 95°C for 5 min, 30 cycles of 92°C for 1.5 min, 50°C for 1 min, and 72°C for 2 min, and a final 10-min extension at 72°C. To avoid potential sample biases, triplicate PCR products for each sample were pooled and purified by 0.8% (w/v) agarose gel electrophoresis. PCR controls with no template were negative.

Purified PCR products were ligated with the pMD19-T vector (TaKaRa, Japan) and cloned into the chemically DH5 competent cells (Tiangen, Beijing, China) according to the manufactures’ instructions. Randomly selected clones were sequenced using a 27F primer (Beijing Genomics Institute, China).
Sequence analysis

After removing sequences of insufficient length or low quality, in total, nearly 400 sequences were retrieved in this study. The length of sequences were about 450-500 bp, covering V1 to V3 hypervariable regions. The sequences were aligned using the NAST aligner at the Greengenes web site and were then taxonomically classified according to the best match with the Greengenes reference database . The presence of chimeras was checked using the Greengenes chimera-check tool (Bellerophon server) . NCBI BLAST was used to find the most closely related 16S rRNA gene sequences in the public database. The sequences unrelated to known MTB (<80% sequence identity) in the database were attributed to non-magnetotactic contaminations and were removed in this study (Supplementary Table 3). In this way, a total of 334 high quality 16S rRNA gene sequences, with 21-47 sequences per sample, were retrieved.

The number of OTUs was estimated using the FastGroupII algorithm with similarity threshold of 98% (Percentage Sequence Identity with Gaps algorithm) . Rarefaction curves were calculated using the FastGroupII algorithm or the freeware program aRarefactWin (available at http://www.uga.edu/~strata/software/Software.html). To estimate the coverage of clone libraries, sequences were collected into OTUs based on 98% sequence identity. We chose the 98% threshold because this is the similarity threshold between two well-studied model MTB organisms Magnetospirillum magnetotacticum MS-1 and M. magnetotacticum AMB-1 . The coverage estimators were calculated by the equation C = [1 - (n/N)]×100, where n is the number of unique clones and N is the total number of clones examined .

A representative sequence from each OTU (98% sequence identity) was aligned using CLUSTAL W software and corrected by manual inspection. The phylogenetic tree was constructed using MEGA version 4.0 through the neighbor-joining method and was linearized assuming equal evolutionary rates in all lineages. The bootstrap resamplings were repeated 1000 times.
Statistical analyses

The unweighted UniFrac approach was used to determine the overall phylogenetic distance between each pair of MTB communities from distinct locations . The UniFrac algorithm estimates the phylogenetic distance between communities and can reflect the occurrence of distinct microbial lineages based on phylogenetic information. In addition to sequences retrieved in the present study, publicly available sequence sets of MTB communities from globally distinct locations were also included in the UniFrac analysis in order to compare their phylogenetic relationships to the MTB communities retrieved here. These 16S rRNA gene sequences were extracted from the most updated Database of Magnetotactic Bacteria (http://database.biomnsl.com/, last check on October 5, 2010) . The selection was restricted to studies that matched each one of the following criteria: (i) investigation of the overall diversity of MTB communities in single locations; (ii) use of bacterial universal primers; (iii) high-quality sequences at least covering the V1 to V3 hypervariable regions. Studies performed at 3 sites were met these criteria, including sequences from Itaipu lagoon (saline) in Rio de Janeiro, Brazil , Jiaozhou Bay (saline) in Shandong Province, China , and Lake Chiemsee (freshwater) near Munich, Germany . A detailed information list of these sequences and environmental sources is given in the Supplementary Table 2. Spearman rank correlation was performed to examine the correlations between the phylogenetic distance of nine MTB communities in China (UniFrac distance matrix) and the measured environmental factors (Euclidean distances). For all statistical analyses, a value of P<0.05 was considered significant.
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