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Supporting information for “The myxobacterial community is a predominant and highly diverse bacterial group in soil niches”
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Xiu-wen Zhou
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, Shu-guang Li
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, Wei Li, De-ming Jiang, Kui Han, Zhi-hong Wu, Yue-zhong
Li *
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State Key Laboratory of Microbial Technology, School of Life Science, Shandong University,
Jinan 250100, China
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Equal contributions to this paper
10 * Corresponding author. Y.Z.L. Tel & Fax: +86 531 88564288; E-mail: lilab@sdu.edu.cn
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16 RNA extraction and purification
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A 0.5 g of the soil sample was crushed with 0.5 g of 0.1 mm diameter beads and then mixed with 300 μl of RNA extraction solution (2% SDS, 3% PVP, 50 mM NaAc, 10
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20 mM EDTA, pH 5.2) using a homogenizer (Bertin, Montigny-le-Bretonneux, France) at a speed of 5,000 × g for 90 s. Following centrifugation, the precipitate was crushed
21 again in 1 ml of fresh extraction solution. The supernatant was extracted twice with
22 phenol-chloroform-isopentanol, and total RNA was precipitated with 0.6 vol of cold

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24 isopentanol, washed with 70% ice-cold ethanol and dissolved in 100 μl of DEPCtreated water. A total of 3 μl of DNase I and 10 μl of 10 × reaction buffer (Fermentas,
25 Shenzhen, China) were added to remove DNA contaminants, followed by extraction
26 two times with phenol-chloroform-isopentanol and precipitation with 0.1 vol of 3 M
27 sodium acetate and 2.5 vol of ethanol. The sample was subsequently washed with
28 70% ice-cold ethanol and the prepared RNA was dissolved in 50 μl of DEPC-treated
29 water and purified on a Sephadex G-75 column.
30 Construction of a 16S crDNA clone library
31 First-strand cDNA was reverse transcribed from the purified RNA using random
32 primers (TaKaRa, Dalian, China), as specified by the manufacturer. The primer sets
33 W1/1492R and W4/1492R were used to amplify Cystobacterineae- and Sorangineae-
34 enriched 16S rRNA gene sequences from the cDNA using a touchdown PCR protocol
35 (Wu et al.
, 2005), respectively. Clone libraries were constructed using the pGEM-T
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37 easy vector and the TOPO-TA cloning kit (Promega, Madison, USA), following the manufacturer’s instructions. For each library, 96 clones were randomly selected from
38 different plates, and the presence of the target small-subunit rRNA gene insert in the
39 colonies was confirmed using the W2 and W5 probes (Wu et al.
, 2005). The positive
40 clones were sequenced at the Shanghai Sangon Sequencing Center (Sangon, Shanghai,
41 China).
42 Phylogenetic analysis
43 The sequence of delta proteobacterium Desulfovibrio desulfuricans (GenBank
44 accession number: MR34113) was chosen as outgroup to root the phylogenetic tree.
45 16S rRNA gene sequences of type strains belonging to each myxobacterial genus
46 were used as references ( Table S7 ) to analyze the phylogenetic relationships.

47 Sequence alignments were performed using MUSCLE version 3.8.31 (Edgar, 2004).
48 The distance matrices between sequences were calculated using the Jukes–Cantor
49 model (Jukes et al.
, 1969). Based on these distance matrices, a neighbor-joining tree
50 was constructed (Saitou et al.
, 1987). Bootstrap values for 500 replicates were
51 calculated, and a consensus tree was built using MEGA 5 software (Tamura et al.
,
52 2011).
53 Genbank accession codes
54 The 16S crDNA sequences have been deposited in GenBank under accession numbers
55 of FJ434994-FJ435014, FJ435016-FJ435049, FJ435051-FJ435065, and FJ435067-
56 FJ435088.
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59 There are two possibilities for myxobacterial community survival: either diverse
60 myxobacterial eco-types are metabolically active contemporaneously, or different
61 myxobacterial species grow successively, i.e., with some work while others rest. To
62 determine the most likely alternative, we extracted total environmental RNA from the
63 soil sample for the subsequent analysis. Because 16S rRNA sequences are also
64 present in low metabolism spores, we determined the appropriate detection threshold
65 for the 16S rRNA sequences in the myxospores using two myxobacterial cultures ( M.
66 xanthus DK1622 of Cystobacterineae and Sorangium cellulosum So0157-2 of
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Sorangineae ). The results indicated that the detection limit of 16S rRNA sequences from myxospores was 1 × 10 4 spores lower than that for vegetative cells (data not
69 shown). Thus, the 16S rRNA sequences in the total environmental RNA were mostly
70 from vegetative cells.

71 Myxobacteria-enriched 16S crDNA libraries were constructed using the
72 myxobacteria-semi-specific primer pairs W2/1492R and W4/1492R, targeting the
73 Cystobacterineae , Sorangineae and Nannocystineae suborders (Wu et al.
, 2005, Jiang
74 et al.
, 2007). Clones from these libraries were randomly selected for sequencing. A
75 total of 83 unique myxobacterial sequences were obtained. Among the 83 sequences,
76 43 sequences belonged to Cystobacterineae , 29 to Sorangineae 2 to Nannocystineae ,
77 and 9 were unclassified Myxococcales . The 83 crDNA sequences were dispersed
78 among different clades or branches of the phylogenetic tree ( Figure S3 ). At a
79 minimum, the results indicated that there were many different myxobacterial species,
80 both cultured and uncultured, which were contemporaneously metabolically active in
81 the soil niche.
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Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput.
Nucleic Acids Res 32 : 1792-1797
Jiang, D.M., Wu, Z.H., Zhao, J.Y. and Li, Y.Z. (2007) Fruiting and non-fruiting myxobacteria: a phylogenetic perspective of cultured and uncultured members of this group.
Mol Phylogenet Evol 44 : 545-552
Jukes, T., Cantor, C. and Munro, H. (1969) Mammalian protein metabolism.
Evolution of protein molecules 3 : 21-132
Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees.
Mol Biol Evol 4 : 406-425
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011)
MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.
Mol Biol Evol 28 : 2731-
2739
Wu, Z.H., Jiang, D.M., Li, P. and Li, Y.Z. (2005) Exploring the diversity of myxobacteria in a soil niche by myxobacteria-specific primers and probes.
Environ
Microbiol 7 : 1602-1610