Download IJS-D-15-00398_Supplementary Material

Terriglobus albidus sp. nov., a novel acidobacterial species of the family
Acidobacteriaceae isolated from Namibian semiarid savannah soil
Javier Pascual1, #, Pia K. Wüst1, #, Alicia Geppert1, Bärbel U. Foesel1, Katharina J. Huber1, and Jörg
Overmann1, 2*
1
Department of Microbial Ecology and Diversity Research, Leibniz-Institute DSMZ-Deutsche Sammlung
von Mikroorganismen und Zellkulturen, Braunschweig, Germany
2
Technische Universität Braunschweig, Braunschweig, Germany
#
these authors contributed equally to the present publication
Running title: Terriglobus albidus sp. nov.
Subject category: New taxa, subsection Other Bacteria
Keywords: Acidobacteria; Acidobacteriaceae; Savannah soil; oligotrophic
* Correspondence: J. Overmann, Leibniz-Institut DSMZ-Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7B, 38124 Braunschweig, Germany.
Tel: +49-531-2616-352. Fax: +49-531-2616-418. Email: [email protected]
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
Ac_26_B10T is KP334258.
1 Supplementary Information
Supplementary Material and Methods
1. Isolation medium SSE/Cmix:
MES
10 mM (final concentration)
SSE (double concentrated)a
500 ml
Distilled water
500 ml
Add to 1000 ml of medium after autoclaving:
Buffer
Trace Element Solution SL-10b
Vitamin Solutionc
Mix of carbohydratesd
Mix of organic acidse
Mix of amino acidsf
Aromatic compoundsg
Inducersh
Adjust pH at 6.0
a
SSE (Soil Solution Equivalent)- double concentrated:
CaCl2 x 2H2O
NH4Cl
MgCl2 x 6H2O
(NH4)2SO4
MgSO4 x 7H2O
CaSO4 x 2H2O
Ca(NO3)2 x 4H2O
NaNO3
KH2PO4 (100 mM solution)
FeSO4 x 7H2O
K2SO4
add distilled water 1000 ml
b
0.2938 g
0.1069 g
0.2036 g
0.1983 g
0.7390 g
0.8606 g
0.2360 g
0.4240 g
0.5000 ml
0.0111 g
0.0870 g
Trace element solution SL-10:
HCl (25%; 7.7 M)
FeCl2 x 4H2O
ZnCl2
MnCl2 x 4H2O
H3BO3
CoCl2 x 6H2O
CuCl2 x 2H2O
NiCl2 x 6H2O
Na2MoO4 x 2H2O
Distilled water
1.0 ml
1.0 ml
20 µM each compound (final concentration)
20 µM each compound (final concentration)
20 µM each compound (final concentration)
20 µM each compound (final concentration)
2 µM each compound (final concentration)
10 ml
1.50 g
70.0 mg
100.0 mg
6.0 mg
190.0 mg
2.0 mg
24.0 mg
36.0 mg
990 ml
2 First dissolve FeCl2 in HCl, then dilute with water, add and dissolve the other salts.
Finally make up to 1000 ml.
c
Vitamin solution:
Biotin
Folic acid
Pyridoxine-HCl
Thiamine-HCl x 2H2O
Riboflavin
Nicotinic acid
D-Ca-pantothenate
Vitamin B12
p-Aminobenzoic acid
Lipoic acid
Distilled water
2.0 mg
2.0 mg
10.0 mg
5.0 mg
5.0 mg
5.0 mg
5.0 mg
0.10 mg
5.0 mg
5.0 mg
1000 ml
d
Mix of carbohydrates: arabinose, fucose, β-gentiobiose, glucose, rhamnose, trehalose,
xylose, N-acetyl-D-galactosamine, glucosamine and mannit.
e
Mix of organic acids: acetate, butyrate, citrate, formate, lactate, succinate, malate,
oxaloacetate, 2-oxoglutarate, propionate, pyruvate and valerate.
f
Mix of amino acids: alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate,
glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine and valine.
g
Aromatic compounds: sodium benzoate and sodium salicylate.
h
Inducers: cyclic adenosine monophosphate (AMP), N-oxohexanoyl-DL-homoserine lactone
and N-butyryl-DL-homoserine lactone.
3 1 2. PCR amplification of the 16S ribosomal RNA gene, sequencing and phylogenetic analysis
2 The almost full-length 16S gene of strain Ac_26_B10T was directly amplified by colony-PCR using
3 primer pair 8f (5´-AGAGTTTGATCCTGGCTCAG-3´ (Turner et al., 1999) and 1492r (5´
4 GGTTACCTTGTTACGACTT-3´ (Lane, 1991). The PCR mixtures included 2.0 µl PCR buffer (10
5 x), 0.8 µl MgCl2 (25 mM), BSA 0.4 µl (20 mg/ml), 0.4 µl dNTPs (10 mM each), 0.08 µl each forward
6 and reverse primers (50 pmol/µl), 0.08 µl Dream Taq DNA polymerase (5 U/µl Thermo Scientific)
7 and 1.0 µl DNA in a total volume of 20 µl. The thermal cycling program consisted of 10 min at 94 ºC,
8 32 cycles of 30 s at 94 ºC, 30 s at 56 ºC and 1 min at 72 ºC and a final elongation step of 7 min at 72
9 ºC. PCR products were purified and sequenced using the above primer pairs and the internal primers
10 1055f (5´-ATGGCTGTCGTCAGCT-3´) (Lane, 1991) and 341r (5´-CTGCTGCCTCCCGTAGG-3´)
11 (Muyzer et al., 1993) by Sanger sequencing employing the AB 3730 DNA analyzer (Applied
12 Biosystems, Foster City, CA) and the AmpliTaq FS Big Dye terminator cycle sequencing kit.
13 Multiple sequence alignment was obtained using the Infernal aligner (INFERence of RNA
14 Alignment) version 1.1rc4 (Nawrocki & Eddy, 2013) as implemented in the Ribosomal Database
15 Project (RDP) release 11 (Cole et al., 2014). Nucleotide sequence alignments were inspected visually
16 to identify positions of uncertain alignment to be corrected or omitted for further analysis.
17 Phylogenetic analysis was performed using the program PAUP* version 4.0b10 (Swofford, 2002).
18 Neighbour-joining (NJ; with Kimura’s two-parameter evolutionary model), maximum-parsimony
19 (MP; heuristic search option) and maximum-likelihood (ML) analyses were done. Since the lengths of
20 the sequences used were uneven, analyses were performed using a pairwise deletion method for gaps
21 and missing sites, using all available comparative data from each sequence pair. For ML, the optimal
22 evolutionary model of nucleotide substitution was estimated through the program jmodeltest2
23 (Darriba et al., 2012) using the Akaike Information Criterion. Bootstrap analyses were performed
24 using 1000 replications.
25 4 25 26 References
27 28 Amann, R. I., Ludwig, W. & Schleifer, K. H. (1995). Phylogenetic identification and in situ
detection of individual microbial cells without cultivation. Microbiol Rev 59, 143-169.
29 30 31 Cole, J. R., Wang, Q., Fish, J. A., Chai, B., McGarrell, D. M., Sun, Y., Brown, C. T., PorrasAlfaro, A., Kuske, C. R. & other authors (2014). Ribosomal Database Project: data and tools for
high throughput rRNA analysis. Nucleic Acids Res 42, D633-642.
32 33 Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. (2012). jModelTest 2: more models, new
heuristics and parallel computing. Nat Meth 9, 772-772.
34 35 Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics,
pp. 115-175. Edited by E. Stackebrandt, Goodfellow, M. . New York: John Wiley and Sons.
36 37 38 Muyzer, G., de Waal, E. C. & Uitterlinden, A. G. (1993). Profiling of complex microbial
populations by denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes coding for 16S rRNA. Appl Environ Microbiol 59, 695-700.
39 40 Nawrocki, E. P. & Eddy, S. R. (2013). Infernal 1.1: 100-fold faster RNA homology searches.
Bioinformatics 29, 2933-2935.
41 42 Swofford, D. L. (2002). PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods).
Version 4.0b10 (Sinauer Associates: Sunderland, MA).
43 44 45 Turner, S., Pryer, K. M., Miao, V. P. & Palmer, J. D. (1999). Investigating deep phylogenetic
relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J Eukaryot
Microbiol 46, 327-338.
46 5 47 Supplementary Table S1. Cellular fatty acid content of strain Ac_26_B10T compared to other
48 validly described Terriglobus species. Strain: 1, Ac_26_B10T; 2, T. aquaticus 03SUJ4T; 3, T. roseus
49 KBS 63T; 4, T. saanensis SP1PR4T; 5, T. tenax DRP 35T. Data for strain 03SUJ4T were taken from
50 Baik et al. (2013), for strains KBS 63T, SP1PR4T and DRP 35T from Whang et al. (2014). Values are
51 percentages of total fatty acids. –, not detectable; tr, trace amount (< 1%).
52 Fatty acid
Saturated
C14:0
C16:0
C20:0
Unsaturated
C14:1 ω5c
C15:1 ω6c
C20:1 ω9c
Methyl-branched
iso-C13:0
iso-C15:0
iso-C17:0
Hydroxy-branched
C10:0 3-OH
Mixed groups
iso-C15:0 3-OH
Summed feature*
3†
8
1
2
3
4
5
tr
3.9
1.0
6.8
9.1
1.1
3.6
10.9
tr
2.3
8.6
-
1.3
3.1
-
Tr
-
2.7
tr
9.0
tr
tr
-
1.9
2.3
-
1.1
tr
-
59.1
tr
2.8
44.5
-
3.7
47.0
2.0
9.9
40.0
1.9
51.2
tr
-
-
-
-
2.1
-
-
1.5
-
-
32.3
1.5
22.5
-
25.7
-
28.5
-
36.7
-
*
53 54 55 56 57 58 59 60 61 62 63 64 65 Summed features represent a set of more than one fatty acid that
could not be resolved by GLC with the MIDI system. Summed
feature 3 contained C16:1 ω7c and/or C16:1 ω6c; summed feature 8
contained C18:1 ω7c and/or C18:1 ω6c.
†
Summed feature 3 was defined as (C16:1 ω7c and/or C16:1 ω6c) in
this study and in Baik et al. (2013) but as (C16:1 ω7c and/or iso-C15:0
2-OH) in Whang et al. (2014). GC-MS data for this summed
feature indicated that in species of the phylum Acidobacteria
subdivision 1, C16:1 ω7c was the dominant fatty acid and that C16:1
ω6c and iso-C15:0 2-OH did not occur (Männistö et al., 2011;
Sinninghe Damsté et al., 2011).
6 66 Supplementary Fig. S1. Phase-contrast photomicrograph of strain Ac_26_B10T grown under
67 optimum conditions. Cells were immobilized on agarose-covered slides (1% w/v) and the image was
68 taken with a Zeiss Axio Imager.M2 microscope (Carl Zeiss) equipped with an AxioCam MRm
69 camera Zeiss Axio microscope (100 x magnification). Bar, 5 µm.
70 71 72 7 73 Supplementary Fig. S2. Growth curves, followed by recording the optical density (OD) at 660 nm,
74 of strain Ac_26_B10T incubated at different pH values and at 28 ºC. pH T0 and Tend designate the pH
75 values at the beginning and at the end of the experiment, respectively.
76 77 8 78 Supplementary Fig. S3. Maximum parsimony (MP) (a) and maximum likelihood (ML) (b) trees
79 illustrating the phylogenetic position of strain Ac_26_B10T and related members of the class
80 Acidobacteriia based on almost full-length 16S rRNA gene sequences. For ML, the optimal
81 evolutionary model of nucleotide substitution applied is GTR+ I (p-inv= 0.3770) + G (gamma shape =
82 0.2120). Bars, (a) estimated substitution per 50 bases positions and (b) 0.02 fixed nucleotide
83 substitution per site. Bootstrap values above 50% (1000 resamplings) are indicated at the branching
84 points. Accession numbers of the respective nucleotide sequences are provided in parentheses.
85 86 87 9 88 Supplementary Fig. S4. Polar lipid profile of strain Ac_26_B10T after two-dimensional two-layer
89 chromatography and detection with molybdatophosphoric acid.
90 91 Abbreviations: DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine;
92 UAPL, unidentified aminophospholipid; UGL, unidentified glycolipid; UPL,
93 unidentified phospholipid. 10