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Microbe-microbe interactions trigger Mn(II)-oxidizing gene
expression
Jinsong Liang1,2,★, Yaohui Bai1,★, Yujie Men3, Jiuhui Qu1
1
Key Laboratory of Drinking Water Science and Technology, Research Center for
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China;
2
University of Chinese Academy of Sciences, Beijing, China;
3
Department of Civil and Environmental Engineering, University of Illinois at
Urbana-Champaign, Urbana, IL, USA
★
These authors contributed equally to this work.
Correspondence: JH Qu, Key Laboratory of Drinking Water Science and Technology,
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18
Shuangqing Road, Haidian District, Beijing 100085, China. Tel: 86 10 62849160. Fax:
86 10 62849160. E-mail: [email protected].
The authors declare no conflict of interest.
This Supplementary Information includes:
Supplementary Methods S1 to S3
Supplementary Figures S1 to S6
Supplementary Tables S1 to S4
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Supplementary Methods
Method S1 Strain identification by 16S rDNA sequencing
Total genomic DNA of the two strains was extracted using TIANamp Bacteria DNA
Kit (TIANGEN) according to the manufacturer’s instructions. The 16S rDNA
fragments were amplified by PCR with the primer set, 27F and 1492R (Weisburg et
al., 1991). The PCR amplification protocol was as follows: one cycle at 95°C for 3
min, and 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, and one
cycle at 72°C for 10 min. The PCR products were purified and then sequenced by
Sanger sequencing. The output sequences from bi-directional sequencing of each
fragment were trimmed and assembled into contigs (> 1300 bp) with DNAMAN
(version 6.0) and then analyzed using BLAST alignment. The closest genus was
assigned to each of the 16S rDNA sequences.
Method S2 Spectroscopic Mn(III) trapping experiments
Mn(III) was also monitored during Mn(II) oxidation by the co-culture of strain
Arthrobacter and strain Sphingopyxis. Ligand-binding complex P2O74- (PP) was
selected to complex Mn(III) by forming stable Mn(III)-PP complex, which has max
absorbance at 258 nm (ε = 6,750 M-1). The co-culture was first cultured in the absence
of Mn(II) for 24 h, and then 200 μM Mn(II) was added to the medium, which was
quickly transferred to five flasks, and PP was sterilely added to each flask to achieve
the final concentrations of 0, 100, 500, 2000, and 10000 μM respectively. Samples of
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1 mL were taken per hour, and filtered through a 0.45 μm pore size filter (JINTENG)
before spectroscopic determination (full wavelength scan). After spectroscopic
determination, the samples were reduced in cuvette by adding 10 μL 10% (w/v)
reducing agent NH2OH·HCl and then subjected to the second spectroscopic
determination.
Method S3 Protein identification with nanoLC-MS/MS
The gel band of interest was excised from the gel, reduced with 25 mM of DTT and
alkylated with 55 mM iodoacetamide. In gel digestion was then carried out with
sequencing grade modified trypsin in 50 mM ammonium bicarbonate at 37°C
overnight. The peptides were extracted twice with 0.1% (v/v) trifluoroacetic acid in
50% (v/v) acetonitrile aqueous solution for 30 min. Extracts were then centrifuged in
a speedvac to reduce the volume.
For nanoLC-MS/MS analysis, the digestion products were separated by a 65 min
gradient elution at a flow rate of 0.250 µL/min with an EASY-nLCII™ integrated
nano-HPLC system (Proxeon, Denmark), directly interfaced with a Thermo
LTQ-Orbitrap mass spectrometer. The analytical column was a home-made fused
silica capillary column (75 µm ID, 150 mm length; Upchurch, Oak Harbor, WA)
packed with C-18 resin (300 Å, 5 µm, Varian, Lexington, MA). Mobile phase A was
Milli-Q water with 0.1% (v/v) formic acid, and mobile phase B was 99.9%
acetonitrile with 0.1% (v/v) formic acid. The LTQ-Orbitrap mass spectrometer was
operated in the data-dependent acquisition mode using Xcalibur 2.0.7 software and
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there was a single full-scan mass spectrum in the orbitrap (400-1800 m/z, 30,000
resolution) followed by 20 data-dependent MS/MS scans in the ion trap at 35%
normalized collision energy (CID). The MS/MS spectra from each LC-MS/MS run
were searched against the selected database using an in-house Proteome Discoverer
searching algorithm.
The search criteria were as follows: full tryptic specificity was required; one missed
cleavage was allowed; carbamidomethylation was set as the fixed modification; the
oxidation (M) was set as the variable modification; precursor ion mass tolerances
were set at 10 ppm for all MS acquired in an orbitrap mass analyzer; and the fragment
ion mass tolerance was set at 0.8 Da for all MS2 spectra acquired in the linear ion
trap.
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Supplementary Figures
Figure S1 Optical density at 600 nm (OD600) in monocultures of Arthrobacter and
Sphingopyxis.
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Figure S2 Mn(III) production in the co-culture of strain Arthrobacter and strain
Sphingopyxis. The max absorbance wavelength of Mn(III)-PP (P2O74-) complex is 258
nm. The dotted curves represent absorbance values of samples (1 mL) after adding 10
μL 10% (w/v) reducing agent NH2OH·HCl.
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Figure S3 Scanning electron microscope of strains Arthrobacter and Sphingopyxis in
monoculture and co-culture. a, Arthrobacter (A); b, Sphingopyxis (S); c, co-culture, 1
h before Mn(II) oxidation initiation; d, co-culture, 1 h after Mn(II) oxidation initiation.
e, 14 out of 64 (22%) Arthrobacter cells were coated with Mn oxides. Bars, 1 μm.
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Figure S4 Alignment of conserved copper-binding regions of the Mn(II)-oxidizing protein (BoxA) in strain Arthrobacter sp. QXT-31 and other
Mn(II)-oxidizing-related multicopper oxidases in other strains. The potential copper-binding sites are indicated as T1, T2, and T3 for three
binding types, i. e., type 1, type 2, and type 3. The accession numbers of CotA, CueO, MofA, MoxA and MnxG are AFL56752, NP_414665,
CAA81037, CAJ19378, and WP_006837219, respectively.
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Figure S5 Phylogenetic analysis illustrating the similarity of BoxA to its closest relatives (search against the NCBI protein database) and the
known Mn(II)-oxidizing protein sequences. The tree was generated by the maximum likelihood method in software MEGA 6 (Tamura et al.,
2013). The numbers (only those greater than 70% are indicated) on the branch nodes demonstrate the percentage of bootstrap support for the
clades based on 1000 bootstrap resampling. Enzymes CotA, MofA, CueO, MoxA and MnxG have been reported as Mn(II)-oxidizing proteins.
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a
b
Figure S6 Abundance estimation of transcripts related to (a) type VI secretion apparatus (T6SS)
and type IV secretion apparatus (T4SS), and (b) phage infection. (a) Transcripts encoding a T6SS
apparatus, a T4SS apparatus and an effector (related to virulence) in Sphingopyxis. (b) Transcripts
encoding a phase shock protein and a peptidoglycan glycosyltransferase in Arthrobacter. Data are
means ± average deviation for two biological replicates, except for 0 h (single sample with no
replicate).
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Supplementary Tables
Table S1 Primers used in PCR and genomic walking.
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Table S2 Top ten candidate proteins for proteins in the ~300-500 kDa protein band based on the analysis using nanoLC-MS/MS.
Accession
Description
Score
Coverage
# Proteins
# Unique Peptides
MW [kDa]
calc. pI
WP_056084337
bilirubin oxidase [Arthrobacter sp. Leaf137]
272.35
8.69
9
1
74.0
7.05
WP_056628334
bilirubin oxidase [Arthrobacter sp. Soil736]
131.82
9.88
9
3
71.9
8.48
WP_003805885
bilirubin oxidase [Arthrobacter globiformis]
105.12
13.69
9
2
73.0
7.64
WP_058810643
MULTISPECIES: serine hydrolase [Sphingopyxis]
54.19
20.63
2
7
41.2
8.34
WP_056343216
hypothetical protein [Sphingopyxis sp. Root1497]
48.06
0.87
1
2
383.8
4.06
WP_058806415
MULTISPECIES: TonB-dependent receptor
37.53
10.45
1
4
68.9
5.39
[Sphingopyxis]
WP_026544903
bilirubin oxidase [Arthrobacter sp. 35/47]
31.13
4.33
2
1
59.4
7.59
WP_054730799
molybdopterin dehydrogenase [Sphingopyxis
28.11
11.39
11
1
33.0
9.52
20.62
12.03
11
1
33.2
9.72
17.43
3.94
2
1
39.0
7.55
macrogoltabida]
WP_058806746
MULTISPECIES: molybdopterin dehydrogenase
[Sphingopyxis]
WP_056347851
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hypothetical protein [Sphingopyxis sp. Root1497]
Table S3 Top ten candidate proteins for proteins in the ~70 kDa protein band based on the analysis using nanoLC-MS/MS.
Accession
Description
Score
Coverage
# Proteins
# Unique
MW [kDa]
calc. pI
Peptides
WP_058809156
hypothetical protein, partial [Sphingopyxis sp. HXXIV]
332.16
36.38
8
13
47.7
4.67
WP_058809698
MULTISPECIES: TonB-dependent receptor [Sphingopyxis]
257.90
19.97
8
15
83.3
5.01
WP_056342572
TonB-dependent receptor [Sphingopyxis sp. Root1497]
107.85
18.00
10
6
84.5
4.92
WP_058809157
MULTISPECIES: TonB-dependent receptor [Sphingopyxis]
84.98
11.45
7
9
98.9
4.69
WP_058456813
MULTISPECIES: TonB-dependent receptor [Sphingopyxis]
74.60
11.32
8
1
84.3
4.77
WP_058807574
MULTISPECIES: hypothetical protein [Sphingopyxis]
63.69
17.77
128
12
81.1
4.94
WP_058807958
MULTISPECIES: hypothetical protein [Sphingopyxis]
60.44
12.08
4
10
108.2
4.89
WP_056084337
bilirubin oxidase [Arthrobacter sp. Leaf137]
34.16
6.63
11
3
74.0
7.05
WP_058810555
MULTISPECIES: ABC transporter ATP-binding protein
30.28
7.43
4
4
88.2
5.16
25.33
5.64
13
4
90.4
4.83
[Sphingopyxis]
WP_056371367
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MULTISPECIES: TonB-dependent receptor [Sphingopyxis]
Table S4 Statistics of RNA-Seq data.
Sample
name
Clean
reads
Clean bases
0h
1.5h_1
1.5h_2
3h_1
3h_2
4h_1
4h_2
7h_1
7h_2
Paired-end
11,862,668
11,739,303
11,533,380
11,548,897
11,649,526
11,779,250
11,859,254
11,768,023
11,712,894
26,192,356
581,270,732
575,225,847
565,135,620
565,895,953
570,826,774
577,183,250
581,103,446
576,633,127
573,931,806
2,357,312,040
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Read
length
(bp)
49
49
49
49
49
49
49
49
49
90
Insert size
(bp)
Q20(%)
GC(%)
Number of reads aligned to
the assembled transcripts
0
0
0
0
0
0
0
0
0
200
97.14
97.60
96.92
96.91
96.79
96.93
97.16
97.10
97.25
96.87
62.59
64.04
64.11
64.02
64.07
63.78
63.70
63.97
61.86
59.86
10900897
10698448
10347700
10547625
10467886
10725710
10954806
10867634
10780192
-
Number of reads aligned to
boxA sequence
166
829
1682
37843
36578
6916
8994
6268
5118
-
References
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. (2013). MEGA6: Molecular evolutionary
genetics analysis version 6.0. Mol Biol Evol 30:2725-2729.
Weisburg WG, Barns SM, Pelletier DA, Lane DJ. (1991). 16S ribosomal DNA amplification for
phylogenetic study. J Bacteriol 173: 697–703.
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