1
2
Supporting information
3
Sequencing. Green leaves were cut from frozen moss specimens with a razor and ground in
4
liquid nitrogen. DNA was prepared with a Nucleospin Plant DNA kit (Clontech) using the kit's
5
PL2 lysis buffer and sent to the Nevada Genomics Center at the University of Nevada Reno. The
6
genomic DNA was used to prepare two libraries (one in 2013 and one in 2014) with an Ion
7
Xpress™ Plus Fragment Library Kit (Life Technologies). The libraries were sequenced with an
8
Ion Torrent Proton Sequencer using the Ion PI Sequencing 200 kit version 2 and Ion PI Chip, (all
9
Life Technologies). The 2013 library yielded 74 million reads with an average length of 143 bp
10
(acc. no. SRX1114535). The 2014 library yielded 85 million reads with an average length of 175
11
bp (acc. no. SRX1143757). The reads were assembled into contigs with CLC Genomics
12
WorkBench. The contigs were annotated by MG-RAST (metagenomics.anl.gov) and MANATEE
13
(http://manatee.sourceforge.net/igs/index.shtml).
14
Databases were constructed with makeblastdb (NCBI) and the reads and contigs were
15
screened for sequences resembling genes of known IBPs, antifreezes and ice-nucleating proteins
16
with blast-2.2.24+ (NCBI). The sequences were aligned manually with BioEdit (Hall, 1999). The
17
presence of N-terminal signal peptides was predicted with SignalP v. 3.0 (Bendtsen et al., 2004).
18
DUF3494 domains of the assembled proteins were identified with NCBI's Conserved Domains
19
site (ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Phylogenetic trees were constructed with Mega 6
20
(Tamura et al., 2011) .
21
1
22
Table S1. Closest matches from named species to DUF3494 domains of 43 putative ice-binding
23
proteins with signal peptides. -1 and -2 refer to first and second DUF3494 domains in double-
24
domain IBPs.
No.1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Acc. no.
KT343917
KT343918
KT343919
KT343920
KT343921
KT343922
KT343923
KT343924
KT343925
KT343926
KT343927
KT343928
KT343929
KT343930
KT343931
KT343932
KT343933
KT343934
KT343935
KT343936
KT343937
KT343938
KT343939
KT343940
KT343941
KT343942
KT343943
KT343944
KT343945
KT343946
KT343947
KT343948
KT343949
KT343950
KT343951
Closest hit based on DUF3494 domain
Species
Phylum2
Acc. No.
Act
Conexibacter woesei
WP_041732645
Act
Marmoricola sp. URHB0036
WP_036224037
Act
Conexibacter woesei
WP_041732645
Act
Micromonospora parva
WP_036411889
Act
Streptomyces sp. NRRL B-5680
WP_037828170
Bact
Flavobacteriales bacterium BRH_c54
WP_045640496
Act
Rhodococcus wratislaviensis
WP_005568966
Bact
Fibrella aestuarina
WP_041258978
Act
Conexibacter woesei
WP_041732632
Plancto
Pirellula staleyi
WP_044183297
Plancto
Singulisphaera acidiphila DSM 18658
AGA28501
Bact
Pirellula staleyi
WP_044183297
Acido
uncultured Acidobacteria bacterium
AAP58537
Act
Leifsonia sp. 109
WP_043742174
Plancto
Singulisphaera acidiphila DSM 18658
AGA28501
Act
Micromonospora lupini
WP_039907259
Bact
Pirellula staleyi
WP_044183297
Act
Conexibacter woesei
WP_041732645
Firm
Pelosinus sp. UFO1
WP_038675319
Act
Nocardioides alkalitolerans
WP_043644455
Act
Marmoricola sp. URHB0036
WP_036224037
Act
Streptomyces sp. NRRL S-481
WP_030955875
Firm
Pelosinus sp. UFO1
WP_038675319
Bact
Haliscomenobacter hydrossis
WP_013768008
Bact
Pirellula staleyi
WP_044183297
Act
Kribbella flavida
WP_041290110
Act
Conexibacter woesei
WP_041732645
Plancto
Singulisphaera acidiphila DSM 18658
AGA28501
Act
Streptomyces sp. NRRL S-481
WP_030955875
Firm
Pelosinus sp. UFO1
WP_038675319
Streptomycetaceae bacterium MP113-05 Act
EST39364
Act
Streptomyces sp. NRRL B-5680
WP_037828170
Act
Agreia bicolorata
WP_044441012
Act
Streptomycetaceae bacterium MP113-05
EST39364
Act
Streptomycetaceae bacterium MP113-05
EST39364
2
E value3
2.2E-84
4.5E-95
2.3E-85
5.8E-91
9.0E-69
3.2E-59
1.8E-89
2.4E-51
1.0E-34
3.3E-73
6.1E-70
1.3E-81
6.9E-78
1.0E-71
5.9E-73
3.1E-96
2.8E-79
3.6E-74
1.2E-65
1.2E-79
4.5E-95
7.4E-51
1.3E-70
3.5E-54
5.3E-73
4.6E-55
5.8E-69
1.9E-73
3.2E-76
1.8E-79
4.1E-66
2.1E-57
5.3E-109
2.9E-65
1.6E-68
36
37
38
39-1
39-2
40-1
40-2
41-1
41-2
42-1
42-2
KT343952
KT343953
KT343954
KT343955
KT343956
KT343957
KT343958
Arsukibacterium sp. MJ3
Pirellula staleyi
Conexibacter woesei
Flavobacteriales bacterium BRH_c54
Epilithonimonas sp. FH1
Flavobacteriales bacterium BRH_c54
Epilithonimonas sp. FH1
Nocardioides sp. CF8
Nocardioides sp. CF8
Flavobacteriaceae bacterium 3519-10
Flavobacterium antarcticum
Proteo
Bact
Act
Bact
Bact
Bact
Bact
Act
Act
Bact
Bact
WP_046552741
WP_044183297
WP_041732645
WP_045640496
WP_034968286
WP_045640496
WP_034968286
WP_036493436
WP_036493436
1.2E-67
8.4E-64
5.2E-72
7.3E-90
2.0E-78
1.1E-79
7.5E-77
2.5E-63
1.9E-65
WP_041252903
1.0E-39
WP_022827004
6.1E-55
25
26
1
-1 and -2 refer to the first and second DUF3494 domains, respectively.
27
2
Act, Actinobacteria; Bact, Bacteroidetes; Firm, Firmicutes; Plancto, Planctomycetes; Proteo,
28
Proteobacteria
29
3
Based on amino acid sequence of DUF3494 domain.
30
31
3
32
Table S2. Numbers of unique reads matching bacterial IBP genes and bacterial glycerol-3-
33
phosphate dehydrogenase (GAPDH) genes (housekeeping genes used as controls) to expect
34
values < 1e-20 in polar and mesophilic bacterial metagenomes. Each metagenome was searched
35
with TBLASTN for sequences matching 1) any of the DUF3494 domains of 14 bacterial and
36
algal genes1 plus 47 DUF3494 domains in the B. argenteum metagenome and 2) any of the
37
complete GAPDH sequences of six bacterial genes.
38
Bacterial metagenome
Bryum leaf
Sea ice
Sugar beet root
Soybean phyllosphere
Sargasso Sea
Rice root
Accession
SRX1114535
SRX1143757
SRX024772
SRX001972
SRX008324
SRX007384
DRX000494
DUF3494
623
GAPDHs
385
Ratio2
161
153
5
2
0
5
320
43
100
186
242
44
11
2
0
2
39
40
1
41
Polaribacter irgensii WP_004570305, Colwellia sp. SLW05 ABH08428, Flavobacteriaceae
42
bacterium 3519-10-1 ACD76102, Streptomyces sviceus WP_007383497 , Conexibacter woesei
43
WP_041732645, Frankia alni WP_041940867, Psychromonas ingrahamii 37 ABM04556,
44
Rhodococcus wratislaviensis WP_005568966, Psychroflexus torquis ATCC 700755 AFU68394,
45
Ferroplasma acidarmanus WP_048074211, Rhodoferax ferrireducens WP_011464175,
46
Navicula glaciei AAZ76251.
47
2
Cytophaga hutchinsonii WP_041932113, Shewanella denitrificans WP_011496354,
No. DUF3494 reads/100 GAPDH reads
48
4
49
Table S3. Cyanobacteria whose 16S rRNA sequences are highly represented in the Bryum
50
argenteum metagenome.
51
Species
Phormidium
sp.
Nostoc sp.
52
53
Leptolyngbya
sp.
Acc. no.
KT343962
KT343963
KT343964
Closest match
Species
Phormidium sp.
CCAP 1462/11
Nostoc sp.
ANT.L52B.8
Leptolyngbya
frigida NT.L53B.2
Acc. no.
%
identi
ty
HF678504
98.7
South Orkney
Islands
AY493593
99.2
Lake benthic mats
AY493576
98.0
Lake benthic mats
5
Habitat
(Antarctica)