Supplementary material
TRFLP profile of sea water and coral mucus
Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis was done for the
same mucus metagenome used in this study and the surrounding sea water metagenome
which showed that the bacterial communities associated with the mucus and the
surrounding sea water were entirely different. For TRFLP analysis, the universal 27F
forward 16S primer was labeled at the 5’ end with phosphoramidite fluorochrome
6-carboxyfluorescein (6-FAM). TRFLP was done according to Klaus et al; 2007 where
the amplicons labeled with 6-FAM were digested with the restriction enzyme MspI. The
Gene scan analysis was done at Macrogen, Korea.
Peak scanner software was used to generate tables of peak area and fragment size data for
each sample.
Peak area and fragment size of Sea water -MspI
Peak area and fragment size of coral mucus -MspI
TRFLP analysis was done using T-RFLP FRAGSORT. T-RFLP FRAGSORT is a
computer program that compares the TRFs obtained from samples processed with widely
used primers (8F, 907R, 27F, 226F, 1111R or EF4) and restriction enzymes (Msp1,
Hha1, Rsa1, HaeIII, and Bfa1) to TRFs from corresponding in silico amplification and
digestions of RDP II alignments (version 8.0).
FRAGSORT input consists of one, two or three data files containing the fragment
sizes and normalized peak areas (or heights) from restriction digestions of environmental
1
DNA amplified with a fluorescent primer. The input file can be in text, csv or genescan
format. The program outputs organisms that match 1, 2 or 3 T-RFLP fingerprints. The
program compiles a list of organisms matching the TRFs from each restriction enzyme,
then determines which organisms match 2 or 3 of the different fragments. The program
output is a list of microorganisms, TRF sizes, and normalized peak areas that correlate
with multiple experimentally generated T-RFLP profiles.
MspI Profile of Sea water
MspI
1.2
Normalized TRF Peak Area (%)
1
0.8
0.6
0.4
0.2
10
9
13
0
13
5
15
5
15
9
16
0
16
0
16
0
16
0
16
0
16
0
16
0
16
1
16
1
16
6
20
2
27
7
33
0
48
4
84
72
65
0
TRF Fragm ent Size (bp)
MspI Profile of Coral mucus
Msp I
1.4
1
0.8
0.6
0.4
0.2
TRF Fragment Size (bp)
2
486
485
485
485
484
448
430
291
184
158
157
157
157
157
149
141
127
126
126
126
126
125
125
125
125
125
122
122
122
88
122
88
88
88
88
87
87
80
71
71
70
0
63
Normalized TRF Peak Area (%)
1.2
Supplementary Table S1.
Representative output list of microorganisms present in the surrounding sea water
Sea water
1 Candidatus Tremblaya princeps.
2 Candidatus Tremblaya princeps.
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
uncultured proteobacterium a2b027 1-2 cm layer of core A.
uncultured bacterium mle1-31.
uncultured bacterium DR9IPCB16SCT4.
Desulfurella acetivorans (T) DSM 5264.
Syntrophus sp..
Syntrophus buswellii (T) DSM 2612.
uncultured bacterium SHA-51.
uncultured bacterium IndB4-24.
Lactococcus lactis lactis K338.
Actinocorallia aurantiaca (T) JCM8201.
Actinocorallia glomerata IMSNU 22179T.
Actinocorallia longicatena IMSNU 22180T.
Mycobacterium sp. IP20010664.
Mycobacterium sp.
Mycobacterium savoniae type strain: E533.
Mycobacterium smegmatis mc2 155.
Thermotoga lettingae (T) TMO.
Propionibacterium freudenreichii JS53.
Corynebacterium sp. 61720.
uncultured bacterium O-F-5.
Corynebacterium sp. CIP102857.
Corynebacterium sp. CIP102645.
Corynebacterium sp. CIP102622.
Corynebacterium sp. CIP102590.
Corynebacterium sp. CIP102346.
Corynebacterium sp. CIP102211.
Corynebacterium sp. CIP102124.
Corynebacterium sp. CIP102076.
Corynebacterium sp. CIP101775.
Corynebacterium sp. CIP107291.
Corynebacterium sp. CIP107067.
Corynebacterium segmentosum CIP107068,
34 (CCUG37878).
3
35
36
37
38
39
40
41
Corynebacterium macginleyi CIP104099T,
(ATCC51787T).
Corynebacterium accolens CIP104783T, (ATCC49724T).
Corynebacterium fastidiosum CIP103808.
Corynebacterium
pseudogenitalium
CIP106714,
(ATCC33035),
uncultured Corynebacterium sp. ACTINO9B.
Gordonia nitida J-1.
Gordonia alkalivorans.
Representative output list of microorganisms present in the coral mucus
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Coral Mucus
bacterium Te32A.
22
uncultured bacterium SOB-13.
bacterium Te45R.
23
uncultured proteobacterium OCS7.
bacterium Te58R.
24
uncultured bacterium O-CF-10.
bacterium Te47R.
25
uncultured bacterium C-F-17.
uncultured bacterium D137.
26
bacterium H15.
bacterium Te37R.
27
uncultured bacterium FB33-34.
bacterium Te49A.
28
uncultured bacterium FB47-42.
Enterococcus sp. GvF410.
29
uncultured bacterium FB49-2b.
Enterococcus faecium E 26.
30
uncultured bacterium 015B-F04.
uncultured isopod gut bacterium
RKPsAG.
31
uncultured bacterium 015C-F06.
Enterococcus faecalis UK873
32
Zoogloea ramigera ATCC 25935.
bacterium Te6A.
33
uncultured bacterium 4f39.
bacterium Te50A.
34
uncultured sludge bacterium A35.
bacterium Te95A.
35
uncultured bacterium SHA-58.
15 Pseudoalteromonas bacterium .
36
gamma proteobacterium HTCC230.
16 Rhodothermus marinus 10.
37
Rhodothermus marinus (T) R-10
17 (DSM 4252).
38
gamma proteobacterium HTCC234.
uncultured Sphingobacteriales bacterium
SF62.
18 uncultured bacterium IheB3-31.
39
uncultured
Sphingobacteriales
19 bacterium SF63.
41
uncultured bacterium KD8-91.
20 unidentified bacterium JTB326.
42
uncultured bacterium FukuN9.
21 Psychrobacter sp (T).
43
Uncultured rumen bacterium F24-F11.
uncultured bacterium KD8-112.
4