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ISMEJ-11-00360OAR
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Supplementary information
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Viral infections stimulate the metabolism and shape prokaryotic assemblages
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in submarine mud volcanoes
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Corinaldesi C., Dell’Anno A., Danovaro R.
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Department of Life and Environmental Sciences, Università Politecnica delle Marche, Via
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Brecce Bianche, 60131 Ancona, Italy
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Supplementary Materials and Methods
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Supplementary Figures S1-S2
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Supplementary References
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Supplementary Materials and Methods
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Biochemical composition of organic matter
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Chlorophyll-a and phaeopigments were extracted from sediments with 90% acetone (24 h in the
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dark at 4°C). After centrifugation (800 × g), the supernatant was used to determine the functional
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chlorophyll-a and acidified with 0.1 N HCl to estimate the amount of phaeopigments.
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Proteins were extracted from sediments with NaOH (0.5 M, 4 h) and, after centrifugation (800 × g),
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the supernatant was analyzed spectrophotometrically according to Hartree (1972) modified by Rice
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(1982) to compensate for phenol interference. Protein concentrations were calculated from
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calibration curves of bovine serum albumin (ranging from 10 to 200 µg mL-1).
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Carbohydrates were determined spectrophotometrically according to Gerchacov and Hatcher
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(1972). The method is based on the same principle as the widely used method of Dubois et al.
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(1956), but is specifically adapted for carbohydrate determination in sediments. Carbohydrate
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concentrations were calculated from calibration curves of D-glucose (from 10 to 200 µg mL-1)
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Lipids were extracted by direct elution with chloroform and methanol (1:1 V:V) and the resulting
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fraction, after evaporation in a dry hot bath at 80-100 °C for 20 min, was quantified according to the
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sulfuric acid-carbonization procedure (Marsh and Weinstein 1966). Lipid concentrations were
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calculated from calibration curves of tripalmitine (from 10 to 100 µg mL-1). For each biochemical
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analysis, blanks were made with the same sediment samples previously treated in a muffle furnace
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(450°C, 4 h). All analyses were carried out in 3-5 replicates, using about 1 g of wet sediment.
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CARD-FISH: Catalyzed Reporter Deposition – Fluorescence In Situ Hybridization
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Sediment samples were centrifuged (16000 ×g for 5 min), washed with PBS, then centrifuged
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(16000 ×g for 5 min) and re-suspended in PBS:96% ethanol. Samples were then treated with
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ultrasounds (three times, 1 minute each; Branson sonifier 2200, 60W), properly diluted and then
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filtered onto 0.2-µm polycarbonate membrane filters (25 mm diameter, Nuclepore). Filters were
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dipped in low-gelling-point agarose (0.1% [wt/vol] in Milli-Q water), dried on a glass Petri dish at
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37°C, and dehydrated in 95% ethanol. For cell wall permeabilization, filters were incubated either
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with lysozyme (Sigma-Aldrich) for bacteria and, in order to permeabilize also archaeal cell walls,
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with proteinase K (Sigma-Aldrich). Subsequently, the filters were washed three times with Milli-Q
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water and incubated in 0.01 M HCl (room temperature, 20 min) to inhibit intracellular peroxidases
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and to inhibit residual proteinase K. After incubation in HCl, filters were washed twice with Milli-Q
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water, dehydrated with 95% ethanol and dried at room temperature. Filters were cut in sections for
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hybridization with the oligonucleotide probes Eub-mix (Eub338, Eub338-II and Eub338-III)
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targeting Bacteria, Arch915 targeting Archaea and Non-338 (non-sense probe). To determine the
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abundance of Crenarchaea and Euryarchaea the oligonucleotide probes Cren537 (5’
and
Eury806
(5’-CACAGCGTTTACACCTAG-3’)
were
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TGACCACTTGAGGTGCTG-3’)
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utilized. Three hundred µl of appropriate hybridization buffer containing also the blocking reagent
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was mixed with HRP (Horseradish Peroxidase)-Probe working solution (50 ng µl-1) and this
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hybridization mix was added to each filter sections. Stringencies were regulated for each probe by
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adjusting the formamide concentration in the hybridization buffer (55% formamide for Eub338 mix
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and 35% for Arch915). The hybridization was performed at 35°C for at least 2 hours in a dynamic
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incubator. Thereafter, the sections were transferred into 50 ml of pre-warmed washing buffer at
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37°C for 15 min. Different washing buffers were prepared for each probe. Sections were then
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placed in PBS (pH 7.6), amended with 0.05% Triton X-100 and incubated at room temperature for
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15 min. After removal of the excess buffer, the filter sections were added with appropriate amounts
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of the substrate mix for tyramide signal amplification, containing amplification buffer and
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tyramide-Cy3, and incubated for 30 min in the dark at 37°C. After amplification, filters were
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washed in PBS (room temperature, 15 min), Milli-Q water, and 95% ethanol. Finally, the filter
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sections were air dried and observed under epifluorescence microscopy (Zeiss Axioskop 2,
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magnification × 1000) using filters appropriate for the fluorochrome utilized (Cy3; filter set #15,
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excitation BP 546/12, beam splitter FT 580, emission LP 590).
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Prokaryotic turnover
0.5
day
-1
0.4
0.3
0.2
0.1
0
MV02
MV03
MV04
MV05
MV06
CTRL1
CTRL2
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Figure S1. Prokaryotic turnover (expressed as day-1) in surface sediments (0-1 cm) of mud volcano
and control stations. Standard deviations are reported.
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a)
Prokaryotic
structure
Prokaryoticassemblage
assemblage structure
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Surface sediments
100%
80%
60%
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40%
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20%
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0%
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MV02
MV03
MV04
MV05
MV06
CTRL1
CTRL2
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b)
Subsurface sediments
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100%
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80%
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60%
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40%
20%
n.d.
n.d.
CTRL1
CTRL2
0%
MV02
MV03
MV04
MV05
MV06
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Bacteria
Crenarchaea
Euryarchaea
Other archaea
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Figure S2. Prokaryotic assemblage structure in surface (a) and subsurface sediments (b). n.d.= not
determined
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References
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Dubois M, Gilles K, Hamilton JK, Rebers PA, Smith F. (1956). Colorimetric method for
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determination of sugars and related substances. Anal Chem 28: 350-356.
Gerchacov SM, Hatcher PG. (1972). Improved technique for analysis of carbohydrates in sediment.
Limnol Oceanogr 17: 938-943
Hartree EF. (1972) Determination of proteins: a modification of the Lowry method that gives a
linear photometric response. Anal Biochem 48:422-427
Marsh JB, Wenstein DB. (1966). A simple charring method for determination of lipids. J Lipid Res
7: 574-576
Rice DL. (1982). The detritus nitrogen problem: new observations and perspectives from organic
geochemistry. Mar Ecol Progr Ser 9: 153-162
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