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