1
Supplementary information
2
Supplementary material and methods
3
Sampling site: the Äspö Hard Rock Laboratory
4
The Äspö Hard Rock Laboratory (HRL) is situated north of Oskarshamn on the east coast of
5
Sweden. It consists of a 3.6-km-long tunnel extending from the ground surface to a depth of 450 m
6
into granitic rock. It was built as a research laboratory and to demonstrate the potential for the
7
geological disposal of spent nuclear waste (Pedersen, 2001). Along the walls of the tunnel,
8
groundwater-containing fractures are intersected by boreholes. The fractures were packed off with
9
packers and the groundwater was under in situ pressure, related to the depth of the borehole.
10
Groundwater could be accessed via valves and tubes that connect the packed-off borehole sections.
11
For this investigation, samples for chloride analysis were diluted to the range of 0.5–70 mL L–1 or
12
70–170 mg L–1 and analyzed using ion chromatography according to the SS-EN ISO 10 304-1 and
13
SIS 02 81 36 standards, respectively. For sulphate estimates, samples were diluted to 0.5–70 mg L–1
14
and analysed using ion chromatography according to SS-EN ISO 10 304-1. The borehole names and
15
sampling dates for SRB and phages are listed in supplementary Table 1.
16
MPN of SRB and identification of SRB isolates
17
Sulphate-reducing bacteria were sampled from seven boreholes (supplementary Table 1). In March
18
and May 2006, the borehole KJ0052F01 circulation and flow cell system was sampled using a
19
pressure vessel sampler as described by Hallbeck and Pedersen (2008). Borehole KA3542G01 was
20
sampled in November 2006, boreholes KA2162B, KA3110A, KA3510A, and KJ0052F03 in
21
September 2007, and borehole KJ0050F01 in June 2008 using in situ pressure and based on
22
Pedersen’s (2001) descriptions. In these boreholes, 2–10 L of groundwater was drained from the
23
sampled sections before sample collection to wash out the tubes connecting the boreholes to the
24
valves. For boreholes KA2162B and KA2198A, the groundwater had been continuously flowing at
25
approximately 1 L per minute for more than a year when sampled. Samples for the estimation of the
26
MPN of SRB were collected by connecting 1/8” poly-ether-ether-ketone (PEEK) tubing (Genetec,
27
Göteborg, Sweden) to the borehole or by filling 20-mL BD Plastipak syringes (VWR, Stockholm,
28
Sweden) with water; samples were then transferred into N2-filled 27-mL anaerobic tubes using a
29
needle. The syringes used in the field were put in an anaerobic jar and flushed twice with N2 using a
1
30
vacuum pump, or, if this was unavailable, were individually flushed three times with N2. Before use,
31
the N2 had been filtered though 32-mm-diameter, 0.2-μm-pore-size Filtropur S syringe filters
32
(No./REF 83.1826.001; Sarstedt, Nümbrecht, Germany). The same day or the morning after
33
sampling, the groundwater samples were inoculated into SRB medium that mimicked groundwater
34
chemistry, using a most probable number technique, in 10-mL portions in 27-mL anaerobic tubes
35
(Hallbeck and Pedersen, 2008). The syringes used in the lab were either flushed once with N2 and
36
once with N2:H2:CO2 (85:10:5) using the airlock of the AAL anaerobic chamber from Coy
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Laboratory Products (Grass Lake, MI, USA) or individually flushed three times with N2. All SRB
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cultivations reported here were done in an anaerobic medium composed of a basal medium with
39
additions, based on Widdel and Bak (1992) and modified as described by Hallbeck and Pedersen
40
(2008).
41
The basal medium used to grow SRB was mixed in double-distilled H2O, autoclaved and contained
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(g L1): 11.9 NaCl, 1.0 CaCl2  2H2O, 0.67 KCl, 1.0 NH4Cl, 0.15 KH2PO4, 0.5 MgCl2  6H2O, and
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0.3 MgSO4  7H2O. The medium was cooled under an N2:CO2 (80:20) atmosphere to obtain an
44
anaerobic environment. The following were added to the medium using sterile N2-flushed syringes
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(L1): 10 mL of trace element solution (Wolin et al. 1963), 60 mL of NaHCO3 solution, 10 mL of
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yeast extract solution, 10 mL of vitamin solution, 1 mL of thiamine solution, 1 mL of vitamin B12
47
solution, 5 mL of iron stock solution, 2 mL of resazurin solution, 10 mL of cysteine hydrochloride, 5
48
mL of lactate, and 10 mL of Na2S solution. The pH was in the 6.87.5 range and adjusted with 1 M
49
NaOH or 1 M HCl if necessary. The medium flask was then over pressurized and dispensed via an
50
outlet tube and a needle in 9-mL portions into 27-mL anaerobic tubes (no. 2048-00150; Bellco
51
Glass, NJ, USA), fitted with butyl rubber stoppers (no. 2048-117800; Bellco Glass), and sealed with
52
aluminium crimp seals (no. 2048-11020; Bellco Glass). Before use, the tubes had been autoclaved,
53
sealed in an anaerobic box containing an N2:H2:CO2 (92–93:2–3:5) atmosphere, flushed twice with
54
N2:CO2 (80:20), and put under a pressure of 0.01 MPa above atmospheric pressure. After 8–12
55
weeks of incubation of cultures at 20°C in the dark, the MPN tubes were analysed for turbidity by
56
ocular examination and for sulphide production using the CuSO4 method according to Widdel and
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Bak (1992) on an Ultraspec 2000 UV visible spectrophotometer (Amersham Pharmacia Biotech,
58
Uppsala, Sweden) to confirm SRB growth. Samples were considered positive if they contained three
59
times more sulphide than the groundwater with same dilution as the sample, filtered though 0.2-μm-
60
pore-size Filtropur S syringe filters (No./REF 83.1826.001; Sarstedt, Nümbrecht, Germany).
2
61
For cultivation on anaerobic agar, the mixed and autoclaved agar solution contained (g L1): 0.5
62
KH2PO4, 7 NaCl, 1 CaCl2  2H2O, 0.67 KCl, 1 NH4Cl, 0.5 MgCl2  6H2O, and 67 agar. The
63
somewhat cooled solution was placed in an anaerobic box, dispensed in 3-mL aliquots in 27-mL
64
anaerobic tubes, and sealed with rubber stoppers and aluminium crimp seals. The tubes were then
65
taken out of the box, flushed twice with N2:CO2 (80:20), and put under a pressure of 0.01 MPa.
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Upon inoculation, the agar was melted and placed in a 60°C water bath. N2-flushed 10-mL syringes
67
were used to mix agar and 7 mL of medium for SRB; the solution was then placed in a water bath at
68
45°C. SRB cultures from MPN tubes originating from borehole KJ0052F01 circulation and flow cell
69
systems, sampled and cultivated as described above, were added using N2-flushed syringes in 1-mL
70
portions and the tubes were carefully mixed. The tubes were allowed to cool down while rotating,
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causing the agar and cells to be dispersed on the sides of the tubes. After incubation in a dark
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environment at 19°C for 6–12 weeks, colonies were picked from the agar inside an anaerobic box
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and transferred to 27-mL anaerobic tubes containing 9 mL of medium for SRB. Four isolates of SRB
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from SRB cultures derived from borehole KJ0052F01 were used to scan for the viable number of
75
viruses and to analyse the host range of the isolated phages. Two SRB isolates for each of boreholes
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KA3510A, KA2162B, and KA3110A were subsequently used to further test the phage host range.
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The isolates were from the highest dilutions of MPN cultures displaying growth and cultivated on
78
anaerobic agar.
79
The DNA of SRB was extracted using the spin-column protocols for the pre-treatment of Gram-
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positive bacteria and the purification of total DNA from animal tissues supplied with the QIAGEN
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DNeasy blood and tissue kit (QIAGEN, Hilden, Germany). For DNA extraction, SRB cultures were
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sampled using 2-mL BD Plastipak syringes (VWR) into 1.5-mL Eppendorf tubes, which were then
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centrifuged for 10 min at 7500 rpm. Pellets were dissolved in 180 µL of enzymatic lysis buffer
84
containing 20 mM Tris-HCl (Sigma-Aldrich, Steinheim, Germany), 2 mM Na EDTA, 1.2% Triton
85
X100 (Sigma-Aldrich), and 20 mg mL–1 lysosyme (Sigma-Aldrich). The DNA was further extracted
86
using the pre-treatment for Gram-positive bacteria and the spin-column protocol (QIAGEN DNeasy
87
blood and tissue kit; Qiagen, Hilden, Germany) for purification of total DNA from animal tissues.
88
Sulphate reducing bacteria isolates were identified by their 16S rRNA gene sequence. Samples of 25
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µL for PCR were amplified using the MyCycler thermal PCR cycler (Bio-Rad Laboratories,
90
Sundbyberg, Sweden with the bacterial primers 27f and 1492r (Lane, 1991; Weisburg et al., 1991).
91
The 16S rRNA gene sequences were then determined by the MWG Biotech Sequencing Team
3
92
(Ebersberg, Germany) using the internal 16S rRNA primers 27f, 357r (5´-
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CTCCTACGGGAGGCAGCA G-3´), 530f (5´-GTGCCAGC(AC)GCCGCGG-3´), 907r (5´-
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CCGTCAATTCCTTTRAGTTT-3´), and 926f (5´-AAACT(CT)AAA(GT)GAATT GACGG-3´),
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which span the whole 16S rRNA gene. The sequences were compared and affiliated with sequences
96
from GenBank using the BLAST tool at NCBI. For PCR of the 16S rRNA gene sequence, the PCR
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mixture contained 0.2 µM of each primer, 20 ng of DNA (except for negative control), and 12.5 µL
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2  iProof Mastermix (Bio-Rad Laboratories, Sundbyberg, Sweden) to a final reaction volume of 25
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µL diluted with RNase/DNAse free water in 100-µL PCR tubes (AH Diagnostics, Skärholmen,
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Sweden). Amplification was carried out using the MyCycler thermal PCR cycler (Bio-Rad
101
Laboratories). The program for PCR was initial denaturation at 98°C for 3 min; this was followed by
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30 cycles of denaturation at 98°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 40 s,
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and a final extension at 72°C for 5 min. PCR products were run on a 1% agarose gel containing
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0.625 µg mL–1 EtBr (cat. no. 161-300; Bio-Rad Laboratories, Sundbyberg, Sweden) for 45 min at 90
105
V. The bands were then analyzed with an SYBR filter in a GelDoc according to the manufacturer’s
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protocol (Bio-Rad Laboratories). The molecular weight standard used ranged from 100–1500 bp in
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length (TaKaRa; Fisher Scientific, Göteborg, Sweden). The PCR product was purified using a
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QIAquick PCR purification kit according to the manufacturer’s protocol (cat. no. 28104; Qiagen).
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Enterobacterial intergenomic repetitive consensus (ERIC) sequences from the genomes were
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amplified by ERIC-PCR using primers ERIC2 and ERIC1r (Debruijn, 1992). The PCR products
111
were run on a 1% agarose gel containing 0.625 µg mL–1 EtBr to detect the size of the fragments. For
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ERIC-PCR, reaction mixtures consisted of 12.5 µL  2 of iProof Mastermix (PCR Mastermix
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containing 1.5 mM MgCl2 and 0.2 mM concentration of each dNTP; Bio-Rad Laboratories), 2 µM
114
of each of the forward and reverse primers, 20 ng of the template (DNA), and water to a total
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volume of 25 µL. Water served as the negative control. Amplifications were performed using the
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MyCycler thermal PCR cycler (Bio-Rad). The program for PCR was initial denaturation at 98°C for
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7 min; this was followed by 30 cycles of denaturation at 98°C for 1 min, annealing at 52°C for 1
118
min, extension at 72°C for 8 min, and a final extension at 72°C for 16 min. PCR products were run
119
on a 1% agarose gel containing 0.625 µg mL–1 EtBr for 60 min at 90 V. The bands were then
120
analyzed with an SYBR filter in a GelDoc according to the manufacturer’s protocol (Bio-Rad
121
Laboratories). A molecular weight standard ranging from 100–1500 bp in length (TaKaRa; Fisher
122
Scientific) was used to detect the size of the fragments.
4
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Determination of total bacterial and viral numbers and viable biomass
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The total number of cells (TNC) and the number of virus-like particles (VLP) were determined using
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a direct count method. Samples were filtered and stained with SYBR Gold according to Noble and
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Fuhrman (1999) and Chen et al. (2001). Cultures containing approximately 107 counts mL–1 or more
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were diluted in 0.9% NaCl solution before filtration. The NaCl solution had been filtered through
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0.02-µm-pore-size Anotop 25 Plus filters (Whatman, Maidstone, England, UK) before use. Between
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0.5 and 1 mL of sample was used for filtration onto 0.02-μm-pore-size, 25-mm-diameter Anodisc
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filters (Whatman) using a GV 025/2 vacuum filter glass apparatus (Whatman). The filters were dried
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from underneath on Kimcare medical wipes (Kimberly-Clark, Surrey, UK) and stained on top of
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100-μL drops of SYBR Gold nucleic acid gel stain (Invitrogen, Eugene, OR, USA) for 15 min in a
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dark Petri dish. The SYBR Gold had been diluted just before staining to a concentration of 25 in
134
0.02-μm-pore-size filtered (Whatman) MQ water. The filters were dried from underneath to remove
135
excess stain, mounted on microscope slides with 30 μL of freshly made 0.1% p-phenylenediamine
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(MP Biomedicals, Solon, OH, USA) antifade solution on top, and covered with cover slips. Drops of
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non-autofluorescent immersion oil (Leica, Wetzlar, Germany) were placed on the slide before
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microscopy. Both cells and viral particles were counted in the same fields of view when the numbers
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allowed it, under blue light using a Leica DMR HC epifluorescence microscope (Leica) equipped
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with an HC Plan 10/25 ocular (Leica) and an HCX PL Fluotar 100/1.30 oil objective (Leica). At
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least 300 microbial and viral particles were counted per filter in up to 30 fields; however, in the
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samples taken from SRB batch cultures at an early growth phase, only approximately 100 cells per
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filter were counted in 30 fields. Each field counted was 0.01 mm2 in size. Viral counts were
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completed within two months and bacterial counts within five months.
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The concentration of viable biomass in cultures was estimated using an ATP assay (Lundin, 2000;
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Lundin et al., 1986). Briefly stated, the ATP biomass kit HS for determining total ATP in living cells
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was used (BioThema, Handen, Sweden). ATP was extracted by mixing 100 μL of B/S extractant
148
with 100 μL of sample; 100 μL of this mixture was added to 400 μL of reagent HS, and light
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emission was measured in an FB12 tube luminometer (Sirius Berthold, Pforzheim, Germany).
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Detection of lysed cultures
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Since viruses need an active host for effective lytic infection, SRB were grown in batch cultures to
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estimate how they grew over time. Cells were cultivated in triplicate in 50 mL of medium in 100-mL
5
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serum flasks (no. 1500; Nordic pack, Södertälje, Sweden) fitted with butyl rubber stoppers (no.
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2048-117800; Bellco Glass, Vineland, NJ, USA) and sealed with aluminium crimp seals (no. 2048-
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11020; Bellco Glass). As in experiments where phages were added to SRB cultures, cells were
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grown in medium containing 7 g L–1 of NaCl, at 21°C at 100 rpm on a KS250 basic orbital shaker
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(Kika Labortechnik, Saufen, Germany). Growth was followed by sampling 1.5 mL of culture to 2-
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mL Eppendorf tubes approximately every 24 h using 2-mL BD Plastipak syringes (VWR). To
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extract ATP from the cultures, 150 µL of sample was mixed with 150 µL of B/S buffer from the
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ATP biomass kit HS in a 1.5-mL Eppendorf tube and kept frozen at –20°C. Samples were later
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thawed, 100 µL was mixed with 400 µL of reagent HS, and further analyzed according to the ATP
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assay to determine the sample amount of viable biomass. The ATP concentration was plotted against
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time for the different SRB to approximate the time it took to reach exponential and stationary
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growth. Samples to be tested for SRB phages were added during the mid-exponential growth of the
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batch cultures, while cell lysis was determined after the cultures were estimated to have reached
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stationary growth.
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Because it was not obvious which technique should be used to detect lysed SRB cultures, several
168
methods were used after samples had been added. First, ocular determination was used, the turbidity
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of cultures being estimated according to a scale of 1–3. Dense cultures, like those to which no
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phages were added, were considered to be 3 on the scale and clearly lysed cultures were considered
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1, or, if more dense, 2. Lysed cultures also typically contained black precipitations. Lysed and
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unlysed cultures were also differentiated using the ATP assay to estimate the viable biovolume in
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samples. The third method was to measure turbidity using spectrophotometry. Approximately 5 min
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after the tubes had been thoroughly shaken, the turbidity was directly measured in the 27-mL
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anaerobic tubes used for cultivating cells and phages, at OD 660 nm using a DR/2500 Odyssey
176
HACH spectrophotometer (HACH, Loveland, CO, USA). Lysed cultures were always detected
177
relative to cultures to which no phages had been added, and sterile medium served as a control for
178
contamination. For turbidity measurements and the ATP assay, a ratio of the amount of ATP (or
179
turbidity) in a culture containing no added phages to the amount of ATP (or turbidity) in the
180
examined culture was calculated. When this ratio exceeded 2, samples were considered positive and
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to contain phages lytic to the cells. The ATP assay could be used to estimate, quickly and reliably,
182
the viable biomass (Lundin, 2000), but was more costly and less time efficient than the
6
183
spectrophotometric turbidity measurement later used. The latter method entailed measuring OD
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directly in 27-mL tubes and was not suitable for measurements of cultivations in 100-mL flasks.
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Since several different methods were used to detect lysed SRB cultures, the methods were tested and
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compared on D. aespoeensis cultures immune and sensitive to the phages. To test the reliability of
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the methods for detecting lysis and the presence of phages, 9-mL cultures of SRB34, SRB35, and D.
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aespoeensis immune to the isolated phage were compared with D. aespoeensis cells sensitive to the
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phage. The average numbers of virus particles counted using the SYBR Gold staining method were
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compared with turbidity measurements made using spectrophotometry and the ATP assay,
191
respectively. This was done eight days after 1 mL of phage culture was added to 9 mL of an
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exponentially growing batch culture. When the cultures were analyzed, the number of VLP was
193
found to have increased in the same cultures in which a decline in OD and ATP could be seen. The
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average number of virus particles counted using the SYBR Gold staining method was 8.55  108 (±
195
SD 1.06  108, n = 3) phages mL–1 in immune cultures and 8.63  109 (± 7.43  107, n = 3) phages
196
mL–1 in lysed cultures. The corresponding turbidity measurements made using spectrophotometry
197
were 0.22 (± 0.02) and 0.00 (± 0.02), and using the ATP assay in the same cultures were 3.1  108 (±
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1.5  108) and 3.1  107 (± 2.5  107) amol ATP mL–1. Hence, as defined for the detection of lysed
199
cultures, the OD and amount of ATP in uninfected cultures were distinctly higher than in infected
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cultures. The three different methods were convenient to use in different experimental set-ups and all
201
produced reliable results, in that they identified the same cultures as infected.
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Phage isolates and phage morphology
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To examine phage morphology, recently lysed D. aespoeensis cultures containing the five phage
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isolates were collected using sterile syringes. Drops of the cultures were transferred onto formvar-
205
and carbon-coated copper grids (Electron Microscopy Sciences, Cedarlane Laboratories, Burlington,
206
ON, Canada) for 30 sec, to let the particles settle, and stained with 1% uranyl acetate (Electron
207
Microscopy Sciences) for 30 sec. Filter paper was used to wick off excess sample. Deionized water
208
filtered through 0.02-µm-pore-size Anodisc filters (Whatmann) was used to prepare the uranyl
209
acetate. Grids were stored in the dark to prevent stain fading. Grids were examined at a
210
magnification of 45 000 or 70 000 times using a Philips 201 TEM (Philips, Eindhoven, Netherlands)
211
operating at 60 kV. Phage sizes in electrographs were measured using ImageJ software (Abramoff et
212
al., 2004). Using the “Set Scale” tool in ImageJ and the size of scale bars, the number of pixels nm–1
7
213
was determined in micrographs. By marking phage structures, the size of these could be estimated
214
using the “Measure” tool.
215
References
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Abramoff MD, Magelhaes PJ, Ram SJ (2004). Image Processing with ImageJ. Biophotonics Int 11:
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Chen F, Lu JR, Binder BJ, Liu YC, Hodson RE (2001). Application of digital image analysis and
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flow cytometry to enumerate marine viruses stained with SYBR gold. Appl Environ Microbiol 67:
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Debruijn FJ (1992). Use of repetitive (repetitive extragenic palindromic and enterobacterial
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genomes of Rhizobium meliloti isolates and other soil bacteria. Appl Environ Microbiol 58: 2180–
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Lundin A (2000). Use of firefly luciferase in ATP-related assays of biomass, enzymes, and
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9
Supplementary Table 1. Dates of sample collection for screening and most probable number (MPN) of phages and SRB from
boreholes in the Äspö Hard Rock Laboratory tunnel. Samples collected for phage cultivation were filtered through 0.2-μm-pore-size
syringe filters into 27-mL anaerobic tubes, before addition to exponentially growing SRB batch cultures.
Borehole
a
Sampling date
Screening for phages using
D. aespoeensisa
MPN of phagesb
MPN of
SRBb
Screening for phages using isolates
SRB2, SRB3, SRB5, and SRB22b
SA1328A
2006-10-31
-
-
2007-09-27
KA2198A
2006-10-30
-
-
2007-09-27
KA2162B
2006-10-30
2006-11-09
2007-09-27
2007-09-27
KA3110
2006-10-30
2007-03-07
2007-09-27
2007-09-27
KJ0052F03
2006-10-30
2006-11-09
2007-09-27
2007-09-27
KJ0052F01
2006-10-30
-
2006-03-23
2007-09-27
KJ0050F01
2006-10-30
-
2008-06-24
2007-09-27
KA3542G01
2006-10-31
-
2006-11-08
2007-09-27
KA3510A
2006-10-30
2006-11-08
2007-09-27
2007-09-27
KF0069A01
2006-10-31
-
-
2007-09-27
Using 50-mL cultures.
b
Using 10-mL culture
10