Supplementary Information:
DNA/RNA extraction - DNA and RNA were extracted from 3 replicates of
each sample using half of each minicore (0.5 cm2; top 2 mm approx 0.25 g dry
weight) by beadbeating, using a Mikro-dismembrator-U (B. Braun, Biotech
International) at 2000 rpm. for 30 s, in 2 ml microcentrifuge tubes containing 1 g of
0.1 mm Zirconia/Silica beads (Biospec products Inc.), 0.5 ml of 1 M sodium
phosphate buffer (pH 8.0), and 0.5 ml of phenol-chloroform-isoamyl alcohol
(25:24:1). After centrifugation (16,100 g for 1 min at 4°C), 450 ml of the upper
aqueous layer was removed and added to tubes containing 0.03 g polyvinylpolypyrrolidone (Sigma), and incubated at 4oC for 1 h to remove inhibitory humic
acids. After centrifugation the supernatant was transferred to tubes containing 1
volume of chloroform-isoamyl alcohol (24:1) and mixed by inversion. After further
centrifugation (16,100 g for 1 min at 4°C), the upper aqueous layer was transferred to
a clean tube and nucleic acids were precipitated on ice for 30 min by adding 0.1
volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol. Nucleic
acids were pelleted by centrifugation (16,100 g for 25 min at 4°C), washed with 70%
ethanol and air-dried before resuspending in 200 l of DEPC-treated water. DNA was
removed from 50 l of the total nucleic acid extraction by DNase digestion, using
Turbo DNA-free™ protocols (Ambion) and a control PCR was performed to confirm
that DNA was completely removed.
Reverse transcription polymerase chain reaction (RT-PCR) - Reverse
transcription was performed using SuperScript™ reverse transcriptase III (Invitrogen)
using protocols as recommended by the manufacturer with 0.1 mM reverse primer
(described below). 2 l of reverse transcription products were subsequently used in
PCR reactions to amplify the variable V3 region of the 16S rRNA gene (Escherichia
coli positions 341–534) using primers to conserved regions (forward primer with GC
clamp 5′ - CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG
GGG GCC TAC GGG AGG CAG CAG - 3′; reverse primer 5′ - ATT ACC GCG
GCT GCT GG - 3′) (Muyzer et al, 1993). Separate reactions were also performed
with Archaea-specific 16S rRNA primers ARC344F (forward primer with GC clamp
5′ - CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GAC
GGG GYG CAG CAG GCG CGA - 3′) (Stahl and Amann, 1991) and ARC915R (5′ GTG CTC CCC CGC CAA TTC CT - 3′) (Raskin et al, 1994). 50 l reactions
contained 0.4 mM of forward and reverse primer, 0.1 mM dNTPs, 2.5 U of Taq
polymerase and 5 ml of the reaction buffer supplied with the enzyme (Qiagen®), and
2 ml of template cDNA. Amplification was performed in a Gene Amp® PCR system
9700 Thermocycler (Applied Biosystems) as follows: 95°C for 30 s, 30 cycles of
94°C for 30 s, 60°C for 30 s and 72°C for 30 s, then 72°C for 10 min. Amplification
of the target cDNA was confirmed by agarose gel electrophoresis (2% w/v in 1 
TAE buffer at 100 V) and ethidium bromide staining.
Denaturing gradient gel electrophoresis - Denaturing gradient gel
electrophoresis analysis of bacterial and archaeal 16S rRNA RT-PCR products was
performed using the Bio-Rad D Code system, on gels consisting of 8% (w/v)
polyacrylamide (acrylamide: bisacrylamide, 37:1) and a denaturing gradient from
40% to 60% [100% denaturant is 7 M urea and 40% v/v formamide], in 1 TAE (40
mM Tris-acetate, 1 mM di-sodium-EDTA, pH 8.0), at 60V and 60°C for 17 h. After
electrophoresis, gels were stained with silver nitrate. DGGE gels were analysed for
relative band intensity using Phoretix 1D Version 5.00. Triplicate DGGE profiles per
sample were imaged, all of which were highly similar. These triplicates were then
pooled (5l RT-PCR product from each replicate) and the combined 15l used to
produce the composite DGGE gel presented in Fig.4.
Q-PCR – Bacterial and Archaeal 16S rRNA gene abundance was quantified
by separate Q-PCR reactions on an ABI 7000 Sequence Detection System (Applied
Biosystems), using the primer pairs (but without the GC clamps) detailed in the
DGGE section. DNA isolated from the sediment minicores was amplified in triplicate
together with no-template controls. Reaction mixtures using SYBR® Green PCR
Master Mix (Applied Biosystems), cycling conditions, standard curve construction
(R2=0.99, E=95%), and quantification were performed as described previously
(McKew et al, 2007).
16S rRNA PCR amplicon libraries - Bacterial community composition and
diversity were assessed from 16S rRNA gene libraries constructed from the DNA
extracts of selected samples (Wet controls from days 1 and 28, desiccated samples
from day 14 and 23, and the reflooded sediment samples from day 28) using Roche
454 pyrosequencing technology at the NERC Molecular Genetics Facility at the
University of Liverpool. Amplicons for the pyrosequencing libraries were produced
by PCR amplification of the V3 region of the 16S rRNA gene using the same target
sequence and PCR cycling conditions as detailed in the RT-PCR section. Samples
were multiplexed on a 1/8 section of the pyrosequencing plate, so fusion primers also
contained a unique 10 base barcode to distinguish each sample (Parameswaran et al,
2007). Sequences analysed were a minimum length of 150 base pairs (mean length
183 base pairs) and were classified using the Greengenes database (DeSantis et al,
2006b) and NAST multiple sequence aligner (DeSantis et al, 2006a). Phylogenies
were constructed with the PHYLIP software package (Felsenstein, 2005), using the
neighbour-joining algorithm, and analysed with UNIFRAC (Lozupone et al, 2006).
Shannon diversity scores (H′) were calculated using the RDP Pyrosequencing
Pipeline’s alignment, complete linkage clustering and Shannon and Chao1 index
tools.
Supplementary references:
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chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ
Microbiol 72:5069-5072.
Felsenstein J. (2005). PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author.
Department of Genome Sciences, University of Washington, Seattle.
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