Supplementary Materials
PCR, cloning and phylogenetic analysis of dsrA genes
The dsrA genes were PCR amplified from diseased lesions using the primers
DSR1-F+ and DSR-R (Leloup et al., 2007; Kondo et al., 2008). PCRs were
performed on a Corbett Palm-cycler (Corbett Research, Australia). as follows; 300
nM of each primer, 250 M of each deoxyribonucleotide triphosphate, 1  PCR
buffer (Tris-Cl, KCl, (NH4)2SO4, pH 8.7), 2 mM MgCl2 and 2.5 Units of iTaq DNA
polymerase (Scientifix, Australia) was combined and adjusted to a final volume of 50
L with sterile water. Cycling conditions were 95ºC for 3 min followed by 35 cycles
of 95ºC for 15 secs, 67ºC for 30 secs and 72ºC for 30 secs; and one final extension at
72ºC for 10 min. Amplified DNA from lesion samples was ligated into pCR2.1-TOPO
Cloning® vector using the protocol of the manufacturer (Invitrogen, California,
USA). Ligation products were transformed into competent Top10 E.coli cells using
the methods of the manufacturer with recombinant transformations selected by blue
and white screening. For each library, clones were randomly selected and each
stabbed into a well of a 96-well microtitre plate containing LB-agar with ampicillin at
50 g ml-1. Plates were sent to Macrogen Inc. (Seoul, South Korea) for sequencing
using the M13 forward primer and a copy library stored in microtitre plates containing
ampicillin and glycerol at -80ºC.
Retrieved dsrA gene clone sequences were visualized and vector sequences
removed with the sequence analysis package Sequencher (Gene Codes Corporation,
MI, USA). Derived dsrA sequences from this study and reference dsrA sequences
were imported into the MEGA software package (Kumar et al., 2008), truncated to
the 221 bp amplified product size and aligned using the Clustal W application
(Thompson et al., 1994) followed by a manual correction of the alignment when
necessary. Phylogenetic trees were constructed using the neighbor-joining (JukesCantor correction) (Saitou and Nei, 1987) algorithms implemented in MEGA. The
short dsrA gene sequences (221 bp) representing individual clones or dominant OTU
groups from each lesion have been deposited in the GenBank database under
accession numbers HM444055-HM444065.
Real-time PCR assay for quantification of dsrA and 16SrRNA genes
Real-time PCR assays were carried out using a Rotor-Gene 3000 real-time
DNA amplification system (Corbett Research, Australia) and the EXPRESS qPCR
Supermix Universal Mix (Invitrogen, USA) in 20 ul final reaction volumes with
primers at a final concentration of 500 nM each and probes at 200 nM each. Cycling
parameters were 50°C for 2 mins, 95°C for 2 min, followed by 40 cycles of 95°C for
15 sec and 65°C for 60 sec.. At the end of the annealing/extension phase of each
thermal cycle, fluorescence was measured in the FAM and JOE channels. For each
sample a 10x dilution series, from 50 ng to 0.05 ng DNA was run in triplicate for each
dilution. PCR products from real-time PCR assays were confirmed to be the target of
correct size using standard agarose gel electrophoresis without the formation of nonspecific products indicating that curves generated were produced by dsrA or 16S
rRNA genes only and could therefore be used to determine dsrA and 16S rRNA gene
copy numbers using Rotorgene software version 6.1.71 (Corbett Research) as detailed
below.
Standard curves were generated for each gene by cloning the amplicon into
pCR2.1-TOPO (Invitrogen), linearising the resulting plasmid with Hind III, and
amplifying in triplicate a 10x serial dilution from 107 to 103 copies. 107 copies in
triplicate were amplified in each sample run. Standard curves for both 16S rRNA and
dsrA genes along with their calculated r2 values can be found in Supplementary
Figure 1. Negative controls which contained no template were also run in triplicate.
Data and copy numbers of dsrA or 16S rRNA gene targets in samples were analyzed
using the Rotor-Gene software version 6.1.71 (Corbett Research) following the
manufacturers guidelines.
Supplementary Figure 1: Standard curves delineating threshold (Ct) values of
fluorescence for indicators of (a) number of dsrA gene copies verses ng of total DNA
and (b) number of 16S rRNA gene copies verses ng of total DNA. Error bars indicate
standard deviation of the mean for three replicate qPCR reactions.
References:
Kondo R, Shigematsu K, Butani J (2008). Rapid enumeration of sulphate-reducing
bacteria from aquatic environments using real-time PCR. Plankton Benthos Res 3:
180-183.
Kumar S, Dudley J, Nei M, Tamura K (2008). MEGA: A biologist-centric software
for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9: 299-306.
Leloup J, Loy A, Knab NJ, Borowski C, Wagner M, Jørgensen BB (2007). Diversity
and abundance of sulfate-reducing microorganisms in the sulfate and methane zones
of a marine sediment, Black Sea. Environ Microbiol 9: 131-142.
Saitou N, Nei M (1987). The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406-425.
Thompson JD, Higgins DG, Gibson TJ (1994). CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence weighting,
position specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 46734680.