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 nMof 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 3min followed by 35cycles of95ºC for 15 secs, 67ºC for 30 secs and 72ºC for 30 secs; and one final extension at 72ºC for 10min.Amplified DNA from lesion samples wasligated into pCR2.1-TOPO Cloning® vector using the protocol of the manufacturer (Invitrogen, California, USA). Ligationproducts were transformed into competent Top10E.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 stabbedinto a well of a 96-well microtitre plate containing LB-agar with ampicillin at 50 gml-1.Plateswere sent to Macrogen Inc. (Seoul, South Korea) for sequencing using the M13 forward primer and a copy library stored in microtitre plates containing ampicillinand 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 (Jukes-Cantor correction) (Saitou and Nei, 1987)algorithms implemented in MEGA. The short dsrAgene 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 and16SrRNA 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 20ul final reaction volumes with primers at a final concentration of 500nM each and probes at 200nM each. Cycling parameters were 50°C for 2 mins, 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and65°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 50ng to 0.05ng 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 non-specific 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 rRNAgene 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 Res3: 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 Bioinform9: 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 Microbiol9: 131-142.

Saitou N, Nei M (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution4: 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 Res22: 4673-4680.