Environmental genomics reveals a functional chlorite dismutase in the nitrite-oxidizing bacterium ‘Candidatus Nitrospira defluvii’

Fig. S1. Illustration of the genome region of N. defluvii that was sequenced and analysed in this study. Arrows indicate the position and orientation of coding sequences (CDS). Colours indicate the assignment of CDS to TIGR role categories, which are listed in the bottom part of the figure. Black arrows indicate that the respective CDS shared sequence similarities only with ORFs of unknown function in other organisms, or contained only short conserved motifs or structural features. White arrows indicate that the respective CDS showed no similarity at all to any sequence in public databases. Scale bars and numbers indicate nucleotide positions.

Fig. S2. Alignment of selected chlorite dismutase (CLD)-like protein sequences based on conserved secondary structure motifs. Positions that are conserved in all analysed sequences of CLD-like proteins (n = 136) are shaded in grey. Vertical arrows point at the histidine residue that is likely to be the haem ligand in these enzymes. Each sequence is coloured according to PSIPRED (Jones, 1999) secondary structure prediction (red: α-helix, blue: β-strand). The coloured bars above the alignment show secondary structure motifs (red: α-helix, yellow: 310-helix, blue: β-strand) as detected in the crystal structure of the CLD-like protein of Thermus thermophilus (Ebihara et al., 2005). The last column in the last row (Id. %) shows amino acid sequence identities to the CLD of N. defluvii.

Fig. S3. Functional validation of the CLD of N. defluvii.
A. The red-coloured E. coli culture, which expressed the recombinant CLD of N. defluvii (left test tube), and the control E. coli culture, which did not contain this gene (right test tube), after cell lysis by sonication but prior to addition of NaClO₂.
B. The same test tubes as in (A) immediately after addition of 200 mM NaClO₂. Formation of gas (presumably O₂) is visible only in the left tube, which contained the E. coli culture expressing the CLD of N. defluvii.
C. The same test tubes as in (B) after subsequent addition of 200 mM AgNO₃. Ag+ forms a white precipitate with Cl^-, but a yellow precipitate with ClO₂^- (Holleman and Wiberg, 1985). The results shown in (B) and (C) clearly demonstrate that the CLD of N. defluvii has chlorite dismutase activity.

Fig. S4. Effects of pH (A) and temperature (B) on the specific activity of the CLD of N. defluvii. The pH optimum was determined in 10 mM sodium phosphate buffer solutions at 30°C. The temperature optimum was determined in 10 mM sodium phosphate buffer (pH 6.0). All experiments were performed with 65 mM chlorite. Error bars indicate the standard error of the mean (n = 3).

Table S1. List of all coding sequences identified on the analysed genome fragment of ‘Candidatus Nitrospira defluvii’ and the corresponding annotations.

Table S2. Sources of environmental sequences of CLD-like proteins that were used for protein phylogeny (see Fig. 5).

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