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emi412116-sup-0001-figs1.tiff3074KFig. S1. Photographs of Streptomyces griseoflavus Tu4000 and Streptomyces sp. HFI6–HFI9 soil isolate colonies on R2A medium plates. S. griseoflavus Tu4000 had the smooth and waxy appearance of a bld (bald) Streptomyces mutant, while strains HFI6–HFI9 formed fuzzy colonies consistent with the presence of aerial hyphae. The pigmentation of strains HFI6–HFI8 was light pink, and strain HFI9 was darker with a brown exudate secreted into the surrounding medium. HFI6–HFI9 strains had the strong scent of geosmin, while S. griseoflavus Tu4000 did not.
emi412116-sup-0002-figs2.tiff3074KFig. S2. Photomicrographs of Streptomyces griseoflavus Tu4000 and Streptomyces sp. HFI6–HFI9 soil isolate cultures on R2A medium plates. The same samples are photographed in Fig. S1. Only substrate mycelia are visible in the S. griseoflavus Tu4000 colony, while HFI6–HFI9 strains had plentiful aerial hyphae.
emi412116-sup-0003-figs3.tif23401KFig. S3. Molecular phylogenetic analysis of hhyL sequences by the maximum likelihood method. The diversity of the high-affinity group 5 [NiFe]-hydrogenase (hhyL) sequences of the strains (bold) we tested for H2 uptake (Table 1) are compared with hhyL sequences from the NCBI microbial genome database in this amino acid tree. The hhyL-containing Streptomyces sequences form two distinct clusters at a deep 99% bootstrap branch: Cluster 1 and Cluster 2. Isolates that have been tested for H2 uptake are marked to indicate whether (*) or not (†) high-affinity H2 uptake was observed. Culture collection strains investigated in this study were selected to broaden representation across the clusters and genera: Streptomyces griseoflavus Tu4000 (Cluster 1), Rhodococcus equi (Actinobacterium, Cluster 1) and Streptomyces cattleya (Cluster 2). Strains HFI6, HFI7, HFI8 and S. griseoflavus Tu4000 hhyL are closely related to Cluster 1 Streptomyces spp. soil isolates that take up H2 (summarized in Constant et al., 2011b), while strain HFI9 hhyL is more closely related to the R. equi hhyL. Cluster 2 S. cattleya hhyL is closely related to Streptomyces sp. AP1 hhyL, which also consumes H2 (Constant et al., 2011b). Other culture collection strains that have been tested for H2 uptake include Ralstonia eutropha H16 (Conrad et al., 1983a) and Mycobacterium smegmatis (King, 2003b). The evolutionary history was inferred by using the maximum likelihood method based on the Whelan and Goldman (2001) model. The hhyL gene from an archaeon Sulfolobus islandicus HVE10/4 was used as the outgroup. The bootstrap values are shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbour-join and BioNJ algorithms to a matrix of pairwise distances estimated using a WAG model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories [+G, parameter = 1.2590]). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 58 amino acid sequences. There were a total of 427 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).
emi412116-sup-0004-figs4.tiff3074KFig. S4. Microscopic observations of the development of Streptomyces sp. HFI8 show that strain HFI8 underwent the full lifecycle from spore to spore in less than 1.8 days, after which nearly all the viable cells existed as spores. Each panel shows a representative micrograph of the culture taken on a different day after the inoculation. Image A shows the substrate mycelium that grows after the germination of inoculated spores. By day 1.8 (B), septated aerial hyphae (punctuated tubular branches) and fully formed spores (round cells) are present. Mainly spores are present from day 2.9 to day 22 (C–H), and the same persists until day 44 (not shown).
emi412116-sup-0005-figs5.tif2871KFig. S5. Photograph of serum vials during the aerial biomass removal experiments (Table 2, Samples 1–6) illustrates the separation of the aerial hyphae and spores from the substrate mycelium: (A) vial containing an whole intact strain HFI8 culture (Table 2, column 5); (B) vial from which the aerial biomass had been isolated using glass beads leaving behind the remaining substrate mycelium (Table 2, column 7); and (C) vial containing the isolated aerial biomass on the surface of the glass beads (Table 2, column 6). In some samples, glass beads were rolled on the whole colony surface (A) and were left in the same vial with no biomass transfer (Table 2, Samples 7–12).
emi412116-sup-0006-figs6.eps10KFig. S6. Scatter plot of the initial H2 oxidation rate vs the reduction in H2 uptake by Streptomyces sp. HFI8 during the glass bead procedure (Tables 2, 5th and 8th columns). The larger the initial H2 oxidation rate, the larger percentage reduction by the glass beads (R2 = 0.93), regardless of culture age or the amount of glass beads used for transfer.
emi412116-sup-0007-figs7.eps13KFig. S7. Determination of H2 uptake kinetic parameters by the Lineweaver–Burk (LB) and Eadie–Hofstee (EH) methods for Streptomyces sp. HFI8. The H2 uptake rate (V) nmol h−1 and initial H2 concentration (S) in ppm are used to generate the LB plot as 1/V vs 1/S and EH as V vs V/S. Km was determined from the LB plot as the Km = −1/x-intercept (38 ppm) and Vmax as Vmax = 1/y-intercept (30 μmol min−1 g−1). Km was determined from the EH plot as Km = -slope (22 ppm). Km and Vmax values were reported for a given strain only if the LB and EH Km values methods agreed within 50%.
emi412116-sup-0007-tables1.docx149KTable S1. The top database matches for strains HFI6–HFI9 16S rRNA gene, and hhyL nucleotide sequences indicate that the strains are Streptomyces spp. containing hhyL sequences. The GenBank accession number is listed for each deposited sequence. The results of NCBI Megablast BLAST search are listed for each sequence, where queries were made for the 16S rRNA sequences against the 16S rRNA gene sequence database and for the hhyL sequences against the entire nucleotide sequence database. The top match for each BLAST search is listed along with the total score, E value and maximum identity of the match. Strains HFI6–HFI9 16S rRNA gene sequences were 100% identical to several different strains of Streptomyces spp. Strains HFI6–HFI9 hhyL sequences are highly similar to published cultured and uncultured hhyL sequences, of which some were submitted to public databases as hydB-like genes, although the hhyL terminology has been more recently adopted (Constant et al., 2011b).

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