Supplemental Figures

Supplemental Figure 1

Supplemental Fig. 1. Effect of nickel concentration on A. macleodii hydrogenase activity. AltDEcells were inoculated in marine broth medium (control) or in the medium supplemented with 10, 50 or 100 M NiCl2. After 24 h growth the cells were collected and in vitro H2 evolution activity in crude extracts was determined.

Supplemental Figure 2

Supplemental Fig. 2. Effect of nickel and iron on AltDE growth. Cells were inoculated in marine broth medium (control) or in the medium supplemented with 100 M NiCl2 (Ni-only), 50 M Fe-EDTA (Fe-only) or both 100 M NiCl2 and 50 M Fe-EDTA (Ni+Fe). Samples were collected every three hours. The optical density (OD) at 600 nm was then determined for each sample.

Supplemental Figure 3

Supplemental Fig. 3. Determination of A. macleodii hydrogenase activity in AltDE cells grown in marine broth supplemented with different iron salts. AltDE cells were inoculated in marine broth containing 50 M ferric sulfate, ferric citrate, ferric ammonium citrate, or ferric nitrate. After 24 h growth the cells were collected and in vitro H2 evolution activity was determined in crude cellular extracts. AltDE cells inoculated in marine broth supplemented with 100 M NiCl2 were used as a positive control. FeAmCit, Ferric ammonium citrate.

Supplemental Figure 4

Supplemental Fig. 4. Analysis of transcription of hynS and hynL genes by RT-PCR. A. RT-PCR to detect co-transcription of hynS and hynL. Templates and specific primers used for RT-PCR or PCR reactions are indicated on the top and left of the panel, respectively. hynS, primers specific for hynS; hynShynL, primers specific for the junction region of hynS and hynL; hynS-RT, hynS-specific primers used in the RT-PCR reaction without reverse transcriptase; RNA, AltDE RNA samples used as templates for the RT-PCR reactions; NTC, No RNA templates used in the RT-PCR reaction; and DNA, AltDE DNA samples used as PCR templates. B. Titration of RNA concentrations for RT-PCR experiments. RT-PCR was performed with hynL-specific primers on RNA samples from Ni-treated (+Ni) or control (-Ni) AltDE cells. The RNA templates were diluted to a series of concentrations as indicated to determine optimal RNA concentrations for exponential amplification of hynL transcripts. “-“ represents a non-RT control. Based on this titration result, 40 ng RNA was used as templates for all RT-PCR reactions in the Panel C. C. Detection of the hynL mRNA levels in AltDE cells following treatment of nickel and iron. Total RNA was isolated from AltDE cells at 3h, 6h and 9 h after treatments of NiCl2, Fe-EDTA, both NiCl2 and Fe-EDTA, and neither NiCl2 nor Fe-EDTA (Control). Specific primers for the hynL gene were used in the RT-PCR reactions. The absence of DNA contamination in the samples was confirmed by performing reactions lacking reverse transcriptase (-RT). The quality of the RNA was determined by examining the 16S and 23S rRNA after electrophoresis on a denaturing formaldehyde ethidium bromide gel.

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