Figure 1.
Growth and nitrate/nitrite reduction of S. oneidensis strains.
A. Growth of S. oneidensis wild type (WT) in a chemostat supplemented with 5 mM NaNO3 (WT/NO3−) or NaNO2 (WT/NO2−) upon inoculation. Oxygen levels (O2-WT/NO3− and O2-WT/NO2−) were set at 20% initially, reduced to 2% and 0% at 36 and 48 h after inoculation, respectively. Similar results were obtained from ΔnapA and ΔnrfA, which are unable to respire on nitrate and nitrite respectively (not shown for clarity). B. Nitrate/nitrite concentrations in the chemostat cultures shown in A. Nitrate or nitrite was added 3, 15, 27, 39 h after the inoculation during growth and assayed at 1, 3, 7, 12 h after each addition using IC. Experiments were repeated with ΔnapA or ΔnrfA for comparison. NO3−/WT and NO3−/ΔnapA represent the wild type and ΔnapA cultures in the presence of nitrate and NO2−/WT and NO2−/ΔnrfA represent the wild type and ΔnrfA cultures in the presence of nitrite. C. Growth of S. oneidensis in batch cultures under aerobic conditions. WT/NO3−, and WT/NO2− represent growth with nitrate and nitrite, respectively. Growth in the absence of either chemical (WT) is included for comparison. D. Nitrate/nitrite respiration. Shown are concentrations in the batch cultures shown in C. E. Nitrate respiration. Shown are nitrate concentrations in the batch cultures with ΔnapA, ΔnrfA or ΔcymA. F. Nitrite respiration. Shown are nitrite concentrations in the batch cultures with ΔnapA, ΔnrfA or ΔcymA. Experiments were performed at least three times independently. Error bars represent the standard deviation (SD) (n = 3–6). In the case of chemostat, error bars are omitted for clarity.
Figure 2.
Expression analysis of nrfA during aerobic growth of S. oneidensis.
A. A lacZ-based reporter analysis of the nap and nrfA promoters. Presented in columns is expression of nap and nrfA in the cells cultured aerobically in the absence of nitrite (/−) and in the presence of either nitrate (/NO3−) or nitrite (/NO2−). The napA mutant instead of the wild type was used here to keep nitrate unreduced. The nrfA mRNAs in the samples growing with NaNO2 were also analyzed by qRT-PCR and presented (dash line, abundance relative to 16 S rRNA). Error bars represent the standard deviation (SD) (n = 3). B. Western analysis of the cell samples used in A. Wild type cells grown in the absence or presence of nitrite at indicated time points were assayed, respectively. The complemented ΔnrfA (ΔnrfAc, carrying PnrfA-nrfA) exhibited over-production of NrfA and ΔnrfA was used as the negative control.
Table 1.
Sequence similarities between E. coli NarXLEc, NarQPEc and S. oneidensis proteins.
Figure 3.
NarQ-NarP two-component system in aerobic respiration of nitrate/nitrite. A.
Trans-phosphorylation of S. oneidensis NarP and SO1860 by NarQ51–585. The trans-phosphorylation assay was performed in the presence of [γ-33P]ATP with NarQ51–585 and 3 µg NarP, 3 µg SO1860, or without either NarP or SO1860. The resulting protein mixture was then resolved on an SDS-PAGE gel and the phosphorylated proteins were visualized by autoradiography. The numbers in the figure are the amount of NarQ51–585 protein used, in µg. Arrows indicate the position of phosphorylated NarQ51–585 or NarP protein. B. Nitrate/nitrite reduction in ΔnarP under aerobic conditions. Cells of tested strains grown aerobically in the presence of nitrate or nitrate were collected at the indicated times. Concentrations of nitrate and nitrite (8 h and after) remaining in cultures were measured. narPc represents ΔnarP complemented by a copy of narP on pHG101. The negative control (ΔnapA and ΔnrfA) and error bars (SD, n = 3) were omitted for clarity.
Figure 4.
Binding analysis of NarP to nap and nrfA promoters.
A. EMSA assay with carbamoyl phosphate (lanes 2–6) and NarQ51–585 (lanes 7–11) treated NarP. NarP# represents NarP carrying a D57N mutation. Binding assays with NarP# treated with NarQ51–585 and ATP are shown in lanes 12–13. All of the binding assays were performed with 2 ng of nrfA upstream fragments in the presence of 2 µg non-specific competitor DNA poly(dI·dC). Lanes 6 and 11 contain 10 µM of unlabeled nrfA upstream fragments as competitor DNA. The concentration of NarP or NarP# is indicated in the figure (µM). B. The EMSA assay was performed with 2 µM phosphorylated NarP and various amounts of 33P end-labeled nrfA (−50 to −200 relative to the translation start codon) and nap (−50 to −200) upstream fragments. Non-specific competitor DNA, 2 µg poly(dI·dC), was added in all lanes. C. Western blotting analysis. Upper panel, analysis of NrfA in ΔnarP. Cells grown in the presence of nitrite at the indicated time points were assayed. Lower panel, analysis of NarP. Cells grown in the presence of nitrate and/or nitrite at the indicated time points were assayed. In both panels, ΔnarPc represents ΔnarP containing pHG102-narP (ParcA-narP), in which narP is over-expressed. D. qRT-PCR analysis of the narQ-narP operon. The wild type cells grown with nitrate or nitrate aerobically were collected at the indicated time points and assayed. Abundance is given relative to 16S rRNA. Error bars represent the standard deviation (SD) (n = 3).
Figure 5.
Crp and Fnr in aerobic nitrate/nitrite respiration of S. oneidensis.
A. qRT-PCR analysis of crp and fnr. The wild type cells grown in the presence or absence of nitrate or nitrate aerobically were collected at the indicated time points and assayed. Expression level of each gene was presented under three conditions: –, no addition of nitrate or nitrite, NO3−, nitrate added, and NO2−, nitrite added. Abundance is given relative to 16 S rRNA. B. Western blotting analysis of Crp. Upper panel, the wild type cells cultured in the absence of nitrate or presence of nitrate at 4, 8, and 12 h were assayed. Lower panel, the wild type cells cultured in the absence of nitrite or presence of nitrite at 8, 12, and 16 h were assayed. Δcrp was used as the negative control. C. qRT-PCR analysis of nap, nrfA, and narP in cells grown with nitrate. The wild type, Δcrp, and Δfnr mutant strains were assayed at indicated time points. Abundance is given relative to 16 S rRNA. D. Nitrate/nitrite assay. Cells of tested strains grown aerobically in the presence of nitrate or nitrate were collected at the indicated times. Concentrations of nitrate and nitrite (8 h and after) remaining in cultures were measured. Δcrpc represented the mutant containing a copy of crp on the complementation plasmid. Solid and dash lines represent cells grown in the presence of nitrate and nitrite, respectively. The wild type and Δfnr were indistinguishable from each other and thus data for the wild type and Error bars (SD, n = 4) were omitted for clarity.
Figure 6.
Model for regulation of nitrate/nitrite respiration in S. oneidensis.
Shown is schematic diagram of regulation of nitrate/nitrite respiration. NarQ responds to both nitrate and nitrite and subsequently phosphorylates NarP, which activates nap, nrfA, and possibly more operons. Crp responds to cellular cAMP levels, whose fluctuation may be affected by an unknown oxygen-sensing protein and/or the redox states of quinol pools. The activated Crp proteins (labeled with a) bind to target genes to activate their transcription. The unconfirmed signal transduction pathways are shown in dash lines.
Table 2.
Strains and plasmids used in this study.