Effect of Copper on Expression of Functional Genes and Proteins Associated with Bradyrhizobium diazoefficiens Denitrification

Abstract

Nitrous oxide (N₂O) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of N₂O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N₂, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N₂O emissions from agricultural soils.

Keywords: Cu-containing nitrite reductase; enzymatic activity; gene expression; nitric oxide reductase; nitrous oxide reductase; periplasmic nitrate reductase.

Figure 1

Growth of B. diazoefficiens 110spc4 in Cu limitation (Cu-L) (●), Cu standard (Cu-S) (■) and high Cu (Cu-H) (▲) BVMN media under oxic (A), anoxic (B) and microoxic (C) conditions. In (B,C), growth in the Cu-S BVM medium was also included (□). Error bars represent standard error between triplicates, and where not visible, these were smaller than the symbols.

Figure 2

Transcriptional expression of denitrification genes monitored as β-galactosidase activity from napE-lacZ (A), nirK-lacZ (B), norC-lacZ (C) and nosR-lacZ (D) fusions chromosomally integrated in B. diazoefficiens 110spc4 grown aerobically in Cu-S (white bars) and microaerobically in Cu-L (light grey bars), Cu-S (dark grey bars) and Cu-H (black bars) BVMN media for 3 days. A post-hoc Tukey HSD test at p ≤ 0.05 was applied in (A–D); same lower-case letters in each figure indicate that values are not statistically different. (E) Expression changes of napE, nirK, norC and nosR genes in B. diazoefficiens 110spc4 grown microaerobically in Cu-L compared with Cu-S measured by qRT-PCR. Data expressed as Miller Units (MU) and Fold Change (FC) are means with standard deviation from at least three independent cultures assayed in triplicate.

Figure 3

Nitrate reductase protein levels and activity. (A) Western-blotted sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) gels of soluble fraction (20 µg) proteins probed with anti-NapA antibodies from P. pantotrophus. Soluble fraction from a napA::Ω mutant strain was used as negative control in the experiments. Apparent mass of NapA (94 kDa) is shown in the left margin. Soluble fraction was isolated from 3-day incubation cultures. (B) Methyl viologen-dependent nitrate reductase (MV⁺-NR) activity in Cu-L (light grey bars), Cu-S (dark grey bars) and Cu-H (black bars) conditions. Data are means with standard error bars from at least two independent cultures assayed in triplicate. (C) Extracellular nitrate concentration in the Cu-L (●), Cu-S (■) and Cu-H (▲) growth media. Error bars represent standard error between triplicates, and where not visible, these were smaller than the symbols. Cells were grown under microoxic conditions in BVMN medium with different Cu concentrations.

Figure 4

Nitrite reductase protein levels and activity. (A) Western-blotted SDS-PAGE gels of periplasmic (21 µg) and cytosolic (12 µg) proteins probed with anti-NirK antibodies from B. diazoefficiens 110spc4. Soluble fraction from a nirK::Ω mutant strain was used as negative control in the experiments. Apparent mass of NirK (37 kDa) is shown in the left margin. Periplasmic and cytosolic fractions were isolated from 3-day incubation cultures. (B) Methyl viologen-dependent nitrite reductase (MV⁺-NIR) activity under Cu-L (light grey bars), Cu-S (dark grey bars) and Cu-H (black bars) conditions. Data are means with standard error bars from at least two independent cultures assayed in triplicate. (C) Extracellular nitrite (NO₂⁻) concentration in the Cu-L (●), Cu-S (■) and Cu-H (▲) growth media. Error bars represent standard error between triplicates, and where not visible, these were smaller than the symbols. Cells were grown in BVMN medium with different Cu concentrations under microoxic conditions.

Figure 5

Nitric oxide reductase expression and activity. (A) Heme-stained proteins (30 µg) of membranes prepared from B. diazoefficiens 110spc4. CycM and NorC cytochromes identified previously are specified in the right margin. Apparent masses of the proteins (kDa) are shown in the left margin. (B) Nitric oxide reductase activity of B. diazoefficiens 110spc4 (WT). The norC::aphII-PSP (norC) mutant strain cultured in Cu-S medium was used as negative control in the experiments. Data represent means with standard error bars from at least two independent cultures assayed in triplicate. Cells were grown microaerobically in Cu-L, Cu-S or Cu-H BVMN medium for 3 days.

Figure 6

Nitrous oxide reductase expression and activity. (A) Western-blotted SDS-PAGE gels of periplasmic (left, 21 µg) and cytosolic (right, 14 µg) proteins probed with anti-NosZ antibodies from P. denitrificans. Apparent masses of NosZ (67 kDa) and truncated NosZ (50 kDa) are shown in the left margin. Periplasmic and cytosolic fractions were isolated from 3-day incubation cultures. (B) N₂O consumption capacity of cells grown for 3 days under Cu-L (light grey bar), Cu-S (dark grey bar) and Cu-H (black bar) conditions. Soluble proteins (A) and cells (B) from a nosZ::Ω mutant cultured microaerobically in Cu-S medium were used as negative controls in the experiments. Data represent means with standard error bars from at least two independent cultures assayed in triplicate. In B, a post-hoc Tukey HSD test at p ≤ 0.05 was applied; same lower-case letters indicate that values are not statistically different. (C) Nitrous oxide (N₂O) accumulation in the headspace of Cu-L (●), Cu-S (■) and Cu-H (▲) growth medium. Error bars represent standard error between triplicates, and where not visible, these were smaller than the symbols. Cells were grown aerobically or microaerobically in BVMN medium with different Cu concentrations.

Figure 7

(A) β-galactosidase activity from the nosR-lacZ transcriptional fusion chromosomally integrated in B. diazoefficiens 110spc4 (white bars) and nosR mutant backgrounds (dark grey bars) grown aerobically in Cu-S or microaerobically in Cu-L, Cu-S and Cu-H BVMN media for 3 days. Data expressed as Miller Units (MU) are means with standard error bars from at least three independent cultures assayed in triplicate. (B) Nitrous oxide reductase activity of B. diazoefficiens 110spc4 (WT) and ΔnosR strains incubated in Cu-L and Cu-S BVMN media under microoxic conditions. The nosZ::Ω mutant strain cultured in the Cu-S medium was used as negative control in the experiments. N₂O was measured in the headspace of the cultures after 3 days of incubation. Data represent means with standard error bars from at least two independent cultures assayed in triplicate.