The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification

Abstract

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

Keywords: Cu-responsive regulator; gene expression; nitric oxide reductase; nitrite reductase.

Figure 1.

Regulatory network of denitrification genes in B. diazoefficiens in response to O₂ and NO. The regulatory protein FixK₂ is the direct activator of nap, nirK, and nos genes in response to low O₂ conditions (≤ 5% O₂). This regulator also activates nnrR, which encodes the transcription factor NnrR, necessary for induction of nor genes in response to NO. FixK₂ is also the link to the RegSR–NifA cascade responsible for activating the nif and fix genes in response to very low O2 conditions (≤ 0.5% O₂).

Figure 2.

Genomic context of the csoR gene in B. diazoefficiens 110spc4 (A), B. liaoningense CCNWSX0360 (B), and M. tuberculosis H37v (C). The nucleotide sequence of the regions marked with a dash square adjacent to csoR are also shown. In each sequence, the CsoR boxes are shaded in grey, with the inverted repeats underlined. In (B) and (C), the −10 and −35 regions of the csoR promoter are indicated with an open black box. (B) and (C) are adapted from Liang et al. (2016) and Liu et al. (2007), respectively.

Figure 3.

(A) Growth of B. diazoefficiens 110spc4 (black symbols) and ΔcsoR strain (open symbols) under Cu limitation (Cu-L, i.e. chelated) (circles), standard Cu (Cu-S, 0.02 M) (squares), or high Cu (Cu-H, 13 M) (triangles) BVMN media under oxic (dash line, 21% O₂) or microoxic conditions (solid line, 2% O₂). Data are means with standard error bars from at least two independent cultures assayed in triplicate, and where not visible, these were smaller than the symbols. (B) Intracellular Cu concentration of B. diazoefficiens 110spc4 and ΔcsoR mutant grown for 3 days under microoxic conditions in Cu-L, Cu-S, or Cu-H BVMN media. Data expressed as g Cu mg protein⁻¹, are means with standard deviation from at least two independent cultures assayed in triplicate.

Figure 4.

Transcriptional expression of denitrification genes monitored as β-galactosidase activity (histograms) from napE-lacZ (A), nosR-lacZ (B), nirK-lacZ (C), and norC-lacZ (D) transcriptional fusions chromosomally integrated in B. diazoefficiens 110spc4 (white bars) or ΔcsoR strain (grey bars) grown microaerobically in Cu-L, Cu-S, or Cu-H BVMN media for 3 days. Data expressed as MU are means with standard deviation bars from at least three independent cultures assayed in triplicate. Same lower- or upper-case letters in each figure indicate that values are not statistically different according to a post hoc Tukey HSD test at P ≤ .05; lower-case letters indicate comparisons between Cu conditions, while upper-case letters mean comparisons between strains. Below each histogram, qRT-PCR expression of each denitrification gene, napE (A), nosR (B), nirK (C), and norC (D), is shown. (E) qRT-PCR values of copA gene in Cu-L, Cu-S, or Cu-H BVMN media. Fold-change (FC) values refer to differences in expression in the ΔcsoR mutant relative to the WT. Data are means with standard deviation in parentheses from at least three independent cultures assayed in triplicate.

Figure 5.

Schematic representation of the possible events occurring inside the cell under Cu limitation in the WT (A) or ΔcsoR (B) grown under microoxic (2% O₂) conditions. In the WT grown under Cu limitation (A), CsoR remains attached to the csoR–copA divergon, preventing copA maximal transcription levels. The synthesized P-type ATPase CopA proteins would be inactive because of the low cytosolic Cu availability. In ΔcsoR grown under Cu limitation (B), CsoR is absent and copA expression would be derepressed making the cytosolic Cu concentration even more limiting (expressed in the figure as ↑ Cu-limitation). The results obtained in this work suggest that RegR could be involved in the control of nor genes under Cu limitation. Question marks indicate nondemonstrated events. Perpendicular lines indicate negative control.