Corynebacterium glutamicum ArnR controls expression of nitrate reductase operon narKGHJI and nitric oxide (NO)-detoxifying enzyme gene hmp in an NO-responsive manner

Abstract

Corynebacterium glutamicum ArnR is a novel transcriptional regulator that represses expression of the nitrate reductase operon narKGHJI and the nitric oxide (NO)-detoxifying flavohemoglobin gene hmp under aerobic conditions. In a previous study, we showed that ArnR-mediated repression is relieved during anaerobic nitrate respiration, but we could not pinpoint the specific signal that ArnR senses. In this study, we show that in the absence of nitrate, ArnR-mediated repression is maintained under anaerobic conditions. The derepression in response to nitrate is eliminated by disruption of narG, suggesting that ArnR senses nitrate derivatives generated during nitrate respiration. Specifically, the hmp gene is upregulated in the presence of nitrite or nitric oxide (NO) in an ArnR-dependent manner, although the response of narK appears to be greatly affected by ArnR-independent regulation. In vitro binding of ArnR to the narK and hmp promoter regions is more strongly inhibited by NO than by nitrite. We previously showed that the UV-visible spectrum of ArnR is typical of a Fe-S cluster-containing protein. Site-directed mutagenesis of each of three cysteine residues, which are possibly involved in coordination of the cofactor in the ArnR protein, results in loss of the binding of this protein to its target promoters in vitro and eliminates the repression of the target genes in vivo under aerobic conditions. These observations suggest that the cofactor coordinated by these three cysteine residues in the ArnR protein plays a critical role in the NO-responsive expression of the narKGHJI operon and the hmp gene.

FIG 1

Relative mRNA levels of the narK and hmp genes determined by quantitative RT-PCR. The mRNA levels of the genes from C. glutamicum wild-type (WT) and arnR-deficient (ΔarnR) strains grown in BT minimal medium under aerobic (white bars) and anaerobic conditions in the absence (gray bars) or presence (black bars) of nitrate are shown. The levels are presented relative to the value of the wild-type strain grown under aerobic conditions. The values represent the means from at least three independent experiments with standard deviations (error bars).

FIG 2

Transcriptional responses of the narK and hmp genes to nitrate, nitrite, and NOC-7 under anaerobic conditions. The C. glutamicum wild-type (filled circles), arnR-deficient (open squares), and narG-deficient (open triangles) strains were grown anaerobically to the exponential phase in BT minimal medium containing 1% glucose and 20 mM acetate and then supplemented with 30 mM sodium nitrate (A and B), 10 mM sodium nitrite (C and D), or 50 μM NOC-7 (E and F). The levels are presented relative to the value obtained for the wild-type cells immediately before each supplementation. The values represent the means from at least three independent experiments with standard deviations (error bars).

FIG 3

Effects of NO on aerobic and anaerobic growth of C. glutamicum strains. (A and B) The wild-type (filled symbols) and hmp-deficient (open symbols) strains were treated with NOC-7 at a concentration of 50 μM (triangles) or 200 μM (squares) under aerobic (A) and anaerobic nitrate respiration (B) conditions. Circles indicate untreated cells. NOC-7 was added to the aerobic cultures at early exponential phase (3 h) and to the anaerobic cultures at the start of cultivation (0 h). (C) The wild-type (filled circles), hmp-deficient (open triangles), and arnR-deficient (open squares) strains, which carry control vector pCRB12, and the hmp-deficient strain carrying plasmid pCRB633 for expression of the hmp gene (filled triangles) were all supplemented with 200 μM NOC-7 during aerobic growth. The wild type carrying pCRB12 (open circles) was untreated. Similar results were obtained from three independent experiments, and representative results are shown. OD₆₁₀, optical density at 610 nm.

FIG 4

Transcriptional responses of the narK and hmp genes to NOC-7 under aerobic conditions. The C. glutamicum wild-type (filled circles) and arnR-deficient (open squares) strains were aerobically grown in BT minimal medium containing glucose to early exponential phase (3 h) and then supplemented with 50 μM NOC-7. The levels are presented relative to the value obtained for the wild-type cells immediately before each supplementation. The values represent the means from at least three independent experiments with standard deviations (error bars).

FIG 5

Effect of NO and nitrite on DNA binding and the absorption spectrum of the ArnR protein. (A and B) Purified ArnR protein was treated with NOC-7 at a concentration of 1, 4, 20, or 100 μM or with sodium nitrite (NaNO₂) at 1, 2.5, 5, 10, or 20 mM for 15 min, prior to in vitro binding to the narK and hmp promoter regions (PnarK and Phmp, respectively). DNA probes (10 nM) were mixed with ArnR proteins (0 to 300 nM), and the mixtures were then subjected to electrophoresis. (C) The UV-visible absorption spectrum of the purified ArnR protein was recorded after treatment with NOC-7 at a concentration of 20 μM (green line) or 100 μM (blue line) or with sodium nitrite at a concentration of 4 mM (red line) or 20 mM (orange line) for 15 min. The black line shows the spectrum of untreated ArnR.

FIG 6

Effect of mutation of three cysteine residues on DNA binding and the absorption spectrum of the ArnR protein. (A) In vitro binding of the wild-type ArnR protein and its variants (C179A, C193A, and C223A) to the promoter regions of the narK and hmp genes (PnarK and Phmp, respectively). DNA probes (10 nM) were mixed with ArnR proteins (0 to 300 nM), and the mixtures were then subjected to electrophoresis. (B) The UV-visible absorption spectra of the purified wild-type ArnR protein and its variants (ArnRC179A, ArnRC193A, and ArnRC223A) were recorded. The black, red, green, and blue lines show the spectra of wild-type ArnR, ArnRC179A, ArnRC193A, and ArnRC223A, respectively.

FIG 7

Influence of expression of ArnR variants in the arnR-deficient strain on transcription of the narK and hmp genes. The arnR deletion mutant (ΔarnR) strain carrying a plasmid for expression of wild-type ArnR (ArnR) or ArnR variants (ArnRC179A, ArnRC193A, or ArnRC223A) was subjected to quantitative RT-PCR analysis. These strains were grown aerobically to the exponential phase in BT minimal medium containing glucose as a carbon source. The values represent the means from at least three independent experiments with standard deviations (error bars).