Nitrate-Responsive Suppression of Dimethyl Sulfoxide Respiration in a Facultative Anaerobic Haloarchaeon, Haloferax volcanii

Abstract

Haloferax volcanii is a facultative anaerobic haloarchaeon that can grow using nitrate or dimethyl sulfoxide (DMSO) as a respiratory substrate under anaerobic conditions. Comparative transcriptome analysis of denitrifying and aerobic cells of H. volcanii indicated extensive changes in gene expression involving the activation of denitrification, suppression of DMSO respiration, and conversion of the heme biosynthetic pathway under denitrifying conditions. The anaerobic growth of H. volcanii by DMSO respiration was inhibited at nitrate concentrations of <1 mM, whereas nitrate-responsive growth inhibition was not observed in the ΔnarO mutant. A reporter assay demonstrated that the transcription of the dms operon was suppressed by nitrate. In contrast, the anaerobic growth of the ΔdmsR mutant by denitrification was little affected by the addition of DMSO. NarO has been identified as an activator of denitrification-related genes in response to anaerobic conditions, and here, we found that NarO is also involved in nitrate-responsive suppression of the dms operon. Nitrate-responsive suppression of DMSO respiration is known in several bacteria such as Escherichia coli and photosynthetic Rhodobacter species. This is the first report to show that a regulatory mechanism that suppresses DMSO respiration in response to nitrate exists not only in bacteria but also in haloarchaea. IMPORTANCE Haloferax volcanii can grow anaerobically by denitrification (nitrate respiration) or DMSO respiration. In facultative anaerobic bacteria that can grow by both nitrate respiration and DMSO respiration, nitrate respiration is preferentially induced when both nitrate and DMSO are available as the respiratory substrates. The results of transcriptome analysis, growth phenotyping, and reporter assays indicated that DMSO respiration is suppressed in response to nitrate in H. volcanii. The haloarchaeon-specific regulator NarO, which activates denitrification under anaerobic conditions, is suggested to be involved in the nitrate-responsive suppression of DMSO respiration.

Keywords: DMSO reductase; DMSO respiration; DmsR; Haloferax volcanii; NarO; denitrification; haloarchaea; nitrate reductase; transcription regulator.

FIG 1

Relative expression levels of genes involved in denitrification and DMSO respiration in denitrifying H. volcanii cells. Gene arrangements of the denitrifying gene cluster (A), the regulator gene narO and the nitrate reductase operon narBCGHDJ (B), and the regulator gene dmsR and the DMSO reductase operon dmsABCD (C) are indicated on the top of each panel. Functional genes for denitrification and DMSO respiration are shown in pale gray, and the regulator genes are in dark gray. The putative recognition motif (CGAA-NNNN-TTCG) for the HTH-type regulator is marked by asterisks in panels A and B. TPM values (left) in denitrifying cells and logFC values (right) of the genes are indicated by white bars and solid bold lines in the panels, respectively.

FIG 2

Effect of nitrate on anaerobic growth of H. volcanii in DMSO medium. (A and B) H. volcanii strain H26 (A) and strain NO02 (B) were cultivated anaerobically in 50 mM DMSO-containing Hv medium in which KNO₃ was not supplemented (open circles) or supplemented at 0.1 mM (squares), 1 mM (diamonds), 10 mM (triangles), and 100 mM (inverted triangles) in a 96-well microplate as described in Materials and Methods. The two strains were also cultivated in medium without DMSO or nitrate (marked by closed circles). The OD₆₀₀ was measured using a microplate reader every 24 h after the start of cultivation. (C) The OD₆₀₀ values of strain H26 (black bars) and strain NO02 (white bars) after 5 days of cultivation under each condition. The mean values and the standard errors (SE) (error bars) were calculated (n = 8).

FIG 3

Effect of nitrate on the transcription activities of the dms operon and the nar operon of H. volcanii cultivated anaerobically in DMSO medium. H. volcanii strain NAP01 (black bars) and strain DMP1 (white bars) were cultivated anaerobically in Hv medium containing 50 mM DMSO and various concentrations of nitrate (0 to 100 mM) in a microplate. After 5 days of cultivation, archaeal cells of both strains were harvested, and the induced β-galactosidase (BgaH) activity in Miller units (m.u.) was measured to estimate the transcription of the nar and dms operons as described in Materials and Methods. The mean values and the SE were calculated from the results of three independent experiments.

FIG 4

Effect of DMSO on denitrifying growth of H. volcanii. (A and B) H. volcanii strain H26 (A) and strain DR1 (B) were cultivated anaerobically in 50 mM nitrate-containing Hv medium in which DMSO was not supplemented (open circles) or supplemented at 0.1 mM (squares), 1 mM (diamonds), 10 mM (triangles), and 100 mM (inverted triangles), as described in Materials and Methods. The two strains were also cultivated in medium without DMSO or nitrate (marked by closed circles). The OD₆₀₀ was measured using a microplate reader every 24 h after the start of cultivation. (C) The OD₆₀₀ values of strain H26 (black bars) and strain DR1 (white bars) after 5 days of cultivation under each condition. The mean values and the SE were calculated (n = 8).