1

Reviced manuscript (JB00833-15) 1

2

Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification 3

Is Controlled by the Transcription Regulator NarO 4

5

Tatsuya Hattori1, Hiromichi Shiba1, Ken-ichi Ashiki1, Takuma Araki2, Yoh-kow Nagashima1, 6

Katsuhiko Yoshimatsu3, and Taketomo Fujiwara1,2* 7

8 1Department of Science, Graduate School of Integrated Science and Technology, 9

2Department of Environment and Energy Systems, Graduate School of Science and Technology, 10 3Research Institute of Green Science and Technology, 11

Shizuoka University, Shizuoka 422-8529, JAPAN 12

13

Running title: Regulation of Haloarchaeal Denitrification 14

15

Keywords: Haloarchaea, Haloferax volcanii, denitrification, nitrate reductase, nitrite reductase, 16

transcription regulator, oxygen sensor, cysteine-rich motif, NarO 17

18

*Corresponding author. 19

Mailing address: Department of Science, Graduate School of Integrated Science and Technology, 20

Shizuoka University, 836 Oh-ya, Suruga-ku, Shizuoka 422-8529, JAPAN 21

Tel: +081-54-238-4776, 22

Fax: +81-54-238-0986, 23

E-mail address: fujiwara.taketomo@shizuoka.ac.jp 24

25

Abbreviations 26

Bat, bacterioopsin activator; BgaH, haloarchaeal β-galactosidase; DMSO, dimethylsulfoxide; HTH, 27

helix-turn-helix; MV, methylviologen; NirK, copper-containing nitrite reductase; NorB, nitric oxide 28

reductase; NosZ, nitrous oxide reductase; ONPG, o-nitrophenyl-β-D-galactopyranoside; PAGE, 29

JB Accepted Manuscript Posted Online 19 January 2016J. Bacteriol. doi:10.1128/JB.00833-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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polyacrylamide gel electrophoresis; S.E., standard error; TMAO, trimethylamine oxide 30

31

ABSTRACT 32

The extremely halophilic archaeon Haloferax volcanii grows anaerobically by denitrification. A putative 33

DNA-binding protein, NarO, is encoded upstream of the respiratory nitrate reductase gene of H. volcanii. 34

Disruption of the narO gene resulted in loss of denitrifying growth of H. volcanii, and expression of the 35

recombinant NarO recovered the denitrification capacity. A novel CXnCXCX7C motif, showing no 36

remarkable similarities with known sequences, was conserved in the N-terminus of the NarO 37

homologous proteins found in the haloarchaea. Restoration of the denitrifying growth was not achieved 38

by expression of any mutant NarO in which any one of the four conserved cysteines were individually 39

replaced by serine. A promoter assay experiment indicated that the narO gene was usually transcribed 40

regardless of whether it was cultivated in an aerobic or anaerobic condition. Transcription of the genes 41

encoding the denitrifying enzymes nitrate reductase and nitrite reductase was activated under an 42

anaerobic condition. A putative cis element was identified in the promoter sequence of haloarchaeal 43

denitrifying genes. These results demonstrated a significant effect of NarO, probably due to its oxygen 44

sensing function, on the transcriptional activation of haloarchaeal denitrifying genes. 45

46

IMPORTANCE 47

Haloferax volcanii is an extremely halophilic archaeon capable of anaerobic growth by denitrification. 48

The regulatory mechanism of denitrification has been well-understood in bacteria, but it still remains 49

unknown in the archaea. In this work we show that the HTH-type regulator NarO activates transcription 50

of the denitrifying genes of H. volcanii under anaerobic conditions. A novel cysteine-rich motif, which 51

is critical for transcriptional regulation, is present in the NarO. A putative cis element is also identified 52

in the promoter sequence of the haloarchaeal denitrifying genes. 53

54

INTRODUCTION 55

Many kinds of microorganisms are facultatively anaerobic, and they carry out energy 56

conversion by anaerobic respiration or fermentation under the condition of low oxygen tension. The 57

capability of denitrification, which is one of the anaerobic respirations utilizing nitrate as the terminal 58

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electron acceptor, is widely distributed among bacteria, archaea, and eukaryotic fungi (1). In archaea, 59

several species of halophilic euryarchaea and thermophilic crenarchaea have been reported to possess 60

this anaerobic capability, carrying out denitrification during the nitrogen cycle in hypersaline or high 61

temperature environments, respectively (2, 3, 4). 62

Recent progress in the molecular and enzymatic characterization of haloarchaeal 63

denitrification has clarified that, like its bacterial counterpart, successive reduction steps catalyzed by 64

the four redox enzymes, i.e., nitrate reductase, nitrite reductase, nitric oxide reductase, and probably 65

nitrous oxide reductase, occur (2, 3, 4). Respiratory nitrate reductase was purified and cloned from three 66

haloarchaea, and was shown to be a unique hybrid enzyme in combination with a molybdenum protein 67

possessing the respiratory cytochrome bc1 (5, 6, 7, 8, 9, 10) (Fig. 1). The copper-containing nitrite 68

reductase NirK has been isolated from Haloarcula marismortui (11). The norB and nosZ genes, which 69

encode nitric oxide reductase and nitrous oxide reductase, respectively, were identified in the H. 70

marismortui genome (12), although neither enzyme has been purified and characterized. 71

In contrast to the progress in the biochemistry of denitrification, the regulatory mechanism of 72

this anaerobic respiration has remained unknown in the haloarchaea. In Escherichia coli, a facultative 73

anaerobic bacterium capable of nitrate respiration, transcription of the nitrate reductase gene is 74

controlled by a global oxygen response regulator FNR, and the two-component nitrate/nitrite 75

sensor-transducer NarXL (13, 14). FNR, a DNA-binding protein that is a member of the CAP family, 76

harbors four conserved cysteines, which bind an oxygen-sensing iron-sulfur cluster and a 77

helix-turn-helix (HTH) DNA-binding motif in its N- and C-termini, respectively (15, 16). Another 78

oxygen-dependent transcription regulator has also been identified in root nodule bacteria: anaerobic 79

metabolism of these bacteria, including denitrification and nitrogen fixation, is activated via the 80

oxygen-sensing two-component FixLJ-dependent regulatory cascade (17, 18). However, homologous 81

proteins with FNR and FixJL are not present in the archaea, suggesting the presence of another 82

regulatory mechanism for denitrification in archaea. 83

In the halophilic archaea, oxygen- and light-dependent induction of bacteriorhodopsin by the 84

bacterioopsin activator (Bat) has been investigated (19). Bat involves both the PAS domain, a possible 85

redox-sensory motif, and the GAF domain, a light-responsive cGMP-binding motif in the sequence (20). 86

Stimulations by light and anaerobicity activate transcription of the bacterioopsin gene and functionally 87

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related genes via the Bat regulator. Halobacterium sp. NRC-1 is capable of anaerobic respiration 88

utilizing DMSO and/or TMAO as an electron acceptor. A novel DNA-binding protein, DmsR, is 89

encoded in the 5’-flank of the dmsEABCD genes that encodes DMSO/TMAO reductase, a terminal 90

enzyme of anaerobic respiration. DasSarma and his coworkers reported that the ΔdmsR mutant lacks the 91

ability of to grow anaerobically by DMSO/TMAO respiration (21, 22) (Fig. 1). DmsR contains a 92

HTH-type DNA-binding motif (Pfam HTH10) that is homologous with that of the Bat regulator in its 93

C-terminus. Based on a gene disruption and cDNA microarray experiment, they proposed that DmsR 94

itself is an oxygen sensor, and directly activates transcription of the dmsEABCD gene in the anaerobic 95

condition (21, 22). 96

Like H. marismortui, Haloferax volcanii is a facultative aerobic microorganism, and can grow 97

by denitrification. A DNA-binding protein, which is homologous with DmsR, is found to be present in 98

the 5’-flank of the putative nitrate reductase gene in H. volcanii. The gene product, named NarO, is 99

expected to be a cytoplasmic protein, and involves a conserved cysteine-rich sequence that shows no 100

notable similarity to known sequences having any assigned function in the N-terminus. 101

Here we investigated the function of NarO in denitrifying growth of a ΔnarO mutant of H. 102

volcanii. The ΔnarO mutant did not grow anaerobically in the presence of nitrate, while the denitrifying 103

growth was recovered by the expression of recombinant NarO. Site-specific mutagenesis of the 104

recombinant NarO demonstrated the significance of the conserved cysteine residues in NarO. A 105

promoter assay indicated that transcription of the nitrate reductase and nitrite reductase genes were 106

activated when the archaeal cells were incubated anaerobically in the absence of nitrate, and the result is 107

consistent with the observation that only the anaerobic condition is essential for induction of 108

nitrate-reducing activity. Transcription of the denitrifying genes was inactivated in the ΔnarO mutant. A 109

promoter assay also revealed that the narO gene was usually transcribed regardless of whether it was 110

cultivated in an aerobic or anaerobic condition. These results suggested that NarO was the transcription 111

regulator possessing an oxygen-sensing function and activated transcription of the denitrifying genes 112

under the anaerobic condition. 113

114

MATERIALS AND METHODS 115

Strains and growth conditions. H. volcanii strain H26 was kindly supplied by Dr. T. Allers (Inst. 116

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Genetics, Nottingham Univ., UK) (23). Growth medium contained 5.0 g·L-1 Bacto-yeast extract (Becton, 117

Dickinson and Company, Sparks, MD), 2.0 g·L-1 Trypton (Oxoid Ltd., Bashingstoke, Hampshire, U.K.), 118

2.0 g·L-1 KCl, 176.0 g·L-1 NaCl, 20.0 g·L-1 MgCl2·6H2O, and 0.1 g·L-1 CaCl2·2H2O, and was adjusted 119

to pH 7.4 before autoclaving. Solutions of chelated iron (100 mg·L-1 FeSO4·7H2O and 100 mg·L-1 120

ethylenediamine tetraacetate EDTA), and trace elements (100 mg·L-1 Na2MoO4·2H2O, 200 mg·L-1 121

MnCl2·6H2O, 2 mg·L-1 CoCl2·6H2O, 100 mg·L-1 ZnSO4·7H2O, and 100 mg·L-1 CuSO4·5H2O), which 122

were also prepared and autoclaved separately, were mixed with the medium at a volume of 1/1,000 each. 123

The resulting Hv medium was used for cultivation of the strain. Aerobic cultivation was performed in 124

the Hv medium at 37ºC in the dark with shaking (120 rpm) for aeration. Cultivation of the strain under 125

anaerobic conditions was carried out by using the medium supplemented with 50 mM KNO3 as a 126

terminal electron acceptor of denitrification. The cultivation vessel, which was 150 mL in volume and 127

contained 40 mL medium, was sealed with butyl rubber: then the gas phase in the vessel was exchanged 128

by gentle bubbling with O2:N2 (0.2:99.8 [vol/vol]) mixed gas (Shizuoka Sanso Co., Shizuoka, Japan) for 129

5 min using a sterile needle. Cultivation vessels were shaken at 80 rpm at 37°C in the dark. The gas 130

phase in the vessel was changed every 24 h. Growth was monitored by measuring the optical density at 131

600 nm (OD600) using a model MPS-2000 spectrophotometer (Shimadzu, Co., Kyoto, Japan) equipped 132

with a cell holder for analysis of the suspension. 133

134

Disruption of narO gene in H. volcanii. Strain H26, an orotate:phosphoribosyl transferase (PyrE2) 135

mutant of the H. volcanii strain DS2 (wild type), was used to disrupt the narO gene by the method of 136

double integration (23) as outlined in supplementary Fig. S1AB. Standard protocols used for DNA 137

handling in E. coli and H. volcanii followed Sambrook and Russell (24) and Dyall-Smith (25), 138

respectively. PCR amplification of a 670 bp DNA fragment upstream of narO (HVO_B0159) was 139

carried out by KOD-plus DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) using set of 140

oligonucleotide primers narOUf and narOUr against the H. volcanii genome as a template. The DNA 141

sequence of the PCR product was determined using a SequeCEQ 8000 Genetic Analysis System 142

(Beckman Coulter, Inc., Brea, CA). An 800 bp DNA fragment downstream of the narO gene was also 143

amplified by using primers narODf and narODr. Both fragments, narOU+narOD, were cloned into a 144

pTA131 plasmid (supplied by Dr. T. Allers) (23), which harbored a functional pyrE2, generating pΔnarO. 145

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Demethylation of the pΔnarO plasmid was carried out using E. coli strain SCS110 (Δdam) for the 146

efficient transformation of the halophilic archaea (26). Sequences of oligonucleotide primers used for 147

PCR amplification are shown in supplementary Table S1. 148

Transformation of the pΔnarO to H. volcanii H26 was carried out according to the previously 149

described protocol (25). A transformant was streaked on casamino acid (Hv-Ca) agar plates that 150

contained no uracil, and incubated at 37°C for about 2 wks (23). One of the integrants that appeared on 151

the agar plate, which had gained uracil prototrophy by pyrE2 integration, was chosen. Then, the pop-in 152

strain, designated NO01, was streaked again on Hv-Ca agar plates supplemented with 10 mg·L-1 uracil 153

and 50 mg·L-1 5’-fluoroorotate (5’-FOA) and incubated at 37°C. The colonies appearing on the plate, 154

which showed tolerance to 5’-FOA by their inability to convert this compound to the toxic analog 155

5-fluorouracil by a pyrE2 pop-out via homologous recombination, were confirmed by PCR 156

amplification using the primers narOUf and narODr as shown in supplementary Fig. S1C. The narO 157

deletion mutant derived from H. volcanii H26 thus obtained was designated NO02. The haloarchaeal 158

strains prepared and used in this study are summarized in Table 1. 159

160

Expression of H. volcanii NarO. An expression plasmid of H. volcanii NarO was constructed by 161

utilizing an H. volcanii-E. coli shuttle vector and promoter sequence of the haloarchaeal constitutive 162

gene encoding KatG catalase-peroxidase (27, 28). The pHKH6 plasmid, which harbors Haloarcula 163

marismortui katG (rrnAC1171) and its promoter sequence (77 bp in length) and the 3’-flanking 114 bp 164

DNA region, into which a (CAC)6 sequence as a His6-tag had been introduced into the 3’ end of katG 165

(28). Using the pHKH6 plasmid as a template, inverse PCR was carried out with oligonucleotide 166

primers HmKPr and HmKTf to remove the DNA region encoding KatG protein from the pHKH6. In 167

addition, a 640 bp DNA fragment encoding narO was amplified using primers narOf and narOr. The 168

amplified narO gene was inserted into the manipulated pHKH6 using a ligation kit (Ligation High ver.2, 169

Toyobo), generating pUCkGnarO. Finally, the pUCkGnarO insert was cut out using BamHI and XbaI, 170

and cloned into a pMLH32EV vector, generating pkGnarO as an expression plasmid for NarO. Here, 171

pMLH32EV is a shuttle vector derived from pMLH32 (kindly supplied by Dr. Dyall-Smith) by EcoRV 172

digestion and self-ligation to demolish the bgaH gene, which encoded haloarchaeal β-galactosidase from 173

Haloferax lucentense (JCM 9276T) and was originally present in the pMLH32 (27). 174

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Point mutagenesis at the positions of five cysteine residues (Cys17, Cys81, Cys83, Cys91, 175

Cys100) in the recombinant NarO was carried out by technical application of PCR. Five sets of 176

oligonucleotide primers, C17Sf and C17Sr, C81Sf and C81Sr, C83Sf and C83Sr, C91Sf and C91Sr, and 177

C100Sf and C100Sr, in which the corresponding Cys codon (TGC or TGT) was replaced with a Ser 178

codon (TCG), were amplified by using the pUCkGnarO plasmid separately as a template. After 179

treatment with DpnI to decompose the template DNA, the linear PCR products were cyclized by 180

homologous recombination between the 5’ and 3’-regions in E. coli JM109 cells. The inserts in the five 181

plasmids were cut out and cloned into pMLH32EV, generating pkGnarOC17S, pkGnarOC81S, 182

pkGnarOC83S, pkGnarOC91S, and pkGnarOC100S, respectively, as expression plasmids for the mutant 183

NarOs. 184

The six constructs, pkGnarO, pkGnarOC17S, pkGnarOC81S, pkGnarOC83S, pkGnarOC91S, 185

and pkGnarOC100S, were introduced into strain NO02. The transformants were selected by tolerance to 186

0.5 mg·L-1 novobiocin, a DNA gyrase inhibitor, on the Hv-agar medium. The colonies appearing on the 187

plate were obtained and designated NO04 (+pkGnarO), NO05 (+pkGnarOC17S), NO06 188

(+pkGnarOC81S), NO07 (+pkGnarOC83S), NO08 (+pkGnarOC91S), and NO09 (+pkGnarOC100S), 189

respectively. A transformation of NO02 with pMLH32EV was also carried out, and designated NO03. 190

191

Reporter plasmid for narO, narA, and nirK gene promoters. The bgaH gene was used for 192

construction of the reporter assay plasmid (27). The 2.11 kbp bgaH gene was amplified using a set of 193

oligonucleotide primers bgaHf and bgaHr. Genome DNA of H. lucentense was used for the template. 194

The 270 bp DNA fragment including the total region of the H. volcanii narO gene promoter was also 195

amplified using a set of oligonucleotide primers, pnarOf and pnarOr, against the H. volcanii genome 196

DNA as template. Both amplified fragments were cloned into the pMLH32EV vector, generating 197

pnarObgaH as a reporter plasmid for measuring the transcription activity of the narO promoter. The 198

pnarObgaH was introduced into strains H26 and NO02, yielding NOP01 and NOP02, respectively. 199

To analyze the transcription activity of the promoter of the putative nitrate reductase gene, a 200

reporter plasmid was prepared from the 5’-flanking region of the narA gene. The 270 bp DNA fragment 201

including the total region of narA gene promoter was amplified using the set of oligonucleotide primers 202

pnarAf and pnarAr. Reporter plasmid pnarAbgaH was constructed using a similar procedure for the 203

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preparation of pnarObgaH. The plasmid was introduced into strains H26 and NO02, yielding NAP01 204

and NAP02, respectively. 205

The 5’-flanking sequence with 249 nucleotides of the H. volcanii nitrite reductase NirK gene 206

(HVO_2141), including the putative promoter, was amplified by PCR using primers pnirKf and pnirKr. 207

The reporter plasmid pnirKbgaH was constructed by the same procedure. The strains H26 and NO02 208

were transformed by the plasmid, yielding NKP01 and NKP02, respectively. 209

Site-directed mutagenesis of the promoter sequence of the nirK gene was carried out 210

according to Kunkel’s method with a slight modification (29). pUCpnirK, which containes the putative 211

promoter sequence of the nirK gene in the pUC119 vector, was transformed to E. coli strain CJ236 (dut1, 212

ung1, thi-1, relA1/pCJ105 (F' camr)). The transformant was infected by an M13KO7 helper phage, then 213

incubated in the kanamycin-supplemented medium. Uracil-substituted single-stranded pUCpnirK was 214

collected from the supernatant of the medium by polyethylene glycol precipitation. After annealing with 215

a nirKPG2C primer, which was designed for a transversion mutation at the 2nd nucleotide (G to C) in the 216

inverted repeat sequence in the nirK promoter, a complementary strand of the single-stranded pUCpnirK 217

was synthesized by using T4 DNA polymerase (Takara). The chimeric double-stranded DNA thus 218

obtained was transformed to the E. coli strain JM109. The plasmid was isolated from the transformant, 219

and the intended substitution was confirmed by DNA sequencing. The mutant pUCpnirKG2C thus 220

obtained was used for preparation of plasmid pnirKG2CbgaH and transformant NKP03 (host strain: H. 221

volcanii H26) for a reporter assay experiment. Transversion mutations at the 3rd (A to T) and 4th (A to T) 222

nucleotides were also individually carried out by using nirKPA3T and nirKPA4T, respectively. Two mutant 223

plasmids, pnirKA3TbgaH and pnirKA4TbgaH, and the corresponding transformants, NKP04 and NKP05, 224

respectively, were also prepared and used for the experiment. 225

226

Assay of transcription activities of gene promoters. BgaH activity was measured by using 227

o-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate according to the previous report (27). 228

Precultured cells of NOP01, NOP02, NAP01, NAP02, NKP01, and NKP02 were prepared for 229

inoculation by aerobic cultivation in the Hv medium supplemented with 0.5 mg·L-1 novobiocin. After 230

inoculation of the precultured medium with a 10% volume of the cultivation medium, the strains were 231

cultivated in the low-oxygen tension described in Strains and growth conditions using the Hv-medium 232

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supplemented with 0.5 mg·L-1 novobiocin and with or without 50 mM KNO3. The medium (1 mL) was 233

sampled every 24 h using a sterile needle. After measuring the OD600 of the medium, the medium was 234

centrifuged at 22,000 × g for 5 min to separate the cell pellet and supernatant using a centrifugal 235

separator model 3700 (Kubota Co., Tokyo, Japan). The cell pellet obtained was suspended in 800 µl of 236

the assay solution containing 50 mM Tris-HCl buffer (pH 7.2), 2.5 M NaCl, 10 µM MnCl2, and 0.1% 237

β-mercaptoethanol. The cells were solubilized by adding 100 µl of 2% Triton X-100 to the suspension 238

and vortexing for 10 sec. The solution was transferred to a cuvette with a 1 cm light path, then the BgaH 239

reaction was started by adding 100 µl of 8 mg/ml ONPG solution. Increasing absorbance at 405 nm of 240

the solution was monitored using a spectrophotometer. BgaH activity (Miller units) was calculated 241

according to the formula (ΔA405 /OD600 /min) × 103. 242

243

Assay of nitrate and nitrite reducing activities. H. volcanii strains H26 and NO02 were cultivated 244

aerobically in the Hv medium. Anaerobic cultivation of the two strains was also carried out using Hv 245

medium supplemented by 50 mM KNO3 or 77 mM DMSO. Anaerobic incubation of the strains was also 246

performed in the medium without supplementation of any respiratory substrates. The harvested archaeal 247

cells were sonicated by supersonic oscillator VP-30S (Taitec Corp., Saitama, Japan). The suspension 248

thus treated was centrifuged at 12,000 × g for 10 min, and the enzymatic activity of the cell-free extract 249

supernatant was measured. Enzymatic activities of nitrate and nitrite reductions in the cell-free extract 250

were measured according to the methods of Yoshimatsu et al. (30) and Ichiki et al. (11), respectively, 251

with a slight modification. The protein concentration was measured by using a BCA protein assay kit 252

(Pierce, Rockford, IL) using bovine serum albumin as the standard. 253

Nitrate reductase activity was detected on polyacrylamide gel as follows: H. volcanii H26 and 254

NO02 (ΔnarO) were cultivated in the Hv medium under aerobic, anaerobic, or denitrifying conditions. 255

The cells were harvested from 6.0 ml medium by centrifugation after 4 d cultivation. Total proteins were 256

extracted by treating the cell pellet with a minimum volume of 10 mM Tris-HCl buffer (pH 8.0) 257

containing 1% (w/v) Triton X-100 (Sigma, St. Louis, MO), then were subjected to polyacrylamide gel 258

electrophoresis (PAGE) according to the method of Davis (31). After electrophoresis, the gel was 259

enclosed in a sealed glass container filled with 0.1 M Na-Pi buffer (pH 7.0) containing 1.0 M NaCl, and 260

0.2 mM methylviologen (MV). Sodium dithionite solution (final concentration: 2.3 mM) was injected 261

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into the container to reduce MV, then the gel was incubated about 10 min until infiltration of enough of 262

the reduced MV had turned the gel blue. The reaction was started by injection of NaNO3 to 10 mM. An 263

image of the colorless spot appearing on the gel due to oxidation of MV catalyzed by nitrate reductase 264

was recorded by a scanning apparatus. 265

266

Other methods. A primer extension experiment for determination of transcription start point of the nirK 267

gene is described in the supplementary materials. The immunological method for detection of the 268

recombinant NarO is also explained there. A homology search was carried out using the BLAST site 269

(http://blast.genome.jp/), and sequence alignment by the neighbor-joining method was performed using 270

ClustalW (http://clustalw.genome.ad.jp/). All chemicals used in the experiments were of the highest 271

grade commercially available. 272

273

RESULTS 274

Phenotype analysis and complementation of ΔnarO mutation. The putative nitrate reductase gene 275

cluster, with a unique structure combining the respiratory quinol oxidase genes (narABC) and 276

membrane-bound nitrate reductase genes (narGHDJ), is shown in Fig. 1. Homologous gene clusters 277

have been identified in the genome of nine haloarchaeal species (H. volcanii, Haloferax mediterranei, H. 278

marismortui, two strains of Haloarcula hispanica ATCC33960 and N601, Haloarcula sp. CBA1115, 279

Halorhabdus utahensis, Halogeometricum borinquense, Halomicrobium mukohataei, and Halorubrum 280

lacusprofundi). NarO, and its homologous proteins were identified in the 5’-flanking the nitrate 281

reductase gene of six species, H. volcanii, H. marismortui, two strains of H. hispanica, Haloarcula sp. 282

CBA1115, H. utahensis, and H. borinquense. Alignment of amino acid sequences of the seven NarOs is 283

shown in Fig. 2. NarOs are expected to be soluble proteins, with molecular weights of about 22,000, and 284

lacking a translocation signal sequence. The C-terminal amino acid sequence with 58 residues (174−201, 285

H. volcanii NarO numbering) showed significant similarity with the HTH-type DNA-binding motif of 286

the Bat regulator that controls the oxygen- and light-dependent activation of the bop gene and its related 287

genes in haloarchaea (17, 20). The four cysteines with the arrangement CXnCXCX7C (n = ~70), 288

corresponding to Cys17, Cys81, Cys83, and Cys91 (H. volcanii NarO numbering), were conserved. The 289

sequence of the N-terminal side of the NarO homologues showed no notable similarity to sequences 290

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with any assigned function involving that of bacterial FNR proteins, the most investigated 291

oxygen-sensing transcription regulator. 292

To elucidate the function of NarO, the narO gene was disrupted by a double integration 293

method using uracil auxotrophic H. volcanii strain H26 (ΔpyrE2) as shown in Supplementary Fig. S1. In 294

the aerobic condition, the narO mutant NO02 grew as well as the parental strain H26, as shown in Fig. 295

3A, B. When the strains of H. volcanii were incubated anaerobically in the absence of a respiratory 296

substrate, a gradual increase of the OD600 of the medium was observed (triangles in the figure). The 297

observation suggests that the H. volcanii might be able to grow slowly by microaerobic respiration as 298

discussed below. Unlike the parental strain, NO02 grew poorly when cultivated anaerobically in the 299

presence of nitrate (Fig. 3B). Notably, strain NO02 maintained the ability of DMSO respiration, 300

indicating that NarO participates only in regulation of denitrification in H. volcanii. 301

Complementation of the growth capability by denitrification was examined by expression of 302

recombinant NarO in NO02. The expression plasmid pkGnarO, was constructed from the shuttle vector 303

pMLH32EV and the promoter of the katG gene. The recombinant NarO was engineered to contain a 304

C-terminal His6-tag, with the aim of immunological detection and purification of the recombinant 305

protein. The pMLH32EV was derived from pMLH32, which is a low-copy vector whose copy number 306

in the H. volcanii cell is about six (27). Cultivation experiments of strain NO04, the transformant of 307

NO02 by pkGnarO, demonstrated that the anaerobic growth by denitrification was restored by 308

expression of the recombinant NarO, as shown in Fig. 3C. 309

310

Functional analysis of conserved cysteines in NarO. Point mutagenesis of the narO gene carried out 311

on pkGnarO was performed to replace each of the four conserved cysteines of the recombinant NarO 312

with serines. Plasmids for the expression of the mutant NarOs were inserted into NO02 to generate 313

strains NO05, NO06, NO07, and NO08, which harbor the expression plasmids for the Cys17-, Cys81-, 314

Cys83-, and Cys91-mutant NarOs, respectively, and were examined in comparison to NO04. Mutation of 315

the non-conserved cysteine (Cys100 in H. volcanii NarO) was also performed, generating strain NO09. 316

As shown in Fig. 4, under aerobic conditions, the five strains with Cys→Ser narO mutations grew as 317

well as strain NO04; however when cultivated under denitrifying conditions four of the five narO 318

mutants, except for the mutation of the unconserved 5th Cys, were unable to grow. The results 319

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demonstrated that all four conserved cysteines are essential for the function of NarO to regulate 320

denitrification. 321

322

Transcription activities of narO gene promoter. The narO gene is present in the 5’-flanking sequence 323

of the narA gene in the reverse direction, and the narO and narA genes are separated by a 264 bp DNA 324

sequence. The genetic function of transcriptional regulation of both the narO gene and the putative 325

nitrate reductase gene would involve the DNA region (Fig. 5A). Transcription activity was analyzed 326

using the bgaH as the reporter gene and with the forward and reverse directions of the 264 bp DNA 327

sequence as the narA and the narO promoter, respectively. 328

Strains NOP01 and NOP02 were derived from H26 and NO02, respectively, by transformation 329

of the pnarObgaH plasmid. Both strains were cultivated in several conditions, and the induced BgaH 330

activities were measured every 24 hours. Fig. 5B shows the BgaH activities at 72 hours after starting 331

cultivation. Here, aerobic cells are in the late-exponential phase, and denitrifying cells were in the 332

mid-exponential phase. Very slow growth was observed in strains NOP01 and NOP02 under anaerobic 333

incubation in the absence of nitrate. BgaH activities were observed in NOP02 under all cultivation 334

conditions (637–963 Miller units (m.u.)), and no significant differences were detected among the 335

activities according to Bonferroni's multiple comparison test. In contrast, enhancement of the BgaH 336

activity (1,863 ± 192 m.u.), which was about three times the activity in the NOP02 cells, was observed 337

in the denitrifying NOP01 cells. A similar result was obtained when using the microbial cells harvested 338

at 48 hours after starting cultivation (data not shown). 339

340

Transcription activities of narA gene promoter and expression of nitrate reductase. Strains NAP01 341

and NAP02 were derived from H26 and NO02, respectively, by transformation of the pnarAbgaH 342

plasmid for assay of the narA promoter activity. The narA promoter was highly activated in the 343

denitrifying NAP01 cells (868 ± 351 m.u.), as shown in Fig. 5C. Low BgaH activity (226 ± 46 m.u.) 344

was observed in the starved NAP01 cells that had been incubated anaerobically in the absence of nitrate. 345

No BgaH activity was detected in the aerobic cells of NAP01. The NAP02 cells showed very low BgaH 346

activities (2.6 ± 2.0 m.u.) in the denitrifying condition. 347

Expression of nitrate reductase in each cultivation condition was also examined. Nitrate 348

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reducing activities induced in the H26 and NO02 cells, which were cultivated anaerobically in the 349

presence or absence of nitrate for 72 hours, were visualized on polyacrylamide gel by activity staining. 350

Aerobic cells were also prepared and used as the experimental control. As shown in Fig. 6, a colorless 351

spot that corresponded to the nitrate-dependent oxidation of reduced MV appeared on the gel where the 352

total proteins solubilized from the denitrifying cells of H26 had been loaded. Relatively weak activity 353

also appeared in the H26 cells cultivated anaerobically in the absence of nitrate. No enzymatic activity 354

was detected in the aerobic cells of H26. Nitrate reductase was not induced in the NO02 cells even 355

under denitrifying growth conditions. Conditional expression of nitrate reductase was consistent with 356

the action of the narA promoter shown in Fig. 5C. 357

358

Transcription activities of nirK gene promoter. The 169-bp long DNA sequence that exists at the 359

5’-flanking region of the nirK gene (HVO_2141) should include elements to regulate transcription 360

activity. The nucleotide sequence of 300 bp in length containing the whole region of the putative nirK 361

promoter was used to construct the pnirKbgaH plasmid (Fig. 5A). The pnirKbgaH plasmid was 362

transformed to the strains H26 and NO02, then the transformants NKP01 and NKP02, respectively, were 363

prepared. As shown in Fig. 5D, BgaH activities were induced in the NKP01 strain when it was 364

cultivated anaerobically. Like the narA promoter in the H26 cell, transcriptional activity of the the nirK 365

gene promoter appeared in the anaerobic condition whether the nitrate was supplemented or not, but a 366

significant difference was found between the two activities: denitrifying cells and starved cells of 367

NKP01 were estimated to be 350 ± 32 m.u. and 237 ± 69 m.u., respectively. Very low BgaH activity, 3.3 368

± 2.0 m.u., was detected in the denitrifying cells of NKP02, while no activities were observed in the 369

aerobic cells. Similar to the conditional activation found in the narA gene promoter, these results 370

provide persuasive evidence that the narO gene plays a critical role in the activation of the nirK 371

promoter. 372

As indicated in supplementary Fig. S2, a primer extension experiment was performed to 373

determine the transcription start point of the nirK gene in Haloferax denitrificans JCM 8864 (=ATCC 374

35960). H. denitrificans is phylogenetically close to H. volcanii, and the nucleotide sequence of its nirK 375

promoter is very similar to that of H. volcanii (supplementary Fig. S2A). It demonstrates that 376

transcription of the H. denitrificans nirK gene was initiated at a C residue, 15 bp upstream of 377

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the translation start ATG codon and 25 bp downstream of a putative TATA box (supplementary Fig. 378

S2B). Due to the 92.7% identity of the nucleotide sequence, it is most likely that transcription of the H. 379

vocanii nirK gene starts at the corresponding C residue located 14 bp upstream of the translation start 380

codon and 25 bp downstream of the putative TATA box, as indicated in Fig. 7A and supplementary Fig. 381

S2. 382

Nucleotide sequences of the 5’-flanking region of the denitrifying genes, narA, nirK, norB, 383

and nosZ, of the haloarchaeal genome were aligned as shown in supplementary Figs. S3, S4, S5, and S6, 384

respectively. The inverted repeat sequence CGAA-X4-TTGC, which seems to be the recognition site of a 385

HTH-type DNA-binding protein, was identified in a large part of the promoter of the denitrifying genes. 386

As revealed in Fig. 7, a single mutation into the inverted repeat at the 2nd guanine caused a dramatic 387

decrease in the transcriptional activity of the nirK promoter, and only 9% of the activity was detected. 388

Substitutions of the 3rd and 4th adenines also decreased the transcription activity of the nirK promoter to 389

about half, retaining 59% and 53%, respectively. These results demonstrated that the inverted repeat 390

plays a significant role in transcriptional regulation of the denitrifying genes. 391

392

DISCUSSION 393

Of the thirty one species of haloarchaea for which the total genomic information is presently 394

available, six species include a putative nitrate reductase gene in combination with the gene encoding 395

the regulatory protein NarO in the 5’ flanking region the gene (Fig. 2). Interestingly, the regulatory gene 396

encoding the protein that was homologous to NarO, named DmsR, is also present in the 5’ flanking 397

region of the DMSO/TMAO reductase gene of ten haloarchaeal species (Halobacterium sp. NRC-1, H. 398

salinarum R-1, H. volcanii, H. mediterranei, H. marismortui, two strains of H. hispanica, Haloarcula sp. 399

CBA1115, Natronobacterium gregoryi, Natrinema sp. J7-2, and H. mukohataei) (Fig. 1). Although the 400

unique cysteine-rich motif is conserved in DmsR, the internal sequence between the 1st and 2nd cysteines 401

is about 20 residues shorter than that of NarO (21, 33). DmsR has been reported to have a significant 402

role in the conditional expression of DMSO/TMAO reductase in Halobacterium sp. NRC-1 (21, 22) and 403

in H. volcanii (33). 404

We performed gene disruption studies on H. volcanii NarO in combination with expression of 405

a recombinant and reporter assay experiment of denitrifying gene promoters. The narO deletion mutant 406

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of H. volcanii strain NO02 retained the ability to grow aerobically with a growth rate similar to that of 407

strain H26, whereas the mutant did not grow anaerobically by denitrification (Fig. 3). The result 408

demonstrated the critical function of NarO in induction of the denitrifying ability in H. volcanii. 409

Anaerobic growth of H. volcanii with supplementation of DMSO instead of nitrate was not affected by 410

disruption of the narO gene. In contrast, the dmsR deletion mutant of H. volcanii, which had been 411

already prepared in our laboratory, lost the ability of anaerobic DMSO/TMAO respiration but did grow 412

by denitrification, indicating that the functions of NarO and DmsR as the transcription regulator are not 413

redundant (33). 414

In this study, anaerobic cultivation of H. volcanii was carried out under mixed N2 gas 415

containing 0.2% O2. The ‘microaerobic’ condition is essential for reproducibility of the anaerobic 416

growth of H. volcanii as reported previously (33). Although the reason for the unstability of the growth 417

under a strict anaerobic condition is not clear, one possibility is that the energy generation by 418

microaerobic respiration might be significant for H. volcanii for smooth adaptation to the anaerobic 419

condition and induction of the denitrifying enzymes. Very slow growth of strain H26 in the anaerobic 420

condition without nitrate and that of strain NO02 (∆narO) in the denitrifying condition, were observed, 421

as shown in Fig. 3. The results might also correspond to a low energy yield by microaerobic respiration 422

of H. volcanii. 423

As shown in Fig. 5, transcription activity of the narO gene promoter was higher than those of 424

the genes of the catabolic enzymes, nitrate reductase and nitrite reductase, which are expressed in the 425

microbial cells in large amounts (11, 30). However, identification of the recombinant NarO expressed in 426

the H. volcanii cells has not been followed by immune blotting experiments using anti-His6 antibody 427

until now. Purification of the recombinant NarO protein by Ni2+-affinity resin also failed. It is probable 428

that the NarO molecule has a very short lifespan and is immediately decomposed by proteolysis in H. 429

volcanii cells. Another explanation is also possible, e.g., the mRNA of the narO gene is very unstable, 430

and therefore, the concentration of recombinant NarO molecules is lower than can be detected 431

immunologically. 432

As indicated in Fig. 5B, transcription of the narO gene was ordinarily activated at similar 433

transcription levels whether cultivated in an aerobic or anaerobic condition, except for the remarkable 434

increase of the activity observed in the denitrifying NOP01 cells. The results indicated that transcription 435

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of the narO gene is usually activated regardless of the anaerobicity of the growing condition, and is 436

enhanced by the NarO protein. In addition, the results shown in Fig. 5CD demonstrated that NarO is 437

critical for activation of transcription of the denitrifying genes under the anaerobic condition. In bacteria, 438

transcription of denitrifying genes is controlled by dual regulatory systems, one being the 439

oxygen/redox-sensing mechanism, and the other depending on nitrogen oxide species as the respiratory 440

substrate (1, 34). Expression of denitrifying enzymes in bacteria, therefore, hardly occurs when they are 441

cultivated anaerobically in the absence of nitrate (35, 36). The present result suggests that the H. 442

volcanii denitrifying genes are mainly controlled by a NarO-dependent regulatory system that responds 443

to an anaerobic cultivation condition. The results also revealed that NarO-dependent activation of 444

transcription of the denitrifying genes was enhanced under the presence of nitrate, while the genetic 445

mechanism is so far unknown. 446

The unique arrangement of the four conserved cysteines as CXnCXCX7C (n = ~70) is a 447

structural characteristic of NarO and its homologous proteins (Fig. 2). Site-directed mutation 448

experiments clearly demonstrated that all four cysteines in NarO were essential for induction of 449

denitrification, as shown in Fig. 4. In addition, transcription of the denitrifying genes required the narO 450

gene, and was activated in the anaerobic condition (Fig. 5CD). The results are most explicable by the 451

hypothesis that NarO participated in regulation of the oxygen and/or redox potential-dependent 452

transcription of the denitrifying genes. The NarO homologs do not comprise a PAS domain, which is 453

one of the oxygen/redox sensor motifs found in haloarchaeal Bat and other redox-dependent regulatory 454

proteins (17, 20). One possible scenario is that, like the bacterial FNR, metal centers bound to the side 455

chains of cysteines respond to oxygen molecules or low redox potential in the environment, causing 456

activation of NarO (15, 16). In this case, it should be noted that the arrangement of the four conserved 457

cysteines in NarO shows no similarity to that of the iron-sulfur binding domain of the bacterial FNR. It 458

is also plausible that formation of a disulfide bridge between the consensus cysteines under the presence 459

of oxygen or high redox potential inactivates NarO, as is likely for the E. coli OxyR, which controls 460

transcription of the genes encoding HPII catalase and other components for protection of the cell against 461

oxidative stress (37). Another hypothesis is that a heme molecule may be a functional center for oxygen 462

sensing. Heme lyase, which takes part in the biosynthesis of c-type cytochrome, comprises the 463

consensus Cys-Pro-Val sequence as a heme-binding motif (38). This Cys-Pro-Val sequence is highly 464

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conserved at the positions of two of four cysteines corresponding to Cys17 and Cys91 in H. volcanii 465

NarO (Fig. 2). 466

An FNR-like regulator protein was not identified by searching the haloarchaeal genomic 467

information, suggesting another regulatory mechanism for denitrification in the haloarchaea. 468

Interestingly, comparative searching of the putative DNA contact site in the haloarchaeal denitrification 469

gene promoters demonstrated that an inverted repeat, CGAA-X4-TTGC, which was expected to interact 470

with the HTH-type regulator in the dimeric state, was commonly present. The DNA sequence of the 471

nirK promoter reveals a typical arrangement of the putative transcriptional elements in the haloarchaeal 472

denitrifying gene promoter. As represented in Fig. 5A, Fig. 7A, and supplementary Fig. S4, 473

CGAAGATGTTCG is centered 39 bp upstream of the haloarchaeal TATA box (concensus: TTTWWW, 474

W = A or T) in the H. volcanii nirK promoter (39). The transcription start point of the H. volcanii nirK 475

gene was expected to be 25 bp downstream of the TATA box, based on the primer extinction analysis of 476

the nirK gene of H. denitrificans whose nirK promoter sequence was very similar (91% identical) to that 477

of H. volcanii (supplementary Fig. S2). A similar arrangement was also found in a large number of the 478

promoters of the haloarchaeal denitrifying genes nirK, norB, and nosZ (supplementary Figs. S4, S5, and 479

S6, respectively). Point mutagenesis at the 2nd nucleotide of the inverted repeat caused drastic a decrease 480

(only 9.1% remaining) in the transcriptional activity of the nirK promoter (Fig. 7). Additionally, 481

mutations at the 3rd and 4th nucleotides of the inverted repeat resulted in 41% and 47% decreases, 482

respectively, in the nirK promoter activity. These results supported our hypothesis that the genetic 483

function of this inverted repeat sequence is the transcriptional regulation of denitrification in 484

haloarchaea. 485

In contrast to the well-ordered structure of transcriptional elements in the nirK, norB, and 486

nosZ gene promoters, the arrangement of the inverted repeat and putative TATA box was not 487

comprehensive in the narA and narO promoters. The inverted repeat sequence was centered from 220 488

bp, 214 bp, and 191 bp upstream of the putative translation start position of the H. volcanii, H. 489

mukohataei, and H. utahensis narA genes, respectively (supplementary Fig. S3). A TATA box-like motif 490

could not be identified due to the A/T-rich nature of the putative promoter regions. The inverted repeat 491

was 135 bp upstream from the translation start position of the narA genes of four Haloarcula species, 492

but was not found to be present in the narA promoter of H. mediterranei and H. lacusprofundi. 493

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Mutagenetic analysis of the reporter assay experiment and identification of the transcription starting 494

point of the denitrfying genes is now underway. 495

Our present study suggests that NarO is a key regulator of denitrifying growth in H. volcanii. 496

The cysteine-rich motif was critical for transcriptional regulation under the anaerobic condition. The 497

inverted repeat CGAA-X4-TTCG was often present in the haloarchaeal denitrifying gene promoters and 498

was shown to play a significant role in the transcription regulation. The most important problem that 499

remains unsolved is whether NarO binds directly with the inverted repeat and activates transcription of 500

denitrifying genes. In addition, the 46 proteins homologous to H. vocanii NarO were identified in 21 501

species of the 31 haloarchaea for which the total genome sequence is now available. However, except 502

for the seven NarOs and 11 DmsRs, the regulatory targets of the remaining 29 homologs have remained 503

uncertain. Further investigations, especially purification of the recombinant NarO protein, identification 504

of the NarO binding site on the genomic DNA, and assignment of the functions of the NarO homologs, 505

are required for total understanding of the regulation of haloarchaeal anaerobic metabolisms. 506

507

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Acknowledgements 608

TH and HS contribute equally to this work. This work was supported by the research grants from the 609

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Noda Institute for Scientific Research, the Japan Space Forum (Exploratory Research for Space 610

Utilization), and the True Nano Research Program of Shizuoka University to TF. 611

612

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Table 1. Strains of H. volcanii used in this study 613

614

Strain Derivation or reference Genotype

H26 Allers et al. (23) ΔpyrE2

NO01 H26/pΔnarO pop-in ΔpyrE2 narO+::[ΔnarO pyrE2+]

NO02 NO01 pop-out ΔpyrE2ΔnarO

NO03 NO02, pMLH32EV transformed ΔpyrE2ΔnarO {novR}

NO04 NO02, pkGnarO transformed ΔpyrE2ΔnarO {pkatG::narO::tag(His6) + novR}

NO05 NO02, pkGnarOC17S transformed ΔpyrE2ΔnarO

{pkatG::narO(C17S)::tag(His6) + novR}

NO06 NO02, pkGnarOC81S transformed ΔpyrE2ΔnarO

{pkatG::narO(C81S)::tag(His6) + novR}

NO07 NO02, pkGnarOC83S transformed ΔpyrE2ΔnarO

{pkatG::narO(C83S)::tag(His6) + novR}

NO08 NO02, pkGnarOC91S transformed ΔpyrE2ΔnarO

{pkatG::narO(C91S)::tag(His6) + novR}

NO09 NO02, pkGnarOC100S transformed ΔpyrE2ΔnarO

{pkatG::narO(C100S)::tag(His6) + novR}

NOP01 H26, pnarObgaH transformed ΔpyrE2 {pnarO::bgaH + novR}

NOP02 NO02, pnarObgaH transformed ΔpyrE2ΔnarO {pnarO::bgaH + novR}

NAP01 H26, pnarAbgaH transformed ΔpyrE2{pnarA::bgaH + novR}

NAP02 NO02, pnarAbgaH transformed ΔpyrE2ΔnarO {pnarA::bgaH + novR}

NKP01 H26, pnirKbgaH transformed ΔpyrE2{pnirK::bgaH + novR}

NKP02 NO02, pnirKbgaH transformed ΔpyrE2ΔnarO {pnirK::bgaH + novR}

NKP03 H26, pnirKG2CbgaH transformed ΔpyrE2{pnirKG2C::bgaH + novR}

pnirKG2C: second G in inverted repeatwas

replaced by C

NKP04 H26, pnirKA3TbgaH transformed ΔpyrE2{pnirKA3T::bgaH + novR}

pnirKA3T: third A was replaced by T

NKP05 H26, pnirKA4TbgaH transformed ΔpyrE2{pnirKA4T::bgaH + novR}

pnirKA4T: fourth A was replaced by T

Plasmids integrated on the chromosome are indicated by brackets ([]), and episomal plasmids are 615

indicated by braces ({}); 616

617

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FIGURE LEGENDS 618

619

Figure 1. Gene structures of nitrate reductase and DMSO/TMAO reductase in H. volcanii. Physical 620

map of the nitrate reductase and the DMSO reductase gene locus in H. volcanii are shown in A and B, 621

respectively. The directions of the open reading frames (ORFs) are indicated by arrows. The narO 622

(HVO_B0159) and dmsR (HVO_B0361), which are the probable transcription regulators of the nitrate 623

reductase and DMSO reductase genes, respectively, are marked by black. In the nitrate reductase gene 624

locus (A), 7 of 11 ORFs, shown by grey, are assigned to narABCGHDJ (HVO_B0160-0166), and their 625

physiological roles in the nitrate reduction were estimated, while the functions of the other four ORFs 626

remain unknown (5, 8, 10). In B, the arrangement of the DMSO reductase gene, dmsEABCD 627

(HVO_B0362−0366), in H. volcanii is identical to that in Halobacterium sp. NRC-1. The five ORFs are 628

transcribed as a single mRNA in Halobacterium sp. NRC-1 (21). 629

630

Figure 2. Sequence alignment of NarO proteins. The deduced amino acid sequence of H. volcanii 631

NarO (product of HVO_B0159) was aligned with those of orthologous proteins from Halogeometricum 632

borinquense (Hbor_34990, 49.8% identity to H. volcanii NarO), Halorhabdus utahensis (Huta_0019, 633

40.4%), H. marismortui (rrnAC1193, 41.7%), Haloarcula hispanica (HAH_1793, 41.4%), and 634

Haloarcula sp. CBA1115 (SG26_16865, 41.3%). The NarO sequences from the two strains of H. 635

hispanica, ATCC33960 (HAH_1793) and N601 (HISP_09150), are identical. The four conserved 636

cysteines are indicated by white characters in the black box. Asterisks reveal the amino acid residues 637

conserved among the seven NarOs. Residues identical to those of the H. volcanii NarO are emphasized by 638

shading. The HTH-type DNA-binding domain is boxed. Residue numbers are shown in the right margin. 639

640

Figure 3. Complementation of denitrifying growth of ΔnarO mutant by recombinant NarO. Strains 641

H26, NO02 (ΔnarO), and NO04 (NO02/pkGnarO) were cultivated as described in Materials and Methods. 642

The increase in optical density of the medium was measured at 600 nm. The OD600 values of H26 643

(indicated by open symbols), NO02 (grey), and NO04 (black), which grew aerobically without nitrate 644

(circles), anaerobically without supplementation of any respiratory substrate (triangles), anaerobically 645

with nitrate (diamonds), and anaerobically with DMSO (squares), are shown. Experiments were 646

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performed independently three times. Error bars represent standard error (S.E.). When H26 and NO02 647

were cultivated by DMSO respiration, the S.E. values were very small, and therefore the error bars are 648

masked by the square simbols. 649

650

Figure 4. Functional analysis of conserved cysteines of the NarO. Strains H26, NO02 (ΔnarO), NO03 651

(NO02/pMLH32EV), NO04 (NO02/pkGnarO), NO05 (Cys17-mutant NarO was expressed in NO02), 652

NO06 (Cys81), NO07 (Cys83), NO08 (Cys91), and NO09 (Cys100) were cultivated under aerobic (white 653

bars), anaerobic with nitrate (grey), and anaerobic without nitrate (black) conditions. The OD600 of each 654

medium after eight days cultivation are indicated. Experiments were performed independently three 655

times. Error bars represent S.E. 656

657

Figure 5. Transcription activities of narO, narA, and nirK promoters. Transcription activities of the 658

narO, narA, and nirK gene promoters were analyzed by a reporter assay using the bgaH gene encoding 659

haloarchaeal β-galactosidase. A schematic representation of the promoter regions used for reporter 660

plasmid construction is indicated in A, where the conserved inverted repeat and putative haloarchaeal 661

TATA box are shown by grey boxes and white diamonds, respectively. Transcription activities of the 662

narO gene promoters were analyzed as shown in B. Strains NOP01 (H26/pnarObgaH) and NOP02 663

(NO02/pnarObgaH) were cultivated under aerobic (O2:+, nitrate:− and +), anaerobic (O2:−, nitrate:−), 664

or denitrifying (O2:+, nitrate:+) conditions for 4 d. Mean values of induced BgaH activities (Miller 665

units) for NOP01 and NOP02 are indicated as white and gray bars, respectively. Experiments were 666

performed independently at least three times. Error bars represent S.E. Different letters denote 667

significantly different means (P<0.05; one-way analysis of variance with Bonferroni`s test for multiple 668

comparisons). Analysis of the transcription activity of the narA and nirK gene promoters was also 669

carried out in the same manner. Results obtained using strains NAP01 (H26/pnarAbgaH, white bars) and 670

NAP02 (NO02/pnarAbgaH, gray bars) are indicated in C, whereas those obtained using strains NKP01 671

(H26/pnirKbgaH, white bars) and NKP02 (NO02/pnirKbgaH, gray bars) are shown in D. 672

673

Figure 6. Nitrate reductase activity. H26 and NO02 (ΔnarO) were cultivated under aerobic (O2:+, 674

nitrate:−), anaerobic (O2:−, nitrate:−), or denitrifying (O2:−, nitrate:+) conditions. Total proteins were 675

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extracted from the cells by treating them with detergent as described in Materials and Methods. After 676

electrophoresis on polyacrylamide gel, nitrate reducing activity on the gel was visualized as a colorless 677

spot that revealed the oxidation of reduced MV catalyzed by the induced nitrate reductase. 678

679

Figure 7. Mutation analysis of inverted repeat of nirK promoter region. The DNA region located 680

upstream of the nirK gene is shown in A. An inverted repeat (CGAA-X4-TTCG) is boxed, and a putative 681

haloarchaeal TATA box (consensus: TTTWWW) is bolded. The transcription start C-residue, indicated by 682

a shaded white character, was inferred from primer extension analysis of the H. denitrificans nirK (see 683

supplementary Fig. S2). The translation start codon of nirK is italicized. The DNA sequence is numbered 684

relative to the translation start point. In (B), transcription activity was examined using a reporter plasmid 685

in which the 2nd–4th nucleotides of the inverted repeat were individually substituted. Experiments were 686

performed independently three times. Error bars represent S.E. Cultivation of the four strains, NKP02, 687

NKP03, NKP04, and NKP05, and measurement of the induced BgaH activities are described in Materials 688

and Methods. 689

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Figure 1 694

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Figure 2 699

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Figure 3 703

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705 Figure 4 706

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Figure 5 710

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Figure 6 715

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Figure 7 719

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