Anaerobic Growth of Haloarchaeon Haloferax volcanii byDenitrification Is Controlled by the Transcription Regulator NarO

Tatsuya Hattori,a Hiromichi Shiba,a Ken-ichi Ashiki,a Takuma Araki,b Yoh-kow Nagashima,a Katsuhiko Yoshimatsu,c

Taketomo Fujiwaraa,b

Department of Science, Graduate School of Integrated Science and Technology,a Department of Environment and Energy Systems, Graduate School of Science andTechnology,b and Research Institute of Green Science and Technology,c Shizuoka University, Shizuoka, Japan

ABSTRACT

The extremely halophilic archaeon Haloferax volcanii grows anaerobically by denitrification. A putative DNA-binding protein,NarO, is encoded upstream of the respiratory nitrate reductase gene of H. volcanii. Disruption of the narO gene resulted in a lossof denitrifying growth of H. volcanii, and the expression of the recombinant NarO recovered the denitrification capacity. Anovel CXnCXCX7C motif showing no remarkable similarities with known sequences was conserved in the N terminus of theNarO homologous proteins found in the haloarchaea. Restoration of the denitrifying growth was not achieved by expression ofany mutant NarO in which any one of the four conserved cysteines was individually replaced by serine. A promoter assay experi-ment indicated that the narO gene was usually transcribed, regardless of whether it was cultivated under aerobic or anaerobicconditions. Transcription of the genes encoding the denitrifying enzymes nitrate reductase and nitrite reductase was activatedunder anaerobic conditions. A putative cis element was identified in the promoter sequence of haloarchaeal denitrifying genes.These results demonstrated a significant effect of NarO, probably due to its oxygen-sensing function, on the transcriptional acti-vation of haloarchaeal denitrifying genes.

IMPORTANCE

H. volcanii is an extremely halophilic archaeon capable of anaerobic growth by denitrification. The regulatory mechanism ofdenitrification has been well understood in bacteria but remains unknown in archaea. In this work, we show that the helix-turn-helix (HTH)-type regulator NarO activates transcription of the denitrifying genes of H. volcanii under anaerobic conditions. Anovel cysteine-rich motif, which is critical for transcriptional regulation, is present in NarO. A putative cis element was alsoidentified in the promoter sequence of the haloarchaeal denitrifying genes.

Many kinds of microorganisms are facultatively anaerobic,and they carry out energy conversion by anaerobic respira-

tion or fermentation under low-oxygen-tension conditions.Denitrifying capability, which is one of the anaerobic respirationsutilizing nitrate as the terminal electron acceptor, is widely distrib-uted among bacteria, archaea, and eukaryotic fungi (1). In ar-chaea, several species of halophilic euryarchaea and thermophiliccrenarchaea have been reported to possess this anaerobic capabil-ity, carrying out denitrification during the nitrogen cycle in hy-persaline or high-temperature environments, respectively (2–4).

Recent progress in the molecular and enzymatic characteriza-tion of haloarchaeal denitrification has clarified that, as with itsbacterial counterpart, successive reduction steps catalyzed by thefour redox enzymes, i.e., nitrate reductase, nitrite reductase, nitricoxide reductase, and probably nitrous oxide reductase, occur (2–4). Respiratory nitrate reductase was purified and cloned fromthree haloarchaea and was shown to be a unique hybrid enzyme incombination with a molybdenum protein possessing the respira-tory cytochrome bc1 (5–10) (Fig. 1). The copper-containing ni-trite reductase NirK has been isolated from Haloarcula marismor-tui (11). The norB and nosZ genes, which encode nitric oxidereductase and nitrous oxide reductase, respectively, were identi-fied in the H. marismortui genome (12), although neither enzymehas been purified and characterized.

In contrast to the progress in knowledge of the biochemistry ofdenitrification, the regulatory mechanism of this anaerobic respi-ration has remained unknown in haloarchaea. In Escherichia coli,

a facultative anaerobic bacterium capable of nitrate respiration,transcription of the nitrate reductase gene is controlled by a globaloxygen response regulator, FNR, and the two-component nitrate/nitrite sensor-transducer NarXL (13, 14). FNR, a DNA-bindingprotein that is a member of the cyclase-associated protein (CAP)family, harbors four conserved cysteines, which bind an oxygen-sensing iron-sulfur cluster and a helix-turn-helix (HTH) DNA-binding motif in its N and C termini, respectively (15, 16). An-other oxygen-dependent transcription regulator has also beenidentified in root nodule bacteria: anaerobic metabolisms of thesebacteria, including denitrification and nitrogen fixation, are acti-vated via the oxygen-sensing two-component FixLJ-dependent

Received 9 October 2015 Accepted 14 January 2016

Accepted manuscript posted online 19 January 2016

Citation Hattori T, Shiba H, Ashiki K-I, Araki T, Nagashima Y-K, Yoshimatsu K,Fujiwara T. 2016. Anaerobic growth of haloarchaeon Haloferax volcanii bydenitrification is controlled by the transcription regulator NarO. J Bacteriol198:1077–1086. doi:10.1128/JB.00833-15.

Editor: W. W. Metcalf

Address correspondence to Taketomo Fujiwara,fujiwara.taketomo@shizuoka.ac.jp.

T.H. and H.S. contributed equally to this work.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00833-15.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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regulatory cascade (17, 18). However, homologous proteins withFNR and FixJL are not present in archaea, suggesting the presenceof another regulatory mechanism for denitrification in archaea.

In halophilic archaea, oxygen- and light-dependent inductionof bacteriorhodopsin by the bacterioopsin activator (Bat) hasbeen investigated (19). Bat involves both the PAS domain, a pos-sible redox sensory motif, and the GAF domain, a light-responsivecyclic GMP (cGMP)-binding motif in the sequence (20). Stimu-lation by light and anaerobicity activate transcription of the bac-terioopsin gene and functionally related genes via the Bat regula-tor. Halobacterium sp. strain NRC-1 is capable of anaerobicrespiration by utilizing dimethyl sulfoxide (DMSO) and/ortrimethylamine oxide (TMAO) as an electron acceptor. A novelDNA-binding protein, DmsR, is encoded in the 5= flank of thedmsEABCD genes that encode DMSO/TMAO reductase, a ter-minal enzyme of anaerobic respiration. Müller and DasSarma(21) and DasSarma et al. (22) reported that the �dmsR mutantlacks the ability to grow anaerobically by DMSO/TMAO respira-tion (Fig. 1). DmsR contains an HTH-type DNA-binding motif(Pfam HTH10) that is homologous with that of the Bat regulatorin its C terminus. Based on a gene disruption and cDNA microar-ray experiment, they proposed that DmsR itself is an oxygen sen-sor and directly activates transcription of the dmsEABCD genesunder anaerobic conditions (21, 22).

Like H. marismortui, Haloferax volcanii is a facultative aerobicmicroorganism and can grow by denitrification. A DNA-bindingprotein, which is homologous with DmsR, is found to be presentin the 5= flank of the putative nitrate reductase gene in H. volcanii.The gene product, named NarO, is expected to be a cytoplasmicprotein and involves a conserved cysteine-rich sequence thatshows no notable similarity to known sequences having any as-signed function in the N terminus.

Here, we investigated the function of NarO in the denitrifyinggrowth of a �narO mutant of H. volcanii. The �narO mutant didnot grow anaerobically in the presence of nitrate, while denitrify-ing growth was recovered by the expression of recombinant NarO.

Site-specific mutagenesis of the recombinant NarO demonstratedthe significance of the conserved cysteine residues in NarO. Apromoter assay indicated that transcription of the nitrate reduc-tase and nitrite reductase genes was activated when the archaealcells were incubated anaerobically in the absence of nitrate, andthe result is consistent with the observation that only anaerobicconditions are essential for inducing nitrate-reducing activity.Transcription of the denitrifying genes was inactivated in the�narO mutant. A promoter assay also revealed that the narO genewas usually transcribed, regardless of whether it was cultivatedunder aerobic or anaerobic conditions. These results suggestedthat NarO is the transcription regulator possessing an oxygen-sensing function and that activated transcription of the denitrify-ing genes under anaerobic conditions.

MATERIALS AND METHODSStrains and growth conditions. H. volcanii strain H26 was kindly sup-plied by T. Allers (Institute of Genetics, Nottingham University, UnitedKingdom) (23). The growth medium contained 5.0 g liter�1 Bacto yeastextract (Becton, Dickinson and Company, Sparks, MD), 2.0 g liter�1 tryp-tone (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom), 2.0 gliter�1 KCl, 176.0 g liter�1 NaCl, 20.0 g liter�1 MgCl2·6H2O, and 0.1 gliter�1 CaCl2·2H2O and was adjusted to pH 7.4 before autoclaving. Solu-tions of chelated iron (100 mg liter�1 FeSO4·7H2O and 100 mg liter�1

EDTA) and trace elements (100 mg liter�1 Na2MoO4·2H2O, 200 mgliter�1 MnCl2·6H2O, 2 mg liter�1 CoCl2·6H2O, 100 mg liter�1

ZnSO4·7H2O, and 100 mg liter�1 CuSO4·5H2O), which were also pre-pared and autoclaved separately, were mixed with the medium at a vol-ume of 1/1,000 each. The resulting Hv medium was used for cultivation ofthe strain. Aerobic cultivation was performed in the Hv medium at 37°Cin the dark with shaking (120 rpm) for aeration. Cultivation of the strainunder anaerobic conditions was carried out by using the medium supple-mented with 50 mM KNO3 as a terminal electron acceptor of denitrifica-tion. The cultivation vessel, which was 150 ml in volume and contained 40ml of medium, was sealed with butyl rubber; the gas phase in the vessel wasthen exchanged by gentle bubbling with O2-N2 (0.2:99.8 [vol/vol]) mixedgas (Shizuoka Sanso Co., Shizuoka, Japan) for 5 min using a sterile needle.The cultivation vessels were shaken at 80 rpm at 37°C in the dark. The gasphase in the vessel was changed every 24 h. Growth was monitored bymeasuring the optical density at 600 nm (OD600) using a model MPS-2000 spectrophotometer (Shimadzu Co., Kyoto, Japan) equipped with acell holder for analysis of the suspension.

Disruption of narO gene in H. volcanii. Strain H26, an orotate phos-phoribosyl transferase (PyrE2) mutant of the H. volcanii strain DS2 (wildtype), was used to disrupt the narO gene by double integration (23), asoutlined in Fig. S1A and B in the supplemental material. Standard proto-cols used for DNA handling in E. coli and H. volcanii were according toSambrook and Russell (24) and Dyall-Smith (25), respectively. PCR am-plification of a 670-bp DNA fragment upstream of narO (HVO_B0159)was carried out using KOD-plus DNA polymerase (Toyobo Co., Ltd.,Osaka, Japan) with a set of oligonucleotide primers, narOUf and narOUr,against the H. volcanii genome as a template. The DNA sequence of thePCR product was determined using a CEQ 8000 genetic analysis system(Beckman Coulter, Inc., Brea, CA). An 800-bp DNA fragment down-stream of the narO gene was also amplified using the primers narODf andnarODr. Both fragments, narOU and narOD, were cloned into a pTA131plasmid (supplied by T. Allers) (23), which harbored a functional pyrE2gene, generating p�narO. Demethylation of the p�narO plasmid wascarried out using E. coli strain SCS110 (�dam) for the efficient transfor-mation of halophilic archaea (26). The sequences of oligonucleotideprimers used for PCR amplification are shown in Table S1 in the supple-mental material.

Transformation of the p�narO to H. volcanii H26 was carried outaccording to a previously described protocol (25). A transformant was

FIG 1 Gene structures of nitrate reductase and DMSO/TMAO reductase in H.volcanii. A physical map of the nitrate reductase (A) and the DMSO reductasegene (B) loci in H. volcanii is shown. The directions of the open reading frames(ORFs) are indicated by arrows. The narO (HVO_B0159) and dmsR(HVO_B0361) genes, which are the probable transcription regulators of thenitrate reductase and DMSO reductase genes, respectively, are shown in black.In the nitrate reductase gene locus (A), 7 of 11 ORFs, shown in gray, areassigned to narABCGHDJ (HVO_B0160-0166), and their physiological rolesin nitrate reduction were estimated, while the functions of the other four ORFsremain unknown (5, 8, 10). (B) The arrangement of the DMSO reductase gene,dmsEABCD (HVO_B0362-0366), in H. volcanii is identical to that in Halobac-terium sp. NRC-1. The five ORFs are transcribed as a single mRNA in Halo-bacterium sp. NRC-1 (21).

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streaked on Casamino Acids (Hv-Ca) agar plates that contained no uraciland incubated at 37°C for about 2 weeks (23). One of the integrants thatappeared on the agar plate, which had gained uracil prototrophy by pyrE2integration, was chosen. Next, the pop-in strain, designated NO01, wasstreaked again on Hv-Ca agar plates supplemented with 10 mg liter�1

uracil and 50 mg liter�1 5=-fluoroorotate (5=-FOA) and incubated at37°C. The colonies appearing on the plate, which showed tolerance to5=-FOA by their inability to convert this compound to the toxic analog5-fluorouracil by a pyrE2 pop-out via homologous recombination, wereconfirmed by PCR amplification using the primers narOUf and narODr,as shown in Fig. S1C in the supplemental material. The narO deletionmutant derived from H. volcanii H26 thus obtained was designated NO02.The haloarchaeal strains prepared and used in this study are summarizedin Table 1.

Expression of H. volcanii NarO. An expression plasmid of H. volcaniiNarO was constructed by utilizing an H. volcanii-E. coli shuttle vector andpromoter sequence of the haloarchaeal constitutive gene encoding KatGcatalase-peroxidase (27, 28). The pHKH6 plasmid harbors H. marismor-tui katG (rrnAC1171) and its promoter sequence (77 bp in length) and the3=-flanking 114-bp DNA region, into which a (CAC)6 sequence as a His6

tag had been introduced into the 3= end of katG (28). Using the pHKH6plasmid as a template, inverse PCR was carried out with oligonucleotideprimers HmKPr and HmKTf to remove the DNA region encoding theKatG protein from pHKH6. In addition, a 640-bp DNA fragment con-taining narO was amplified using the primers narOf and narOr. The am-plified narO gene was inserted into the manipulated pHKH6 using a liga-tion kit (Ligation high version 2; Toyobo), generating pUCkGnarO.Finally, the pUCkGnarO insert was cut out using BamHI and XbaI andcloned into a pMLH32EV vector, generating pkGnarO as an expressionplasmid for NarO. Here, pMLH32EV is a shuttle vector derived frompMLH32 (kindly supplied by D. M. Dyall-Smith) by EcoRV digestion andself-ligation to demolish the bgaH gene, which encoded haloarchaeal�-galactosidase from Haloferax lucentense (JCM 9276T) and was origi-nally present in pMLH32 (27).

Point mutagenesis at five cysteine residues (Cys17, Cys81, Cys83,Cys91, and Cys100) in the recombinant NarO was carried out by technicalapplication of PCR. Five sets of oligonucleotide primers, C17Sf andC17Sr, C81Sf and C81Sr, C83Sf and C83Sr, C91Sf and C91Sr, andC100Sf and C100Sr, in which the corresponding Cys codon (TGC orTGT) was replaced with a Ser codon (TCG), were amplified by using

the pUCkGnarO plasmid separately as a template. After treatment withDpnI to decompose the template DNA, the linear PCR products werecyclized by homologous recombination between the 5= and 3= regions inE. coli JM109 cells. The inserts in the five plasmids were cut out and clo-ned into pMLH32EV, generating pkGnarOC17S, pkGnarOC81S,pkGnarOC83S, pkGnarOC91S, and pkGnarOC100S, respectively, as ex-pression plasmids for the mutant NarO proteins.

The six constructs, pkGnarO, pkGnarOC17S, pkGnarOC81S,pkGnarOC83S, pkGnarOC91S, and pkGnarOC100S, were introducedinto strain NO02. The transformants were selected by tolerance to 0.5 mgliter�1 novobiocin, a DNA gyrase inhibitor, on the Hv agar medium. Thecolonies appearing on the plate were obtained and designated NO04(�pkGnarO), NO05 (�pkGnarOC17S), NO06 (�pkGnarOC81S),NO07 (�pkGnarOC83S), NO08 (�pkGnarOC91S), and NO09(�pkGnarOC100S). A transformation of NO02 with pMLH32EV wasalso carried out and designated NO03.

Reporter plasmid for narO, narA, and nirK gene promoters. ThebgaH gene was used for construction of the reporter assay plasmid (27).The 2.11-kbp bgaH gene was amplified using a set of oligonucleotideprimers, bgaHf and bgaHr. Genome DNA of H. lucentense was used forthe template. The 270-bp DNA fragment, including the total region of theH. volcanii narO gene promoter, was also amplified using a set of oligo-nucleotide primers, pnarOf and pnarOr, against the H. volcanii genomeDNA as the template. Both amplified fragments were cloned into thepMLH32EV vector, generating pnarObgaH as a reporter plasmid for mea-suring the transcription activity of the narO promoter. pnarObgaH wasintroduced into strains H26 and NO02, yielding NOP01 and NOP02,respectively.

To analyze the transcription activity of the promoter of the putativenitrate reductase gene, a reporter plasmid was prepared from the 5=-flank-ing region of the narA gene. The 270-bp DNA fragment, including thetotal region of the narA gene promoter, was amplified using the oligonu-cleotide primer set pnarAf and pnarAr. The reporter plasmid pnarAbgaHwas constructed using a procedure similar to that for the preparation ofpnarObgaH. The plasmid was introduced into strains H26 and NO02,yielding NAP01 and NAP02, respectively.

The 5=-flanking sequence with 249 nucleotides of the H. volcanii ni-trite reductase NirK gene (HVO_2141), including the putative promoter,was amplified by PCR using primers pnirKf and pnirKr. The reporterplasmid pnirKbgaH was constructed by the same procedure. Strains H26

TABLE 1 Strains of H. volcanii used in this study

Strain Derivation (reference) Genotypea

H26 Allers et al. (23) �pyrE2NO01 H26/p�narO pop-in �pyrE2 narO�::[�narO pyrE2�]NO02 NO01 pop-out �pyrE2 �narONO03 NO02, pMLH32EV transformed �pyrE2 �narO {nov}NO04 NO02, pkGnarO transformed �pyrE2 �narO {pkatG::narO::His6 tag � nov}NO05 NO02, pkGnarOC17S transformed �pyrE2 �narO {pkatG::narO(C17S)::His6 tag � nov}NO06 NO02, pkGnarOC81S transformed �pyrE2 �narO {pkatG::narO(C81S)::His6 tag � nov}NO07 NO02, pkGnarOC83S transformed �pyrE2 �narO {pkatG::narO(C83S)::His6 tag � nov}NO08 NO02, pkGnarOC91S transformed �pyrE2 �narO {pkatG::narO(C91S)::His6 tag � nov}NO09 NO02, pkGnarOC100S transformed �pyrE2 �narO {pkatG::narO(C100S)::His6 tag � nov}NOP01 H26, pnarObgaH transformed �pyrE2 {pnarO::bgaH � nov}NOP02 NO02, pnarObgaH transformed �pyrE2 �narO {pnarO::bgaH � nov}NAP01 H26, pnarAbgaH transformed �pyrE2{pnarA::bgaH � nov}NAP02 NO02, pnarAbgaH transformed �pyrE2 �narO {pnarA::bgaH � nov}NKP01 H26, pnirKbgaH transformed �pyrE2{pnirK::bgaH � nov}NKP02 NO02, pnirKbgaH transformed �pyrE2 �narO {pnirK::bgaH � nov}NKP03 H26, pnirKG2CbgaH transformed �pyrE2{pnirKG2C::bgaH � nov} pnirKG2C; second G in inverted repeat

was replaced by CNKP04 H26, pnirKA3TbgaH transformed �pyrE2{pnirKA3T::bgaH � nov} pnirKA3T; third A was replaced by TNKP05 H26, pnirKA4TbgaH transformed �pyrE2{pnirKA4T::bgaH � nov} pnirKA4T; fourth A was replaced by Ta Plasmids integrated on the chromosome are indicated by brackets, and episomal plasmids are indicated by braces. nov, novobiocin resistance.

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and NO02 were transformed by the plasmid, yielding NKP01 and NKP02,respectively.

Site-directed mutagenesis of the promoter sequence of the nirK genewas carried out, according to Kunkel’s method (29), with a slight modi-fication. pUCpnirK, which contains the putative promoter sequence ofthe nirK gene in the pUC119 vector, was transformed to E. coli strainCJ236 (dut1 ung1 thi-1 relA1/pCJ105 [F= Cmr]). The transformant wasinfected by an M13KO7 helper phage and then incubated in the kana-mycin-supplemented medium. Uracil-substituted single-strandedpUCpnirK was collected from the supernatant of the medium by poly-ethylene glycol precipitation. After annealing with a nirKPG2C primer,which was designed for a transversion mutation at the 2nd nucleotide (Gto C) in the inverted repeat sequence in the nirK promoter, a complemen-tary strand of the single-stranded pUCpnirK was synthesized by using T4DNA polymerase (TaKaRa). The chimeric double-stranded DNA thusobtained was transformed to E. coli strain JM109. The plasmid was iso-lated from the transformant, and the intended substitution was confirmedby DNA sequencing. The mutant pUCpnirKG2C thus obtained was usedfor the preparation of plasmid pnirKG2CbgaH and transformant NKP03(host strain, H. volcanii H26) for a reporter assay experiment. Transver-sion mutations at the 3rd (A to T) and 4th (A to T) nucleotides were alsoindividually carried out by using nirKPA3T and nirKPA4T, respectively.Two mutant plasmids, pnirKA3TbgaH and pnirKA4TbgaH, and the corre-sponding transformants, NKP04 and NKP05, respectively, were also pre-pared and used for the experiment.

Assay of transcription activities of gene promoters. BgaH activitywas measured by using o-nitrophenyl-�-D-galactopyranoside (ONPG) asthe substrate, according to a previous report (27). Precultured cells ofstrains NOP01, NOP02, NAP01, NAP02, NKP01, and NKP02 were pre-pared for inoculation by aerobic cultivation in the Hv medium supple-mented with 0.5 mg liter�1 novobiocin. After inoculation of the precul-tured medium with a 10% volume of the cultivation medium, the strainswere cultivated in low-oxygen-tension conditions described in “Strainsand growth conditions” above, using the Hv medium supplemented with0.5 mg liter�1 novobiocin with or without 50 mM KNO3. The medium (1ml) was sampled every 24 h using a sterile needle. After the OD600 of themedium was measured, the medium was centrifuged at 22,000 � g for 5min to separate the cell pellet and supernatant using a centrifugal separa-tor model 3700 (Kubota Co., Tokyo, Japan). The cell pellet obtained wassuspended in 800 �l of the assay solution containing 50 mM Tris-HClbuffer (pH 7.2), 2.5 M NaCl, 10 �M MnCl2, and 0.1% �-mercaptoetha-nol. The cells were solubilized by adding 100 �l of 2% Triton X-100 to thesuspension and vortexing for 10 s. The solution was transferred to a cu-vette with a 1-cm light path, and the BgaH reaction was started byadding 100 �l of an 8-mg/ml ONPG solution. Increasing absorbance at405 nm of the solution was monitored using a spectrophotometer.BgaH activity (in Miller units) was calculated according to the formula(�A405/OD600/min) � 103.

Assay of nitrate- and nitrite-reducing activities. H. volcanii strainsH26 and NO02 were cultivated aerobically in the Hv medium. Anaerobiccultivation of the two strains was also carried out using Hv medium sup-plemented with 50 mM KNO3 or 77 mM DMSO. Anaerobic incubation ofthe strains was also performed in the medium without supplementation ofany respiratory substrates. The harvested archaeal cells were sonicated bythe VP-30S supersonic oscillator (Taitec Corp., Saitama, Japan). The sus-pension thus treated was centrifuged at 12,000 � g for 10 min, and theenzymatic activity of the cell extract supernatant was measured. The en-zymatic activities of nitrate and nitrite reductions in the cell extract weremeasured according to the methods of Yoshimatsu et al. (30) and Ichiki etal. (11), respectively, with a slight modification. The protein concentra-tion was measured by using a bicinchoninic acid (BCA) protein assay kit(Pierce, Rockford, IL) using bovine serum albumin as the standard.

Nitrate reductase activity was detected on polyacrylamide gel, as fol-lows: H. volcanii H26 and NO02 (�narO) were cultivated in the Hv me-dium under aerobic, anaerobic, or denitrifying conditions. The cells were

harvested from 6.0 ml of medium by centrifugation after 4 days of culti-vation. Total proteins were extracted by treating the cell pellet with aminimum volume of 10 mM Tris-HCl buffer (pH 8.0) containing 1%(wt/vol) Triton X-100 (Sigma, St. Louis, MO), followed by polyacryl-amide gel electrophoresis (PAGE), according to the method of Davis (31).After electrophoresis, the gel was enclosed in a sealed glass container filledwith 0.1 M sodium phosphate buffer (pH 7.0) containing 1.0 M NaCl and0.2 mM methyl viologen (MV). Sodium dithionite solution (final concen-tration, 2.3 mM) was injected into the container to reduce MV, and thenthe gel was incubated for about 10 min until infiltration of enough of thereduced MV had turned the gel blue. The reaction was started by theinjection of NaNO3 to a concentration of 10 mM. An image of the color-less spot appearing on the gel due to the oxidation of MV catalyzed bynitrate reductase was recorded by a scanning apparatus.

Other methods. A primer extension experiment to determine thetranscription start point of the nirK gene is described in the supplementalmaterial. The immunological method for detection of the recombinantNarO is also explained in the supplemental material. A homology searchwas carried out using the BLAST site (http://blast.genome.jp/), andsequence alignment by the neighbor-joining method was performedusing Clustal W (http://clustalw.genome.ad.jp/). All chemicals used in theexperiments were of the highest grade commercially available.

RESULTSPhenotype analysis and complementation of �narO mutation.The putative nitrate reductase gene cluster, with a unique struc-ture combining the respiratory quinol oxidase genes (narABC)and membrane-bound nitrate reductase genes (narGHDJ), isshown in Fig. 1. Homologous gene clusters have been identified inthe genomes of nine haloarchaeal strains (H. volcanii, Haloferaxmediterranei, H. marismortui, Haloarcula hispanica ATCC 33960and N601, Haloarcula sp. strain CBA1115, Halorhabdus utahensis,Halogeometricum borinquense, Halomicrobium mukohataei, andHalorubrum lacusprofundi). NarO and its homologous proteinswere identified in the 5= flank of the nitrate reductase genes of sixstrains: H. volcanii, H. marismortui, two strains of H. hispanica,Haloarcula sp. CBA1115, H. utahensis, and H. borinquense. Thealignment of the amino acid sequences of the seven NarO proteinsis shown in Fig. 2. NarO proteins are expected to be soluble pro-teins, with molecular weights of about 22,000, and they lack atranslocation signal sequence. The C-terminal amino acid se-quence with 58 residues (174 to 201, using H. volcanii NarO num-bering) showed significant similarity with the HTH-type DNA-binding motif of the Bat regulator that controls the oxygen- andlight-dependent activation of the bop gene and its related genes inhaloarchaea (17, 20). The four cysteines with the arrangementCXnCXCX7C (n �70), corresponding to Cys17, Cys81, Cys83,and Cys91 (H. volcanii NarO numbering), were conserved. Thesequence of the N-terminal side of the NarO homologues showedno notable similarity to sequences with any assigned function in-volving that of bacterial FNR proteins, the most-investigated ox-ygen-sensing transcription regulator.

To elucidate the function of NarO, the narO gene was dis-rupted by a double integration method using uracil auxotrophicH. volcanii strain H26 (�pyrE2), as shown in Fig. S1 in the supple-mental material. Under aerobic conditions, the narO mutantNO02 grew as well as the parental strain H26, as shown in Fig. 3Aand B. When the strains of H. volcanii were incubated anaerobi-cally in the absence of a respiratory substrate, a gradual increase inthe OD600 of the medium was observed (Fig. 3, triangles). Theobservation suggests that the H. volcanii might be able to growslowly by microaerobic respiration, as discussed below. Unlike the

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parental strain, NO02 grew poorly when cultivated anaerobicallyin the presence of nitrate (Fig. 3B). Notably, strain NO02 main-tained the ability of DMSO respiration, indicating that NarO par-ticipates only in the regulation of denitrification in H. volcanii.

Complementation of the growth capability by denitrificationwas examined by the expression of recombinant NarO in NO02.The expression plasmid pkGnarO was constructed from the shut-tle vector pMLH32EV and the promoter of the katG gene. The

FIG 2 Sequence alignment of NarO proteins. The deduced amino acid sequence of H. volcanii NarO (product of HVO_B0159) was aligned with that oforthologous proteins from H. borinquense (Hbor_34990, 49.8% identity to H. volcanii NarO), H. utahensis (Huta_0019, 40.4%), H. marismortui (rrnAC1193,41.7%), H. hispanica (HAH_1793, 41.4%), and Haloarcula sp. CBA1115 (SG26_16865, 41.3%). The NarO sequences from the two strains of H. hispanica, ATCC33960 (HAH_1793) and N601 (HISP_09150), are identical. The four conserved cysteines are indicated in white letters. The asterisks reveal the amino acidresidues conserved among the seven NarO proteins. Residues identical to those of the H. volcanii NarO are emphasized by gray shading. The HTH-typeDNA-binding domain is boxed. Residue numbers are shown on the right.

FIG 3 Complementation of denitrifying growth of �narO mutant by recombinant NarO. Strains H26 (A), NO02 (�narO) (B), and NO04 (NO02/pkGnarO) (C)were cultivated as described in Materials and Methods. The increase in optical density of the medium was measured at 600 nm. The OD600 values of H26, NO02,and NO04, which grew aerobically without nitrate (circles), anaerobically without supplementation of any respiratory substrate (triangles), anaerobically withnitrate (diamonds), and anaerobically with DMSO (squares), are shown. The experiments were performed independently three times. The error bars representthe standard error (SE). When H26 and NO02 were cultivated by DMSO respiration, the SE values were very small; therefore, the error bars are masked by thesquare symbols.

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recombinant NarO was engineered to contain a C-terminal His6

tag, with the aim of immunological detection and purificationof the recombinant protein. The pMLH32EV vector was de-rived from pMLH32, which is a low-copy-number vector,whose copy number in the H. volcanii cell is about six (27).Cultivation experiments of strain NO04, the transformant ofNO02 by pkGnarO, demonstrated that the anaerobic growthby denitrification was restored by expression of the recombi-nant NarO, as shown in Fig. 3C.

Functional analysis of conserved cysteines in NarO. Pointmutagenesis of the narO gene carried out on pkGnarO was per-formed to replace each of the four conserved cysteines of the re-

combinant NarO with serines. Plasmids for the expression of themutant NarO proteins were inserted into NO02 to generatestrains NO05, NO06, NO07, and NO08, which harbor the expres-sion plasmids for the Cys17, Cys81, Cys83, and Cys91 mutantNarO proteins, respectively, and were examined in comparison toNO04. Mutation of the nonconserved cysteine (Cys100 in H. vol-canii NarO) was also performed, generating strain NO09. Asshown in Fig. 4, under aerobic conditions, the five strains withCys¡Ser narO mutations grew as well as strain NO04; however,when cultivated under denitrifying conditions, four of the fivenarO mutants, except for that with a mutation of the unconserved5th Cys, were unable to grow. The results demonstrated that allfour conserved cysteines are essential for the function of NarO toregulate denitrification.

Transcription activities of narO gene promoter. The narOgene is present in the 5=-flanking sequence of the narA gene in thereverse direction, and the narO and narA genes are separated by a264-bp DNA sequence. The genetic function of the transcriptionalregulation of both the narO gene and the putative nitrate reduc-tase gene would involve the DNA region (Fig. 5A). Transcriptionactivity was analyzed using the bgaH as the reporter gene and withthe forward and reverse directions of the 264-bp DNA sequence asthe narA and narO promoter, respectively.

Strains NOP01 and NOP02 were derived from H26 and NO02,respectively, by transformation of the pnarObgaH plasmid. Bothstrains were cultivated under several conditions, and the inducedBgaH activities were measured every 24 h. Figure 5B shows theBgaH activities at 72 h after starting cultivation; here, aerobic cellswere in the late exponential phase, and denitrifying cells were inthe mid-exponential phase. Very slow growth was observed instrains NOP01 and NOP02 under anaerobic incubation in theabsence of nitrate. BgaH activities were observed in NOP02 underall cultivation conditions (637 to 963 Miller units [m.u.]), and no

FIG 4 Functional analysis of conserved cysteines of NarO. Strains H26, NO02(�narO), NO03 (NO02/pMLH32EV), NO04 (NO02/pkGnarO), NO05 (Cys17mutant NarO was expressed in NO02), NO06 (Cys81), NO07 (Cys83), NO08(Cys91), and NO09 (Cys100) were cultivated under aerobic (white bars), anaero-bic with nitrate (gray), and anaerobic without nitrate (black) conditions. TheOD600 of each medium after 8 days of cultivation is indicated. The experimentswere performed independently three times. The error bars represent the SE.

FIG 5 Transcription activities of the narO, narA, and nirK gene promoters were analyzed by a reporter assay using the bgaH gene encoding haloarchaeal �-galactosidase.(A) Schematic representation of the promoter regions used for reporter plasmid construction, where the conserved inverted repeat and putative haloarchaeal TATA boxare shown by gray boxes and a diamond, respectively. (B) Transcription activities of the narO gene promoters were analyzed. Strains NOP01 (H26/pnarObgaH) andNOP02 (NO02/pnarObgaH) were cultivated under aerobic (O2, �; nitrate, � and �), anaerobic (O2, �; nitrate, �), or denitrifying (O2, �; nitrate, �) conditions for4 days. The mean values of induced BgaH activities (in Miller units) for NOP01 and NOP02 are indicated by white and gray bars, respectively. The experiments wereperformed independently at least three times. The error bars represent the SE. Different letters denote significantly different means (P0.05, one-way analysis of variancewith Bonferroni’s test for multiple comparisons). Analysis of the transcription activity of the narA and nirK gene promoters was also carried out in the same manner. (Cand D) The results obtained using strains NAP01 (H26/pnarAbgaH, white bars) and NAP02 (NO02/pnarAbgaH, gray bars) (C) and those obtained using strains NKP01(H26/pnirKbgaH, white bars) and NKP02 (NO02/pnirKbgaH, gray bars) (D).

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significant differences were detected among the activities, accord-ing to Bonferroni’s multiple-comparison test. In contrast, en-hancement of BgaH activity (1,863 192 m.u. [mean standarderror]), which was about three times the activity in the NOP02cells, was observed in the denitrifying NOP01 cells. A similar resultwas obtained when using the microbial cells harvested at 48 h afterstarting cultivation (data not shown).

Transcription activities of narA gene promoter and expres-sion of nitrate reductase. Strains NAP01 and NAP02 were derivedfrom H26 and NO02, respectively, by transformation of thepnarAbgaH plasmid for the narA promoter activity assay. ThenarA promoter was highly activated in the denitrifying NAP01cells (868 351 m.u.), as shown in Fig. 5C. Low BgaH activity(226 46 m.u.) was observed in the starved NAP01 cells that hadbeen incubated anaerobically in the absence of nitrate. No BgaHactivity was detected in the aerobic cells of NAP01. The NAP02cells showed very low BgaH activity (2.6 2.0 m.u.) under deni-trifying conditions.

The expression of nitrate reductase under each cultivationcondition was also examined. Nitrate-reducing activities inducedin the H26 and NO02 cells, which were cultivated anaerobically inthe presence or absence of nitrate for 72 h, were visualized onpolyacrylamide gel by activity staining. Aerobic cells were alsoprepared and used as the experimental control. As shown in Fig. 6,a colorless spot that corresponded to the nitrate-dependent oxi-dation of reduced MV appeared on the gel where the total proteinssolubilized from the denitrifying cells of H26 had been loaded.Relatively weak activity also appeared in the H26 cells cultivatedanaerobically in the absence of nitrate. No enzymatic activity wasdetected in the aerobic cells of H26. Nitrate reductase was notinduced in the NO02 cells even under denitrifying growth condi-tions. The conditional expression of nitrate reductase was consis-tent with the action of the narA promoter, as shown in Fig. 5C.

Transcription activities of nirK gene promoter. The 169-bp-long DNA sequence that exists at the 5=-flanking region of the nirKgene (HVO_2141) should include elements to regulate transcrip-tion activity. The nucleotide sequence of 300 bp in length contain-ing the whole region of the putative nirK promoter was used toconstruct the pnirKbgaH plasmid (Fig. 5A). The pnirKbgaH plas-mid was transformed to strains H26 and NO02, and then thetransformants NKP01 and NKP02, respectively, were prepared.As shown in Fig. 5D, BgaH activity was induced in the NKP01strain when it was cultivated anaerobically. Like the narA pro-

moter in the H26 cell, transcriptional activity of the nirK genepromoter appeared under anaerobic conditions whether the ni-trate was supplemented or not, but a significant difference wasfound between the two activity levels: the activities of denitrifyingcells and starved cells of NKP01 were estimated to be 350 32m.u. and 237 69 m.u., respectively. Very low BgaH activity, at3.3 2.0 m.u., was detected in the denitrifying cells of NKP02,while no activity was observed in the aerobic cells. Similar to theconditional activation found in the narA gene promoter, theseresults provide persuasive evidence that the narO gene plays acritical role in the activation of the nirK promoter.

As indicated in Fig. S2 in the supplemental material, a primerextension experiment was performed to determine the transcrip-tion start point of the nirK gene in Haloferax denitrificans JCM8864 (�ATCC 35960). H. denitrificans is phylogenetically close toH. volcanii, and the nucleotide sequence of its nirK promoter isvery similar to that of H. volcanii (see Fig. S2A in the supplementalmaterial). The result suggests that the transcription of the H. deni-trificans nirK gene was initiated at a C residue located 15 bp up-stream of the translation start ATG codon and 25 bp downstreamof a putative TATA box (see Fig. S2B in the supplemental mate-rial). Due to the 92.7% identity of the nucleotide sequence, it ismost likely that transcription of the H. volcanii nirK gene starts atthe corresponding C residue located 14 bp upstream of the trans-lation start codon and 25 bp downstream of the putative TATAbox (Fig. 7A; see also Fig. S2 in the supplemental material).

Nucleotide sequences of the 5=-flanking region of the denitri-fying genes narA, nirK, norB, and nosZ of the haloarchaeal genomewere aligned, as shown in Fig. S3 to S6 in the supplemental mate-rial, respectively. The inverted repeat sequence CGAAX4TTGC,which seems to be the recognition site of an HTH-type DNA-binding protein, was identified in a large part of the promoter ofthe denitrifying genes. As revealed in Fig. 7, a single mutation intothe inverted repeat at the 2nd guanine caused a dramatic decreasein the transcriptional activity of the nirK promoter, and only 9%of the activity was detected. Substitutions of the 3rd and 4th ade-nines also decreased the transcription activity of the nirK pro-moter to about half, with 59% and 53% activity retained, respec-tively. These results demonstrated that the inverted repeat plays asignificant role in the transcriptional regulation of the denitrifyinggenes.

DISCUSSION

Of the 31 species of haloarchaea for which total genomic informa-tion is presently available, six species include a putative nitratereductase gene in combination with the gene encoding the regu-latory protein NarO in the 5=-flanking region of the gene (Fig. 2).Interestingly, the regulatory gene encoding the protein that washomologous to NarO, named DmsR, is also present in the 5=-flanking region of the DMSO/TMAO reductase gene of 10 halo-archaeal strains (Halobacterium sp. NRC-1, Halobacterium salina-rum R-1, H. volcanii, H. mediterranei, H. marismortui, two strainsof H. hispanica, Haloarcula sp. CBA1115, Natronobacterium grego-ryi, Natrinema sp. J7-2, and H. mukohataei) (Fig. 1). Although theunique cysteine-rich motif is conserved in DmsR, the internalsequence between the 1st and 2nd cysteines is about 20 residuesshorter than that of NarO (21, 32). DmsR has been reported tohave a significant role in the conditional expression of DMSO/TMAO reductase in Halobacterium sp. NRC-1 (21, 22) and in H.volcanii (32).

FIG 6 Nitrate reductase activity. H26 and NO02 (�narO) were cultivatedunder aerobic (O2, �; nitrate, �), anaerobic (O2, �; nitrate, �), or denitrify-ing (O2, �; nitrate, �) conditions. Total proteins were extracted from the cellsby treating them with detergent, as described in Materials and Methods. Afterelectrophoresis on polyacrylamide gel, the nitrate-reducing activity on the gelwas visualized as a colorless spot that revealed the oxidation of reduced MVcatalyzed by the induced nitrate reductase.

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We performed gene disruption studies on H. volcanii NarO incombination with expression of a recombinant and a reporterassay experiment of denitrifying gene promoters. The narO dele-tion mutant of H. volcanii strain NO02 retained the ability to growaerobically, with a growth rate similar to that of strain H26,whereas the mutant did not grow anaerobically by denitrification(Fig. 3). The result demonstrated the critical function of NarO inthe induction of the denitrifying ability in H. volcanii. Anaerobicgrowth of H. volcanii with supplementation of DMSO instead ofnitrate was not affected by disruption of the narO gene. In con-trast, the dmsR deletion mutant of H. volcanii, which already hadbeen prepared in our laboratory, lost the ability of anaerobicDMSO/TMAO respiration but did grow by denitrification, indi-cating that the functions of NarO and DmsR as the transcriptionregulator are not redundant (32).

In this study, anaerobic cultivation of H. volcanii was carriedout under N2 gas containing 0.2% O2. The microaerobic condi-tion is essential for the reproducibility of the anaerobic growth ofH. volcanii, as reported previously (32). Although the reason forthe instability of the growth under strict anaerobic conditions isnot clear, one possibility is that the energy generation by mi-croaerobic respiration is significant for H. volcanii for smoothadaptation to the anaerobic conditions and induction of the deni-trifying enzymes. Very slow growth of strain H26 under anaerobicconditions without nitrate and strain NO02 (�narO) under deni-trifying conditions was observed, as shown in Fig. 3. The resultsmight also correspond to a low energy yield by microaerobic res-piration of H. volcanii.

As shown in Fig. 5, the transcription activity of the narO genepromoter was higher than that of the genes of the catabolic en-zymes nitrate reductase and nitrite reductase, which are expressedin the microbial cells in large amounts (11, 30). However, untilnow, identification of the recombinant NarO expressed in the H.volcanii cells has not been followed by immunoblotting experi-ments using anti-His6 antibody. Purification of the recombinantNarO protein by Ni2�-affinity resin also failed. It is probable thatthe NarO molecule has a very short life span and is immediately

decomposed by proteolysis in H. volcanii cells. Another explana-tion is also possible, e.g., the mRNA of the narO gene is veryunstable, and therefore, the concentration of recombinant NarOmolecules is lower than what can be detected immunologically.

As indicated in Fig. 5B, transcription of the narO gene wasordinarily activated at similar transcription levels whether culti-vated under aerobic or anaerobic conditions, except for the re-markable increase in the activity observed in the denitrifyingNOP01 cells. The results indicated that transcription of the narOgene is usually activated, regardless of the anaerobicity of thegrowing conditions, and it is enhanced by the NarO protein. Inaddition, the results shown in Fig. 5C and D demonstrate thatNarO is critical for activation of the transcription of the denitri-fying genes under anaerobic conditions. In bacteria, the transcrip-tion of denitrifying genes is controlled by dual regulatory systems,one being the oxygen/redox-sensing mechanism, and the otherdepending on nitrogen oxide species as the respiratory substrate(1, 33). The expression of denitrifying enzymes in bacteria, there-fore, hardly occurs when they are cultivated anaerobically in theabsence of nitrate (34, 35). The present result suggests that the H.volcanii denitrifying genes are mainly controlled by a NarO-de-pendent regulatory system that responds to anaerobic cultivationconditions. The results also revealed that NarO-dependent activa-tion of transcription of the denitrifying genes was enhanced underthe presence of nitrate, while the genetic mechanism is unknownso far.

The unique arrangement of the four conserved cysteines asCXnCXCX7C (n �70) is a structural characteristic of NarO and itshomologous proteins (Fig. 2). Site-directed mutation experi-ments clearly demonstrated that all four cysteines in NarO wereessential for the induction of denitrification, as shown in Fig. 4. Inaddition, transcription of the denitrifying genes required the narOgene and was activated under anaerobic conditions (Fig. 5C andD). The results are most explicable by the hypothesis that NarOparticipated in regulation of the oxygen and/or redox potential-dependent transcription of the denitrifying genes. The NarO ho-mologs do not comprise a PAS domain, which is one of the oxy-

FIG 7 Mutation analysis of the inverted repeat of the nirK promoter region. (A) The DNA region located upstream of the nirK gene. An inverted repeat(CGAAX4TTCG) is boxed, and a putative haloarchaeal TATA box (consensus TTTWWW) is in bold type. The transcription start C residue, indicated by a shadedwhite letter, was inferred from primer extension analysis of the H. denitrificans nirK (see Fig. S2 in the supplemental material). The translation start codon of nirKis italicized. The DNA sequence is numbered relative to the translation start point. (B) Transcription activity was examined using a reporter plasmid in which the2nd to 4th nucleotides of the inverted repeat were individually substituted. The experiments were performed independently three times. The error bars representthe SE. Cultivation of the four strains, NKP02, NKP03, NKP04, and NKP05, and measurement of the induced BgaH activities are described in Materials andMethods.

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gen/redox sensor motifs found in haloarchaeal Bat and otherredox-dependent regulatory proteins (17, 20). One possible sce-nario is that, like the bacterial FNR, metal centers bound to theside chains of cysteines respond to oxygen molecules or low redoxpotential in the environment, causing the activation of NarO (15,16). In this case, it should be noted that the arrangement of thefour conserved cysteines in NarO shows no similarity to that of theiron-sulfur binding domain of the bacterial FNR. It is also plausi-ble that the formation of a disulfide bridge between the consensuscysteines in the presence of oxygen or high redox potential inac-tivates NarO, as is likely for the E. coli OxyR, which controls thetranscription of the genes encoding HPII catalase and other com-ponents for protection of the cell against oxidative stress (36).Another hypothesis is that a heme molecule is a functional centerfor oxygen sensing. Heme lyase, which takes part in the biosyn-thesis of c-type cytochrome, comprises the consensus Cys-Pro-Valsequence as a heme-binding motif (37). This Cys-Pro-Val se-quence is highly conserved at the positions of two of four cysteinescorresponding to Cys17 and Cys91 in H. volcanii NarO (Fig. 2).

An FNR-like regulator protein was not identified by searchingthe haloarchaeal genomic information, suggesting another regu-latory mechanism for denitrification in haloarchaea. Interestingly,comparative searching of the putative DNA contact site in thehaloarchaeal denitrification gene promoters demonstrated that aninverted repeat, CGAAX4TTGC, which was expected to interactwith the HTH-type regulator in the dimeric state, was commonlypresent. The DNA sequence of the nirK promoter reveals a typicalarrangement of the putative transcriptional elements in the halo-archaeal denitrifying gene promoter. As represented in Fig. 5Aand 7A, and in Fig. S4 in the supplemental material, CGAAGATGTTCG is centered 39 bp upstream of the haloarchaeal TATA box(consensus TTTWWW, where W is A or T) in the H. volcanii nirKpromoter (38). The transcription start point of the H. volcaniinirK gene was expected to be 25 bp downstream of the TATA box,based on the primer extinction analysis of the nirK gene of H.denitrificans, whose nirK promoter sequence was very similar(91% identical) to that of H. volcanii (see Fig. S2 in the supple-mental material). A similar arrangement was also found in a largenumber of the promoters of the haloarchaeal denitrifying genesnirK, norB, and nosZ (see Fig. S4 to S6 in the supplemental mate-rial, respectively). Point mutagenesis at the 2nd nucleotide of theinverted repeat caused a drastic decrease (only 9.1% remaining) inthe transcriptional activity of the nirK promoter (Fig. 7). Addi-tionally, mutations at the 3rd and 4th nucleotides of the invertedrepeat resulted in 41% and 47% decreases, respectively, in the nirKpromoter activity. These results support our hypothesis that thegenetic function of this inverted repeat sequence is the transcrip-tional regulation of denitrification in haloarchaea.

In contrast to the well-ordered structure of transcriptional el-ements in the nirK, norB, and nosZ gene promoters, the arrange-ments of the inverted repeat and putative TATA box were notcomprehensive in the narA and narO promoters. The invertedrepeat sequence was centered from 220 bp, 214 bp, and 191 bpupstream of the putative translation start position of the H. volca-nii, H. mukohataei, and H. utahensis narA genes, respectively (seeFig. S3 in the supplemental material). A TATA box-like motif wasnot identified due to the A/T-rich nature of the putative promoterregions. The inverted repeat was 135 bp upstream from the trans-lation start position of the narA genes of four Haloarcula species,but it was not found to be present in the narA promoter of H.

mediterranei and H. lacusprofundi. Mutagenetic analysis of thereporter assay experiment and identification of the transcriptionstarting point of the denitrifying genes are now under way.

Our present study suggests that NarO is a key regulator ofdenitrifying growth in H. volcanii. The cysteine-rich motif wascritical for transcriptional regulation under anaerobic conditions.The inverted repeat CGAAX4TTCG was often present in the ha-loarchaeal denitrifying gene promoters and was shown to play asignificant role in transcription regulation. The most importantproblem that remains unsolved is whether NarO binds directlywith the inverted repeat and activates the transcription of denitri-fying genes. In addition, the 46 proteins homologous to H. volcaniiNarO were identified in 21 species of the 31 haloarchaea for whichthe total genome sequence is now available. However, except forthe seven NarO proteins and 11 DmsR proteins, the regulatorytargets of the remaining 29 homologs have remained uncertain.Further investigations, especially purification of the recombinantNarO protein, identification of the NarO binding site on thegenomic DNA, and assignment of the functions of the NarO ho-mologs, are required for total understanding of the regulation ofhaloarchaeal anaerobic metabolisms.

ACKNOWLEDGMENTS

This work was supported by research grants from the Noda Institute forScientific Research, the Japan Space Forum (Exploratory Research forSpace Utilization), and the True Nano Research Program of ShizuokaUniversity to T.F.

FUNDING INFORMATIONNoda Institute of Scientific Research provided funding to Taketomo Fu-jiwara. Japan Space Forum (Exploratory Research for Space Utilization)provided funding to Taketomo Fujiwara.

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