JotrRN~ OF I~I~NTATIONANDBIOENGINEERING Vol. 76, No. 2, 82-88. 1993
Cloning and Sequencing of a Gene Encoding Nitrite Reductase from Paracoccus denitrificans and Expression of the Gene in Escherichia coli TAKAYUKI O H S H I M A , MAKOTO SUGIYAMA, NOBUYUKI UOZUMI, SHINJI IIJIMA, AND TAKESHI KOBAYASHI*
Department of Biotechnology, Faculty of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan Received 24 March 1993/Accepted 21 May 1993 A structural gene for nitrite reductase (nirS) was cloned from a denitrifying bacterium Paracoccus denitriflcans into Escherichia coli MVl184. The coding sequence of nits consisted of 1788 nucleotides and the value of the G + C content was 68Yo. This gene seemed to be in an operon structure. The size of nitrite reductase (NIR) was predicted to be 65.5 kDai from the amino acid sequence, which was similar to the value determined with purified NIR by SDS-polyacrylamide gel electrophoresis analysis. From hydrophathy analysis, the NIR from P. denitriflcans seemed to be a periplasmic enzyme. Crude extract from the recombinant E. coli harboring nirS and about 10 kbp of its downstream flanking region had significant activity of NO and N20 formation from nitrite (NO2-), whereas crude extract from E. coli harboring only nits had only weak activity. This result suggested that the downstream region includes the gene responsible for the protein which involved in NIR activation. In addition, the downstream region seemed to have a NO reductase gene, because NO to N20 conversion activity was also detected in the crude extract from the recombinant E. coil harboring nirS and about 10 kbp of its downstream flanking region.
phically with a great variety of multicarbon compounds under aerobic conditions. In addition, it can grow under anaerobic conditions with nitrate, nitrite, or nitrous oxide as a terminal electron acceptor (11, 12) during oxidization of organic nutrients or hydrogen gas (5). During anaerobic growth, the expression of nitrate (13) and nitrite (14) reductases is induced in the presence of nitrate and nitrite. These enzymes have already been purified, and their biological properties studied (15-17). A nitrite reductase (NIR) which has either heme cdl (EC 22.214.171.124) or copper (EC 126.96.36.199) is known to be the key enzyme in yielding the first gaseous product. NIRs have been isolated from various denitrifying bacteria (18-21). Cytochrome cdl from Pseudomonas aeruginosa has been studied extensively (18). This protein is known as a periplasmic enzyme and catalyses the formation of nitric oxide (NO) from nitrite accompanying the oxidation of cytochrome c-551 (19) or azurin (20). The structural gene of nitrite reductase (nitS) from Ps. aeruginosa has been cloned, and the primary structure determined (21). However, heterologous expression of active NIR has not been reported, making the further development of a denitrifying system difficult. In addition, other nits genes from different denitrifying bacteria have not been cloned yet. In the present paper, we report the cloning and DNA sequence of the gene encoding nitrite reductase from P. denitrificans, and the expression of this gene in Escherichia
Denitrification is a respiratory mode of energy conversion, used by facultative anaerobic bacteria and some of fungi, that sequentially transforms ionic nitrogenous oxides to nitrogen gas: NO3---}NO2--+NO-->NEO-~N2 . The process is a part of the global nitrogen cycle and is important in preventing the release of excess nitrogenous oxides in industrial wastewater (1, 2). In addition, nitrate contamination in ground water has led to the initiation of a search for an effective reduction procedure using microorganisms (3-5), because it has been reported that many sources of drinking water, especially in areas of intensive agriculture, contain intolerably high nitrate ion concentrations (6). However, biological denitrification by microorganisms generally requires water enriched with nutrients to support their growth (7, 8), which results in contamination by these other nutrients. Furthermore, these microorganisms are difficult to maintain, and the overall process of denitrification is slow and often incomplete, which results in the production of nitrous oxides which then becomes air pollutants. To overcome these drawbacks, a bacteriafree bioreactor system for denitrification using denitrifying enzymes has been developed for nitrate removal from ground water (9). It is, however, necessary to study their features in detail to achieve an effective denitrifying system through the improvement of denitrifying bacteria and enzymes; at present, the biological properties, especially the genetic features, of most denitrifying enzymes are not well understood. Paracoccus denitrificans is a Gram-negative bacterium capable of growing under various conditions. The bacterium can grow autotrophically on hydrogen and carbon dioxide as well as methylotrophically with methanol or methylamine using an oxygen as a terminal electron acceptor (10). The microorganism also can grow heterotro-
coil MATERIALS AND METHODS Bacterial strains and vector P. denitrificans (IFO 12442) was used in this work. E. coli C600 (supE44, hsdR, thi-1, thr-1, leuB6, lacY1, tonA21) or MVl184 (ara, A(lac-proAB), rpsL, thi, ((D80 IacZAM15), A(srl-recA)306 :: Tn10(tetr)/F'[traD36, proAB ÷, laclq, lacZAM15]) was
* Corresponding author. 82
VoL. 76, 1993 used as the recombinant plasmid host strain, and E. coli P2392 (supE44, supF58, hsdR514, galK2, gaiT22, metB1, trpR55, lacYl, [P2]) as the recombinant phage host strain. The pOCT2, and ,~-DASH II were used for the construction of P. denitrificans genomic libraries. E. coli MV1184 and p U C l l 9 were also used for the expression of recombinant genes. Medium and growth condition P. denitrificans was grown at 30°C in a complex medium (1% Polypepton, 1% meat extract, 0.3% NaC1, 0.3% KNO3, pH7.0) under anaerobic conditions. For NIR purification, this microorganism was grown in a 101 jar-fermentor (Able Co., Tokyo) at 30°C for 72h using N2 gas as a carrier and KNO3 was added intermittently during cultivation so as not to be depleted. Recombinant E. coli was grown at 37°C in LB medium (23) containing 100 ~g/ml of ampicillin (LA medium) in a shake-flask, or on a 2% agar plate. To determine the activity of the recombinant NIR, LA medium containing 3.74/~g/ml of cupric sulfate and 10 /~g/ml of isopropyl-~-D-thiogalactopyranoside (IPTG) was used. Purification of NIR and protein sequencing techniques NIR from P. denitrificans was purified as described by Lain and Nicholas (16). The extent of purification was checked by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (22), and NIR was confirmed by nitrite reducing activity (16) and spectorophotometric analysis (17). To determine the amino acid sequence, the purified NIR was cleaved with trypsin (Boehringer Mannheim Yamanouchi Co., Tokyo) for 1 h at 37°C. The resultant peptide fragments on SDS-PAGE analysis were transferred onto a PVDF (polyvinylidene difluoride) membrane (0.45/~m pore size; Nihon Millipore Co., Tokyo), and their N-terminal amino acid sequences were determined by a protein sequencer (Applied Biosystems Japan, Tokyo). DNA manipulation and screening of P. denitrificans libraries DNA manipulations were performed essentially as described by Maniatis et al. (23). The P. denitrificans genomic DNA libraries were constructed in the pOCT2 HindIII site and ~-DASH II SalI and XhoI sites. The libraries were screened with two kinds of 23-mer oligonucleotides synthesized on the basis of the internal amino acid sequences of purified NIR by a 381A DNA synthesizer (Applied Biosystems Japan). The libraries were spread on LA medium agar plates at a dilution of 1,000~ 3,000 colonies. After incubation at 37°C for 12 h, colonies were transferred onto a nitrocellulose membrane (0.45 mm pore size; Schleicher & Schuell, Dassel, Germany). After lysis of bacteria and binding of the liberated DNA to the nitrocellulose membrane were carried out, these blots were used for hybridization experiments. The hybridization probes were labeled at the 5' end with [r-32p]ATP (>3,000 Ci/mmol; Du Pont, Boston, USA) using a commercial kit (MEGALABEL TM kit; Takara Shuzo Co., Kyoto), and free isotope was removed by DE52 (Whatman Biosystems Co., Maidstone, England) chromatography. The hybridization was carried out with the following modifications; the formamide concentration was 10%, the hybridization solution contained 6 × SSC, and the incubation temperature was 37°C considering the melting temperature of oligonucleotides. These conditions were determined experimentally. After overnight incubation with gentle shaking, the membrane was washed four times with a solution of 6 × SSC supplemented with 0.1% of SDS at 42°C for 10min, and twice with a solution of 3 × SSC supplemented with 0.1% of SDS at 45°C for 10min.
CLONING OF NITRITE REDUCTASE GENE
After the washing steps, the membranes were exposed onto X-ray film (Fuji Film Co., Tokyo) for 2 or 3 d at - 7 0 ° C . Clones that showed a positive signal with both probes were picked up from the original plate and used for further studies. A HindIII-SalI DNA fragment which was labeled with [a-32p]dCTP (>3,000 Ci/mmol; Du Pont) by using a commercial kit (Random Primer DNA Lebeling Kit; Takara Shuzo Co.) was prepared as a DNA probe for the screening of ,t phage DNA libraries (Fig. 4). The recombinants screened by colony and plaque hybridizations were checked by Southern hybridization (24). The hybridization condition when an oligonucleotide was used as the probe DNA was the same as the screening of the DNA library. DNA sequence determination A 2.3 kbp DNA fragment, which has enough length for the entire nirS, was deleted using pUC118, p U C l l 9 , and a commercial kit (Kilo-sequencing deletion kit; Takara Shuzo Co.). Singlestranded template DNA was prepared using M13KO7 helperphage, and double-stranded template DNA was purified using PEG-precipitation and lithium chloride-precipitation. DNA sequencing was performed by the method of Sanger and Coulson (25) with fluorescent - 2 1 M 1 3 or M13 reverse primers, and analyzed on a model 373A automated DNA sequencing system (Applied Biosystems Japan). The sequencing data were processed and analyzed using the GENETYX program (Software Development Co., Tokyo). Preparation of cell extract Cells of recombinant E. coli strain were grown overnight at 37°C and collected by centrifugation at 4°C. They were washed once with 20 mM potassium phosphate buffer (pH 7.5) and disrupted by sonication (Ohtake Work Co., Tokyo) at 50 W for 5 min on ice. They were then centrifuged at 4°C, and the supernatant was collected as the crude extract for enzymatic and immunochemical assays. NIR assay The NIR activity of P. denitrificans or recombinant E. coli was assayed by measuring the residual nitrite concentration in the liquid phase as described by Lam and Nicholas (16). The concentrations of the product (NO and N20) in the gas phase were also determined directly by a C O / N O x analyzer (Best-Sokki Co., Tokyo) and gas chromatograph G2800 (Yanaco Co., Kyoto). One enzyme unit was defined as the activity required for the reduction of 1/zmol of nitrite, or the formation of 1/~mol of NO or N20 per min. Immunochemical techniques To prepare an antiserum against the NIR, mice (BALb 2C) were inoculated with 100 ~g of purified NIR with an equal volume of Freund complete adjuvand. After 2 weeks, each mouse was given a subcutaneous booster injection of 50 pg of purified NIR suspended in Freund complete adjuvand. After 2 weeks, each mouse was given an additional booster injection of 50/zg of purified NIR suspended in Freund incomplete adjuvand. At 3 d after the last booster injection, the mice were bled. After the blood samples had been allowed to clot overnight at 4°C, the serum was obtained by centrifugation and stored at - 7 0 ° C . After the serum was incubated with denatured E. coli MV1184 crude extract for 30 rain at room temperature, the supernatant was used for Western analysis. After crude extracts in SDS-polyacrylamide gel were transferred onto a nitrocellulose membrane (0.45/~m pore size; Schleicher & Schuell) by an electrophoretic transfer unit (LKB-produkter AB, Bromma, Sweden), recombinant NIRs were detected using a commercial kit (Blotting detection kit; Amersham
OHSHIMA ET AL.
J. FERMENT. BIOENO.,
C 1 2
B 1 2
A 1 2
kb "-11.5 "-6
FIG. 1. SDS-PAGE analysis of NIR. Lane 1, NIR peptides digested with trypsin; lanes 2 and 3, purified NIR (the amount applied was different in lanes 2 and 3); M, molecular standard mixture. The gel was stained with Coomassie brilliant blue R-250. J a p a n Co., Tokyo). Chemicals Restriction enzymes and other enzymes for gene m a n i p u l a t i o n s were o b t a i n e d from T a k a r a Shuzo Co. Molecular weight s t a n d a r d for S D S - P A G E were obtained f r o m Sigma Chemical Co. (St. Louis, M O , USA). All other reagent-grade chemicals were obtained f r o m W a k o Pure Chemicals Inc. (Tokyo).
RESULTS Purification of N I R f r o m P. denitriflcans and amino acid sequence determination Seventy kilodaltons o f N I R was purified as a h o m o g e n e o u s state judging by SDSP A G E used to analyze the a m i n o acid sequence (Fig. 1). Because the N-terminal a m i n o acid was blocked, the A I.-YLLDPAAPG~ESTQVHV--EDRP-QQI. 2.-TTEIEAERFLHDGGLDGS-CYFIYA--A-
FIG. 3. Southern analysis of the recombinant plasmid DNAs (A and B) and the recombinant phage DNA (C). A and B, Two plasmids (lanes 1 and 2) isolated from positive clones were completely digested with HindIII and hybridized with the Nir-I (A) and Nir-II (B) probes shown in Fig. 2. C, Two phage DNAs isolated from positive clones were digested with SalI (lane 1) and XhoI (lane 2), but hybridized with the HindIII-SalI fragment as a DNA probe (see Fig. 4). purified N I R was digested by trypsin to determine the internal a m i n o acid sequence o f the N I R . Figure 2A shows the a m i n o acid sequences o f three representative peptides. Two kinds o f oligonucleotide sequences (Nir-I and II) were synthesized on the basis o f the a m i n o acid sequences, as shown in Fig. 2B. These oligonucleotides were used as a mixture to screen the P. denitrificans genomic D N A libraries. Cloning and D N A sequence o f n i r S gene The N I R structural gene (nirS) was screened f r o m the P. denitrificans H i n d I I I D N A library with the oligonucleotides (Nir-I and II) as the p r o b e D N A . By colony hybridization, an 11.5 k b p H i n d I I I fragment which hybridized with both o f the probes was obtained, as shown in Figs. 3A and B. A l t h o u g h a 6 kbp fragment seemed to possess a sequence h o m o l o g o u s with Nit-I, the fragment was not related to nirS. This result was confirmed by genomic Southern hybridization from P. denitrificans (data not shown). Western analysis by using anti-serum against N I R A
3.-FNVYNTMT-TY II. i HindllI 11.5kbp
Nir-I GAATTTGGAATGAAAGAAATGGG G C T G G G C
Nir-II TT TAAT GTATATAATACAATGAC C C T C C T G G C C
FIG. 2. N-Terminal amino acid sequences of tryptic peptides (A) and oligonucleotide sequences used as probes for screening (B). Underlines indicate the amino acid sequences corresponding to the respective oligonucleotide sequences.
Sal 13kbp :: ::
!lac i i ~c
,. iltl Ulll'l~llHHllllllllliHllli]lillllllllllllilllllllllHlllfl HIIIIHHIllllliifltlllllHHHIHI~""-:: pNOH13
FIG. 4. Relative positions and restriction endonuclease map of DNA fragments around nirS (A) and construction of recombinant plasmids for the expression of NIR (B). Abbreviations: B, BamHI; H, HindIII; P, PstI; Sa, SalI; Sm, Sinai; X, XhoI.
VoL. 76, 1993
CLONING OF NITRITE REDUCTASE GENE
-241 G C A T C G G G G G C G G C A A A C G G C T C G G G C C G C G C C G G C A G G G C C A G C A G G A T C A G T G T C A G C -181 A G C G C C A G C A G C A G G C G G C T A G G C G C A T G A G A G G A T T T T C C G ~ G C A T T G C C A T C C G C G T C - 121 C C A G G G T1T C G T C C C G C T G A T C G C G C A A A T G C C G C A C C T C G G C ~ T T . ~ A A A G G T C A ~7G C _ . . -61 ~ C C G C G C G C ~ a A ~ G C G C C C A G G A A C G G C G T G A A C G C G C G C C A G C A A A ~ G C C A G 1 ~ E ] ~ G A C A A A G G A C C C C A T T C G C C A G A C C C G G C C T A C T G G C C T C G G C A G C C C T G G C C C TT ,~ ,~ Q a ~ P F ,~ ,~ P G L ,~ ,~ s ^ A L ,~ L 20 61 G T C C T T G G G C C G C T T G C G G T C G C C G C A C A G G A A C A G G C C G C C C C G C C A A A G G A T C C T G C C V L G P L A V A A Q E Q A A P P K D P A 40 121 G C C G C A C T C G A G G A T C A C A A G A C C A A G A C G G A C A A C C G C T A T G A A G C C C TC G C T G G A C A A A A L E D H K T K T D N R Y E A L A G Q 60 181 C C T T G C A C A G C A G G A C G T A G C G G C G C T A G G C G C C C C A A G G G C A T C C C G G C C C T G T C C G A C P C T A G R S G A R R P K G I P A L S D 80 241 G C C C A A T A C A A C G A A G C C A A C A A G A T C T A T T T C G A A C G C T G C G C C G G T T G C C A C G G C G T C A Q Y N E A N K I Y F E R C A G C H G V I00 301 C T G C G C A A G G G C G C G A C C G G C A A G G C G C T G A C C C C C G A C C T G A C C C G C G A C C T G G G C T T C L R K G A T G K A L T P D L T R D L G F 120 361 G A C T A C C TGCAAAC-C T T C A T C A C C T A C G G C T C G C C G G C G G G G A T G C C G A A C T G G G ~ A C C D Y L Q S F I T Y G S P A G M P N W G T 140 421 T C G G G C C A G C T G A C C G C C G A G C A G G T C G A C C T G A T G G C G A A C T A C C T G C T T C T G G A C C C G 481 G S c G G G c c Q A c L c G i T A T c ~ A Q T G ~ G G D G A L G c M c G ~ T C C T G G ~ A G G L a e L ~ G D ' G ~ G
_,_,_,_,_,_,__,_,_,_,_,._,_,_,_,_._,_,_,.,_ 541 C C G G A A G A C C G G C C G A C C C A G C A G G A A A A C G A C T G G G A T C T G G A A A A C C T G T T C A G C G T C ..P__F,._D__B_2_3__Q._Q. E N D W D L E N L F S V 601 A C G C T G C G C G A C G C C G G C C A G A T C G C G C T G A T C G A C G G G A C C A C C T A T G G G A T C A A G T C G T L R D A G Q I A L I D G T T Y G I K S 661 G T T C T C G A C A A C G G C T A T G C G G T G C A T A T C A G C C G C A T G T C C G C C T C G G G C C G C T A C C T G V L D N G Y A V H I S R M S A S G R Y L 721 T T C G T C A T C G G C C G C G A C G G C A A G G T C A A T A T G A T C G A C C T T T G G A T G A A G G A A C C C G C C F V I G R D G K V N M I D L W M K E P A 781 A C C G T G G C C G A G A T C A A G A T C G G C T C G G A A G C G C G T T C C A T C G A G A C C T C G A A G A T G G A G T V A E I K I G S E A R S I E T S K M E 841 G G C T G G G A G G A A A A A T A C G C T A T T G C C G G C G C C T A T T G G C C G C C G A A A T A C G T C A T C A T G G W E E K Y A I A G A Y W P P K Y V I M 901 T A C G G C A A C A C G C T G G A G C C G A T G A A G A T C C A G T C C A C G C G C G G C A T G A T C T A C G A C G A G Y G N T L E P M K I Q S T R G M I Y D E 961 C A G G A A T A C C A C C C C G A G C C G C G C G T A C C G G C G A T C C T G G C C A G C C A T T A C C G G C C C G A G Q E Y H P E P R V P A I L A S H Y R P E 1021 T T C A T C G T G A A C G T C A A G G A A A C G G G C A A C A T C C T G C T G G T C G A C T A C A C C G A C C T C A A G F I V N V K E T G N I L L V D Y T D L K 1081 A A C C T C A A G A C C A C C G A G A T C G A G G C A G A A C G C T T C C T G C A C G A C G G C G G C C T G G A C G G C
18o 200 220 240 260 280 300 320 340 360
1141 --S -H..R-...Y ..F --I - T - - - . .A - - _N_ . AA R N K L V V I D T 1201 A A G G A A G G C A A G C T G G T C G C G A T C G A G G A T A C C G G C G G C C A G A C C C C G C A T C C G G G C C G C K E G K L V A I E D T G G Q T P H P G R 1261 G G C G C G A A C T T C G T C C A C C C G A C C T T C G G G C C G G T C T G G G C G A C C T T G C A C A T G G G C G A C G A N F V H P T F G P V W A T L H M G D 1321 G A T T C G G T G G C G C T G A T C G G C A C C G A T C C C G A G G G C C A T C C C G A C A A T G C C T G G A A G A T C D S V A L I G T D P E G H P D N A W K I 1381 C T G G A C A G C T T C C C G G C G C T G G C C G T T G G C T C G C T C T T C A T C A A G A C G C A T C C G A A T T C G L D S F P A L A V G S L F I K T H P N S 1441 C A A T A T C T C T A T G T C G A C G C C A C G C T G A A C C C C G A G C C C G A G A T C T C G G G C T C G G T C G C G Q Y L Y V D A T L N P E P E I S G S V A 1501 G T C T T C G A T A C C A A G G C G A T T A C G G C C G A C G G T T C G G A C C C G G A G T T C A A G A C G C T G C C G V F D T K A I T A D G S D P E F K T L P 1561 A T C G C C G A A T G G G C C G G C A T C G C C G A G G G C C A G C C C C G C G T C G T G C A G G G C G A G T T C A A C I A E W A G I A E G Q P R V V Q G E F N 1621 A A G G A C G G C A C C G A G G T C T G G T T C A G C G T C T G G A A C G G C A A G G A C C A G G A A T C G G C G C T G K D G T E V W F S V W N G K D Q E S A L 1681 G T C G T G G T G G A C G A C A A G A C C C T C G A A C T G A A G C A C G T C A T C A A G G A C G A G C G G C T G G T G V V V D D K T L E L K H V I K D E R L V 1741 A C C C C C A C C G G C A A G T T C A A C G T C T A C A A C A C T A T G A C C G A C A C C T A T T G A 1792 G T c G A P G c ~ T T G G ~ F ~ N ' G ~ G G G Y ~ G ~ C ' G ~ G M ~ c c G c c ' c ~ G G - Y A G T A T T T C A A T G G
400 420 440 460 480 500 520 540 560 580
1852 GGGTTCGGACATGC4:CGGCAAGACGGTGACGAACGGGGCCGCGCAAG "GAAAGGGCCCGCC" 1912 G C A G C G G C G C G G A C G G T G C G G T G C G G G G C A ~ ~ - - - - G ~ G ~ G C C G G G T - C G A T C T G T A T C 1972 G G C G C A G G C C C G G G C G A C C G G A A T T G C T G A C G C T G C G G C 4 ~ T T T G C G G C T T T T G C A G C A G G
FIG. 5. DNA sequence of the gene encoding nirS from P. denitrificans. The deduced amino acid sequence is shown below. The solid underlines indicate the initial and stop codons. The boxed sequences are the putative ribosome-binding sites. The thin arrow lines indicate palindromic sequences for the possible transcription termination. The bold arrows indicate open reading frames in the 5' and 3' flanking region of nirS. The amino acid sequences of the isolated tryptic peptides are underlined with broken lines.
i00 3-~ 2- ~
J. FERMENT. BIOENG.,
- -2 - -3
i00 200 300 400 500 FIG. 6. Hydrophobicity profileofNIR.Thesolidlineindic~es
A 2 3 4 5
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thepossiblesignalpeptidesequenceregion. and the DNA sequence (see below) revealed that the 11.5 kbp HindlII fragment did not contain the entire nirS gene. To obtain the lacking structural region and its upstream region, new SalI and XhoI libraries of P. denitrificans were prepared using the ~ phage vector, and were screened by plaque hybridization with a 60 bp SalI-HindlII fragment as a probe. This small fragment was at the 5' terminal region of the 11.5 kbp HindlII fragment. In these 2 phage DNA libraries, overlapping DNA fragments, a 3 kbp fragment of Sail and an 8 kbp fragment of XhoI, were isolated from hybridized clones (Fig. 3C), and their relative locations were determined by restriction mapping, as shown in Fig. 4A. Figure 5 shows the DNA sequence of the nirS structural region, which consists of 1788 nucleotides; the value of the G + C content is 68%. Because the promoter consensus sequence in E. coli and B. subtilis could not be found in the upstream region of nirS but a putative ribosome binding site was observed, the nirS gene was presumed to be in an operon structure. In fact, parts of ORFs were found in the up- and downstream regions of nirS. On the basis of the deduced amino acid sequence, the size of NIR was presumed to be 65.5 kDal, which coincided with that of the purified NIR (Fig. 1). Figure 6 shows the hydrophobicity profile of NIR. The first 25 amino acids which made up the hydrophobic region suggested the existence of a signal sequence. NIR activity in recombinant E. coil Three different plasmids were constructed to analyze recombinant NIR in E. coli using pUCI19 as a vector, as shown in Fig. 4B. These recombinant E. coli produced a protein which cross-reacted with anti-NIR antibody, as shown in Fig. 7. Although the positive protein produced in E. coli/ pNOHB was of relatively low molecular weight (about 50 kDal) in Western analysis, both recombinant proteins produced in E. coli/pNOS3 a n d / p N O H 1 3 , which were at a similar level in gene expression, showed the same (about 70 kDal) molecular weight as the purified NIR from P. denitrificans. Table I shows the nitrite reducing activity, and NO and N20 forming activities. The crude extract from recom-
FIG. 7. Westernanalysis of crude extracts from E. coli harboring various recombinant plasmids. A, Western analysis using antiserum against NIR; B, SDS-PAGE profiles of purified NIR from P. denitrificans (lane 1), crude extract from E. coli/pNOH13 (lane 2), E. coli/pNOS3 (lane 3), E. coli/pNOHB (lane 4), and E. coli/pUC119 (lane 5), respectively. binant E. coli/pNOH13, which contains nirS and its downstream flanking region, showed the highest nitrite reducing activity toward NO2- as a substrate, evaluated from the decrease of nitrite concentration. However, it was difficult to evaluate the activity accurately from the decrease of nitrite concentration, since the host E. coli strain also had nitrite reducing activity (1.6 U/g-protein). The nitrite reducing activity of the host strain may be due to some assimilatory nitrite converting activity. The crude extract from recombinant E. coli/pNOHl3 showed significant activities of NO and N20 formation toward NO2- as a substrate, whereas the crude extract from E. coli/pUC119, or /pNOS3 which has the entire coding region of nirS, showed very weak activities of NO and N20 formation. These results suggested that only the nirS gene product could not have the nitrite reducing activity, and the downstream flanking region would support the activation of NIR. In addition, the downstream region seemed to have a nitric oxide (NO) reductase gene, because the crude extract from E. coli/pNOH13 had the highest NO reducing activity and N20 formation activity from NO as a substrate, as shown in Table 1. Since NO2- exists in the reaction solution and NO gas must diffuse into the solution through the gas-liquid interface, the activity of N20 formation from NO as a substrate may be detected as being lower compared with the case of NO and N20 formation from NO2 as a substrate.
TABLE 1. Specificactivities of crude extracts from various recombinant E. coli using nitrite (NO2-)or nitric oxide (NO) as a substrate Strain E. coli MV1184/pNOH13 E. coli MVllg4/pNOS3 E. coli MVII84/pUCI19 ND, Not detectable.
NO~-reduction (substrate) 2.02 1.58 1.60
Specific activity (U/g-protein) evaluated from the amount of NO formation N20 formation NO reduction (product) (product) (substrate) 0.26 0.17 0.22 0.05 0.04 0.12 0.03 0.03 0.10
N20 formation (product) 0.05 ND ND
VOL. 76, 1993
CLONING OF NITRITE REDUCTASE GENE
DISCUSSION The NIR structural gene of denitdfying Is. aeruginosa has already been cloned and the primary structure has been determined (21). Figure 8 shows a comparison of NIR from P. denitrificans and from Is. aeruginosa. High homology was observed between two NIRs throughout the entire coding region except for the N-terminal region, and the overall homology was calculated to be 62.2%. The size of NIR from P. denitrificans is 65.5 kDal (596 amino acids containing signal peptide sequences), which .is slightly larger than that from Is. aeruginosa (567 amino acids). Though the N-terminal amino acid of the mature NIR and the signal peptide length from P. denitrificans could not be determined exactly because of the modification of the Nterminal amino acid, the analysis of the hydrophobicity provided a representative profile of the periplasmic protein, as shown in Fig. 6. The c-heme binding peptides (21) nearby the N-terminal ends were well conserved in the two NIRs: there are two cysteines at positions 94 and 97 from the N-terminal end which are covalently bound to the protoporphyrin vinyl groups, and methionine at position 135, which was proposed as the sixth heme ligand on the basis of the amino acid alignment with the other bacterial heine c (Kalkkinen, N. and Ellfolk, N.: IUPAC l l t h Int. Symp. Chem. Natl. Prod., p. 79-82, 1978). Although the internal amino acid sequence of NIR from 1
P. denitrificans was highly homologous with that from Is. aeruginosa, the up- and downstream sequences of nirS were different between the two genomic DNAs. The gene encoding for cytochrome c-551, which is known as a direct electron donor of NIR, exists just downstream of nirS and these genes form an operon structure in Is. aeruginosa (19). However, no sequence like the cytochrome c-551 gene was observed in the 1.5 kbp downstream region of nirS in P. denitrificans (data not shown). Further determination of the DNA sequence of the flanking region around nirS is now in progress. Heterologous expressions of active metalloproteins which have heme, Mn, Zn, or other metal ions have scarcely been reported. Dikshit and Webster reported the expression of bacterial hemoglobin which contains the heme o from Vitreoscilla, the total heme content of the protein produced by recombinant E. coli was different from that of authentic bacterial hemoglobin (26). Recently, some genes of denitrifying enzymes were isolated. The gene of nitrous oxide (N20) reductase which contains a copper from denitrifying Pseudomonas stutzeri was isolated and the recombinant N20 reductase produced in E. coli was investigated by Viebrock and Zumft (27). They found that the product of the N20 reductase structural gene (nosZ) in recombinant E. coli was only an apoenzyme, which has no activity. They also reported that the insertional mutagenesis in the downstream region of nosZ
MRQRTPFARPGLLASAALALVLGPLAVAAQEQAAPPKDPAAALEDHKTKTDNR-YEALAG • ,..
SSHLYVDTTFNPDARISQSDAVFDLKNL ..... DAKYQVLPIAEWADLGEGAKRVVQPEY
• * * o . * . * . * . . * * * * * *
* * * * * * * * . . * * * * . *
* * * * * * * * * * * * * * * * * * * * * * * *
* ' * * . * ' ' * * * * * * . . * * .
. . . . . . .
* * * * * * * * * *
FIG. 8. Comparison of amino acid sequence of NIR from P. denitr~cans with that from I s . ae~ginosa. Underfines indicate the c-heme binding peptide (21).
OHSHIMA ET AL.
generated apoenzyme-synthesizing strains and this region consisted o f three gene loci (nosD, F, Y) which would contribute to the copper acquisition or copper processing for nitrous oxide reductase (28). NIR, which has the heme c d l instead of copper, seems to have a similar mechanism for the activation of NIR. Jiingest et al. investigated the function o f the genes a r o u n d nirS of P. stutzeri using insertional mutagenesis, and revealed the presence of a functional locus (or loci) to activate NIR at the 5 kbp downstream from nirS; the mutagenesis in this locus generates the n i r - m u t a n t strains which produce a p o - N I R (29). Therefore, some genes which contribute to the activating or processing o f heme or other metals seem to be necessary for the formation of active denitrifying enzymes, because all kinds of denitrifying enzymes contain either heme or other metals. In the present paper, the heterologous expression o f NIR from P. denitr~ficans was studied, and it was shown that E. c o l i / p N O H 1 3 , which contains nirS and its 10 kbp downstream flanking region, produced active NIR; however, E. c o l i / p N O S 3 , which contains only nirS, could not produce the active NIR, as shown in Table 1. These results strongly suggest that the activator gene of N I R is located in the downstream region of nirS, a n d that this gene has a similar function to that in Ps. stutzeri. Furthermore, the crude extract from E. c o l i / p N O H 13 showed NO reductase activity. It has been reported that NIRs containing heme c d l catalyze only the reduction of nitrite to NO (16, 18). Thus, NO reductase is presumed to be encoded in the 10 kbp downstream region o f nirS, but this hypothesis is not confirmed at this time. The sequence analysis and purification of this protein are now under study in order to reveal the function of the genes contained in pNOH13. ACKNOWLEDGMENTS This research was supported in part by a Grant-in-Aidfor Scientific Research (No. 04202109) from the Ministry of Education, Science and Culture of Japan, by Tokai Industrial Foundation, and by Chubu Electric Power Co. The authors wish to express their sincere thanks to the Gene Research Center of Nagoya University for the determination of the amino acid sequences, the synthesis of oligonucleotides, and technical advice in DNA sequencing. REFERENCES 1. Holi6, J., Weeinbrenner, Z., Czak6, L., and T6th, J.: Application of denitrifying microorganisms in waste water treatment. Biotechnol. Lett., 2, 87-92 (1980). 2. Prakasam, T. B.S. and Krnp, M.: Denitrification. J. Water Pollut. Control Fed., 52, 1195-1205 (1980). 3. Adam, J. W. H.: Health aspects of nitrate in drinking water and possible means of denitrification. Water SA, 6, 79-84 (1980). 4. Richard, Y., Leprinee, A., Martin, G., and Leblane, C.: Denitrification of water for human consumption. Prog. Water Technol., 12, 173-191 (1980). 5. Kurt, M., Dunn, I. J., and Bourne, J. R.: Biological denitrification of drinking water using autotrophic organisms with H2 in a fluidized-bed biofilm reactor. Biotechnol. Bioeng., 29, 493-501 (1987). 6. Mirvish, S.: Gastric cancer and salivary nitrate and nitrite. Nature, 315, 461-462 (1985). 7. Michalski, W. P. and Nicholas, D. J. D.: Molecular characterization of a copper-containing nitrite reductase from Rhodopseudomonas sphaeroides forma sp. denitrificans. Biochim. Biophys. Acta, 828, 130-137 (1985). 8. Michalski, W.P., Heiu, D.H., and Nicholas, D. J. D.: Purification and characterization of nitrous oxide reductase from
Rhodopseudomonas sphaeroides f. sp. clenitrificans. Biochim. Biophys. Acta, 872, 50-60 (1986). 9. Mellor, R.D., Ronnenberg, J., Campbell, W.H., and Dickmann, S.: Reduction of nitrate in water by immobilized enzymes. Nature, 355, 717-719 (1992). 10. Cox, R.B. and Quayle, J.R.: The autotrophic growth of Micrococcus denitrificans on methanol. Biochem. J., 150, 569571 (1975). 11. Haddock, B. A. and Jones, C. W.: Bacterial respiration. Bacteriol. Rev., 41, 47-99 (1977). 12. van Verseveld, H. W., Braster, M., Boogerd, F. C., Chance, B., and Stouthamer, A. H.: Energetic aspects of growth of Paracoccus denitrificans: oxygen-limitation and shift from anaerobic nitrate-limiting to aerobic succinate-limitation. Evidence for a new alternative oxidase, cytochrome al. Arch. Microbiol., 135, 229-236 (1983). 13. Calder, K., Burke, K. A., and Lascells, J.: Induction of nitrate reductase and membrane cytochromes in wild type and chlorate resistant Paracoccus denitrificans. Arch. Microbiol., 126, 149153 (1980). 14. van Verseveld, H.W., Meijer, E.M., and Stouthamer, A.H.: Energy conservation during nitrate respiration in Paracoccus denitrificans. Arch. Microbiol., 112, 17-23 (1977). 15. Forget, P.: Les nitrate-r~ductases bact&iennes: solubilisation, purification et propri6t6s de I'enzyme A de Micrococcus denitrificans. Eur. J. Biochem., 18, 442-450 (1971). 16. Lain, Y. and Nicholas, D. J. D.: A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans. Biochim. Biophys. Acta, 180, 459-472 (1969). 17. Newton, N.: The two-heam nitrite reductase of Micrococcus denitrificans. Biochim. Biophys. Acta, 185, 316-331 (1969). 18. Henry, Y. and Bessi~res, P.: Denitrification and nitrite reduction: Pseudomonas aeruginosa nitrite-reductase. Biochimie, 66, 259289 (1984). 19. Aral, H., Sanbongi, Y., Igarashi, Y., and Kodama, T.: Cloning and sequencing of the gene encoding cytochrome c-551 from Pseudomonas aeruginosa. FEBS Lett., 261, 196-198 (1990). 20. Canters, G. W.: The azurin gene from Pseudomonas aeruginosa codes for a pre-protein with a single peptide: cloning and sequencing for azurin gene. FEBS Lett., 212, 168-172 (1987). 21. Silvestrini, M.C., Galeotti, C.L., Gervais, M., Schinina, E., Barra, D., Bossa, F., and Brunori, M.: Nitrite reductase from Pseudomonas aeruginosa: sequence of gene and the protein. FEBS Lett., 254, 33-38 (1989). 22. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685 (1970). 23. Maniatis, T., Fritscb, E. F., and Sambrook, J.: Molecular cloning. A laboratory manual. Cold Spring Habor Laboratory Press, Cold Spring Habor, New York (1982). 24. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98, 503-517 (1975). 25. Sanger, F. and Couison, A. R.: DNA sequences with chain terminating inhibitors. Proc. Nalt. Acad. USA, 74, 5463-5467 (1977). 26. Diksbit, K.L. and Webster, D.A.: Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coil Gene, 70, 377-386 (1988). 27. Viebrock, A. and Zumft, W.G.: Molecular cloning, heterologous expression, and primary structure of the structural gene for the copper enzyme nitrous oxide reductase from Pseudomonas stutzeri. J. Bacteriol., 170, 4658-4668 (1988). 28. Zumft, W.G., Viebrock-Sambale, A., and Braun, C.: Nitrous oxide reductase from denitrifying Pseudomonas stutzeri; genes for copper-processing and properties of the deduced products, including a new member of the family of ATP/GTP-binding proteins. Eur. J. Biochem., 192, 591-599 (1990). 29. Jiingest, A., Braum, C., and Zumft, W.F.: Close linkage in Pseudomonas stutter of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification. Moi. Gen. Genet., 225, 241-248 (1991).