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 OHSHIMA, 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 reduc- tase (NIR) was predicted to be 65.5 kDai from the amino acid sequence, which was similar to the value deter- mined 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 activ- ity. 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. Denitrification is a respiratory mode of energy conver- sion, 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 microor- ganisms (3-5), because it has been reported that many sources of drinking water, especially in areas of intensive agriculture, contain intolerably high nitrate ion concentra- tions (6). However, biological denitrification by microor- ganisms generally requires water enriched with nutrients to support their growth (7, 8), which results in contamination by these other nutrients. Furthermore, these microorgan- isms 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 bacteria- free bioreactor system for denitrification using denitrify- ing 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 en- zymes; 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 bacte- rium can grow autotrophically on hydrogen and carbon dioxide as well as methylotrophically with methanol or methylamine using an oxygen as a terminal electron ac- ceptor (10). The microorganism also can grow heterotro- * Corresponding author. 82 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 anaer- obic 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 1.9.3.2) or copper (EC 1.7.2.1) is known to be the key en- zyme in yielding the first gaseous product. NIRs have been isolated from various denitrifying bacteria (18-21). Cyto- chrome cdl from Pseudomonas aeruginosa has been studied extensively (18). This protein is known as a peri- plasmic 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). How- ever, heterologous expression of active NIR has not been reported, making the further development of a denitrify- ing 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 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