Nitrificacion De Enzimas

JOURNAL OF BACTERIOLOGY, May 2002, p. 2557–2560 0021-9193/02/$04.00 0 DOI: 10.1128/JB.184.9.2557–2560.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 184, No. 9

Nitrite Reductase of Nitrosomonas europaea Is Not Essential for Production of Gaseous Nitrogen Oxides and Confers Tolerance to Nitrite
Hubertus J. E. Beaumont,1 Norman G. Hommes,2 Luis A.Sayavedra-Soto,2 Daniel J. Arp,2 David M. Arciero,3 Alan B. Hooper,3 Hans V. Westerhoff,1 and Rob J. M. van Spanning1*
BioCentrum Amsterdam, Department of Molecular Cell Physiology, Vrije Universiteit, NL-1081 Amsterdam, The Netherlands1; Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331-29022; and Department of Biochemistry, Molecular Biology and Biophysics,University of Minnesota, St. Paul, Minnesota 551083
Received 27 November 2001/Accepted 1 February 2002

A gene that encodes a periplasmic copper-type nitrite reductase (NirK) was identified in Nitrosomonas europaea. Disruption of this gene resulted in the disappearance of Nir activity in cell extracts. The nitrite tolerance of NirK-deficient cells was lower than that of wild-type cells.Unexpectedly, NirK-deficient cells still produced nitric oxide (NO) and nitrous oxide (N2O), the latter in greater amounts than that of wild-type cells. This demonstrates that NirK is not essential for the production of NO and N2O by N. europaea. Inactivation of the putative fnr gene showed that Fnr is not essential for the expression of nirK. Oxidation of ammonia (NH3) to nitrite (NO2 ) is the first step inthe nitrification process, which plays an essential role in the global nitrogen cycle. Nitrification involves the oxidation of NH3, via NO2 , to nitrate (NO3 ) by the combined actions of NH3- and NO2 -oxidizing bacteria. The NH3-oxidizing bacteria are lithoautotrophs that harvest their vital free energy in the oxidation of NH3 to NO2 . This process is carried out by the sequential actions ofmembrane-bound ammonia monooxygenase, which oxidizes NH3 to hydroxylamine (NH2OH), and periplasmic hydroxylamine oxidoreductase (HAO), which oxidizes NH2OH to NO2 (24). NH3-oxidizing bacteria utilize molecular oxygen both for the formation of NH2OH from NH3 and as the terminal acceptor of electrons from the respiratory chain (24). Alternatively, NO2 and nitric oxide (NO) may also be used as terminalelectron acceptors via a putative denitrification pathway. Homologues of genes encoding the denitrification enzymes nitrite reductase (Nir) and nitric oxide reductase (Nor) are present in the genome of the NH3-oxidizing bacterium N. europaea (22). NH3-oxidizing bacteria produce NO, nitrous oxide, and, in some cases, N2 during growth on NH3. The mechanisms that underlie the production of these nitrogenousgases include the dissimilatory reduction of NO2 (15–17). An enzyme with Nir activity has been isolated from N. europaea (3, 6, 12, 13, 18). The ability of N. europaea to use NO2 as an alternative electron acceptor suggests that the pathway may act as an alternative mode of respiration, as in the “true” denitrifying bacteria (1, 19). Alternatively, the denitrifying enzymes may serve to protectNH3-oxidizing bacteria from the negative effects of NO2 produced during growth (16, 20). On the other hand, the nitrification pathway may also be involved in the production of NO and N2O by NH3-oxidizing bacteria. HAO of N. europaea
* Corresponding author. Mailing address: Department of Molecular Cell Physiology, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, NL-1081 HV Amsterdam, TheNetherlands. Phone: 31-204447179. Fax: 31-20-4447229. E-mail: spanning@bio.vu.nl. 2557

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has been shown to produce NO and N2O during the oxidation of NH2OH in vitro (6, 8). Whether HAO also contributes to the production of these gases in vivo is unclear We disrupted the putative nirK gene of N. europaea to learn whether it encodes a functional...