Physiological and Molecular Biological Characterization of Ammonia Oxidation of the Heterotrophic Nitrifier Pseudomonas putida Michael Daum, 1 Wolfgang Zimmer, 1 Hans Papen, 1 Karin Kloos, 2 Kerstin Nawrath, 2 Hermann Bothe 2 1 Fraunhofer Institut fu ¨r Atmospha ¨rische Umweltforschung, Kreuzeckbahnstr. 19, D-82467 Garmisch-Partenkirchen, Germany 2 Botanisches Institut, Universita ¨t zu Ko ¨ln, Gyrhofstr. 15, D-50923 Ko ¨ln, Germany Received: 9 April 1998 / Accepted: 15 May 1998 Abstract. The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxyl- amine, nitrite, and nitrate. Product formation was accompanied by a small but significant release of NO, whereas N 2 O evolution could not be detected under the assay conditions employed. The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions. Aerobically grown cells utilized -aminobutyrate as a carbon source and as a N-source by ammonification. The physiological experiments, in particular the inhibition pattern by C 2 H 2 , indicated that P. putida expressed an ammonia mo- nooxigenase. DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers. In nitrification, bacteria oxidize ammonia to nitrite and nitrate. These end products are easily drained out to the ground water, assimilated by plants and bacteria, or converted to gaseous N-compounds by denitrification. Classically, nitrifying bacteria are chemoautotrophs [24]. Relatively few of these bacteria can be grown under laboratory conditions, and their growth rates are usually low. In soil, nitrifying bacteria may represent an essential part of the unculturable population. Scattered in the literature are reports that many soil nitrifiers are not restricted to an autotrophic mode of life but can catabo- lize organic soil compounds. Such heterotrophic nitrifiers have been characterized only poorly, although some are culturable [18, 19, 25]. In the present study, nitrification of the heterotrophic nitrifier Pseudomonas putida was investigated. This bacterium was isolated from the upper soil layer of a Norway spruce forest (‘‘Ho ¨glwald’’ between Munich and Augsburg). The present communication will report that this bacterium oxidizes NH 4 Cl in the presence of diverse organic compounds. Product formation of ammonia oxi- dation will also be described. The isolate also reduces nitrate anaerobically. DNA isolated from P. putida hybrid- ized with an amoA probe coding for the smaller subunit of the ammonia monooxigenase from Nitrosomonas europaea. The physical map of the region and the sequence data of the gene from P. putida will be presented. Materials and Methods Origin of Pseudomonas putida isolate. Bacteria from the organic soil layer (upper 5 cm) of a Norway spruce (Picea abies) forest (Ho ¨glwald, 40 km northwest of Munich) were separated aerobically on peptone meat extract (5 g peptone + 3 g meat extract per L, pH 7.0) with agar plates. Single colonies were transferred into liquid media of the same composition and selected for their capability to form nitrite aerobically [18]. The heterotrophically nitrifying isolate chosen for the present study was classified as Pseudomonas putida DSMZ-1088-260 by the Deutsche Sammlung fu ¨r Mikroorganismen (Braunschweig, Germany) because of its morphologic and physiological properties (e.g., growth on acetate, caprate, citrate, phenylacetate, L-arabinose, D-galactose, fructose, glucose, gluconate, D-alanine, L-serine, aerobic acid produc- tion from glucose, fructose, and xylose, nitrite formation from nitrate) and its missing production of a fluorescent dye on Pseudomonas-F- Agar. Growth conditions. The P. putida isolate was grown aerobically with shaking at 25°C in peptone meat extract (5 g peptone + 3 g meat extract per L, pH 7.0) or on mineral medium containing the following salts, in mM: 4.6 NH 4 Cl, 0.16 MgSO 4 , 0.5 CaCl 2 , 0.036 FeSO 4 as EDTA complex, 0.0001 CuSO 4 , 2.0 K-phosphate buffer pH 7. Glucose, glutamine, citrate, malate or -aminobutyrate (20 mM), respectively, Correspondence to: W. Zimmer CURRENT MICROBIOLOGY Vol. 37 (1998), pp. 281–288 An International Journal  Springer-Verlag New York Inc. 1998