Ammonium induces differential expression of methane and nitrogen metabolism-related genes in Methylocystis sp. strain SC2 Bomba Dam, 1,2†§ Somasri Dam, 1,2‡§ Yongkyu Kim 1,2 and Werner Liesack 1,2 * 1 Max Planck Institute for Terrestrial Microbiology, 2 Center for Synthetic Microbiology (SYNMIKRO), Philipps-Universität Marburg, D-35043 Marburg, Germany. Summary Nitrogen source and concentration are major deter- minants of methanotrophic activity, but their effect on global gene expression is poorly studied. Methylocystis sp. strain SC2 produces two isozymes of particulate methane monooxygenase. These are encoded by pmoCAB1 (low-affinity pMMO1) and pmoCAB2 (high-affinity pMMO2). We used RNA-Seq to identify strain SC2 genes that respond to standard (10 mM) and high (30 mM) NH 4 + concentrations in the medium, compared with 10 mM NO3 – . While the expression of pmoCAB1 was unaffected, pmoCAB2 was significantly downregulated (log2 fold changes of -5.0 to -6.0). Among nitrogen metabolism-related pro- cesses, genes involved in hydroxylamine detoxifica- tion (haoAB) were highly upregulated, while those for assimilatory nitrate/nitrite reduction, high-affinity ammonium uptake and nitrogen regulatory protein PII were downregulated. Differential expression of pmoCAB2 and haoAB was independently validated by end-point reverse transcription polymerase chain reaction. Methane oxidation by SC2 cells exposed to 30 mM NH 4 + was inhibited at ≤ 400 ppmv CH4, where pMMO2 but not pMMO1 is functional. When trans- ferred back to standard nitrogen concentration, methane oxidation capability and pmoCAB2 expres- sion were restored. Given that Methylocystis contrib- utes to atmospheric methane oxidation in upland soils, differential expression of pmoCAB2 explains, at least to some extent, the strong inhibitory effect of ammonium fertilizers on this activity. Introduction Aerobic methanotrophic bacteria, or methanotrophs, are crucial players in the global cycle of the greenhouse gas methane. They attenuate methane emission from major sources and constitute the only biological sink for atmos- pheric methane in upland soils. Their activity in soil is highly sensitive to nitrogen amendment. Whether the amendment has inhibitory or stimulatory effects depends largely on the diversity, structure and activity of the methanotrophic community, as well as the type and amount of nitrogen fertilizer applied and the exposure time (Schnell and King, 1994; Dunfield and Knowles, 1995; King and Schnell, 1998; Bodelier and Laanbroek, 2004; Mohanty et al., 2006; Stein et al., 2012). The appli- cation of ammonium fertilizers to various soils and sedi- ments has been shown to inhibit methanotrophic activity (Van Der Nat et al., 1997; Bodelier and Laanbroek, 2004). Long-term effects were observed particularly for atmospheric methane oxidation in various upland soils (Gulledge and Schimel, 1998; Bodelier and Laanbroek, 2004). The key players of atmospheric methane oxidation are thought to be uncultured groups of poorly character- ized methanotrophs named Upland Soil Cluster alpha and gamma (Knief et al., 2003; Dunfield, 2007). Methylocystis spp. are also widely distributed in upland and hydromorphic soils and presumably make a major contribution to the oxidation of atmospheric methane (Knief and Dunfield, 2005; Knief et al., 2005; 2006; Baani and Liesack, 2008). On the other hand, methanotrophs, like all bacteria, require nitrogen for growth. Therefore, contrary to the inhibitory effects observed, ammonium fertilization may stimulate methane oxidation activity in some cases (Bodelier and Laanbroek, 2004; Stein et al., 2012). For example, methanotrophic activity and growth were found to be stimulated after ammonium fertilization in the root zone of rice plants (Bodelier et al., 2000; Krüger and Frenzel, 2003). Later studies suggested that nitro- genous fertilizers stimulate consumption of methane and Received 6 September, 2013; accepted 14 December, 2013. *For correspondence. E-mail liesack@mpi-marburg.mpg.de; Tel. (+49) 6421 178 720; Fax (+49) 6421 178 809. Present addresses: † Microbiology Laboratory, Department of Botany, Visva-Bharati, Santiniketan – 731235, West Bengal, India; ‡ Department of Biomedi- cal Laboratory Science and Management, Vidyasagar University, Midnapore – 721102, West Bengal, India. § These authors contributed equally to the study. Environmental Microbiology (2014) 16(10), 3115–3127 doi:10.1111/1462-2920.12367 © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd