The primer sequences were as follows: napA (forward, 5′-CCGGCTATCGTGGCAAGA-3′; reverse, 5′-CGGGAAGCTGTCGACATTG-3′); nirK

(forward, 5′-CCGCGCGACGCAAA-3′; reverse, 5′-TCGAGCGTATCGGCATAGG-3′); norC (forward, 5′-AGCTCACAGAGCAGGAACTGAAC-3′; reverse, 5′-TGATGCGGCTCGTCCATT-3′); and nosZ (forward, 5′-CGAGGATCTCACGCATGGAT-3′; reverse, 5′-GCGGTGCAACCTCCATGT-3′). sMC00128 was used as an internal standard [49, 50] (forward, 5′-ACGAGATCGAGATCGCCATT-3′; reverse, 5′-CGAACGAGGTCTTCAGCATGA-3′). Each PCR reaction contained 7.5 μl of SYBR Green PCR master mix (PE Applied Biosystems), 5 μl of cDNA and various final concentrations of each primer depending on the studied gene. This concentration was 0.2 μM for norC and sMC00128 and 0.4 μM for napA, nosZ and nirK. The final volume of the PCR reactions MK-8776 was 15 μl. The real-time PCR reactions were

performed on a 7300 Real Time PCR System (PE Applied Biosystems). The initial denaturing time of 10 min was followed by 40 PCR cycles consisting of 95°C for 15 s and 60°C for 60 s. A melting curve was run after MEK162 chemical structure the PCR cycles. During real-time PCR, the efficiency of nirK gene amplification was approximately equal to that of the housekeeping (internal standard) gene; in this case, the comparative CT method (also called ∆∆CT method) was applied for relative quantification. For the other genes, the amplification efficiencies were different from that of the housekeeping gene; the comparative CT method could not be applied, and it was necessary to use the standard curve method. The data were analysed ioxilan using the 7300 System Software (PE Applied Biosystems). The gene expression values under different conditions were expressed relative to the values of cells incubated under an initial O2 concentration of 2% in the absence of nitrate. Acknowledgments This work was supported by a Fondo Europeo

de Desarrollo Regional (FEDER)-co-financed grant (AGL2010-18607) and grant click here AGL2009-10371 from the Ministerio de Economía y Competitividad (Spain). Grant S2009/AMB-1511 from the Comunidad de Madrid and support from the Junta de Andalucía to Group BIO-275 are also acknowledged. We thank G. Tortosa for technical support and A. Becker for providing the E. meliloti mutants. MJT was supported by a fellowship from the Consejo Superior de Investigaciones Cientificas I3P Programme. References 1. Bates BC, Kundzewicz ZW, Wu S, Palutikof JP: Climate Change and Water.Technical Paper of the Intergovernmental Panel on Climate Change. Geneva, Switzerland: IPCC Secretariat; 2008:210. 2. Gonzalez PJ, Correia C, Moura I, Brondino CD, Moura JJ: Bacterial nitrate reductases: molecular and biological aspects of nitrate reduction. J Inorg Biochem 2006,100(5–6):1015–1023.PubMedCrossRef 3. Kraft B, Strous M, Tegetmeyer HE: Microbial nitrate respiration–genes, enzymes and environmental distribution. J Biotechnol 2011,155(1):104–117.PubMedCrossRef 4.