An ammonium transporter of was characterized. cereals (68). Many studies have indicated that promotes plant growth, but the exact mechanism of growth promotion has not been fully characterized. Like most organisms, buy 130641-38-2 uses ammonium salts as a preferred nitrogen source (53). In the absence of combined nitrogen and under microaerobiosis conditions, the nitrogenase enzyme complex is synthesized and converts atmospheric N2 buy 130641-38-2 to NH4+. Unprotonated NH3 is predicted to diffuse out of bacterial cells due to a concentration gradient across the plasma membrane (36). The pH gradient (generally slightly more alkaline inside the bacteria) enhances this process. Therefore, an active ammonium uptake system is required to retain the intracellular fixed nitrogen, acquired at high energy cost by the nitrogenase. Hartmann and Kleiner (24) have shown that ammonium uptake in spp. is energy dependent, follows the Michaelis-Menten kinetics, and is repressed by ammonium. No functional characterization of genetic components of this system has yet been reported. Recently, genes encoding ammonium buy 130641-38-2 transporter proteins and putative ammonium transporter proteins have buy 130641-38-2 been reported for (43), (52), and (tomato) (39). In the gene, whose corresponding amino acid sequence is homologous to those of NH4+ transporter proteins, is part of the dicistronic operon (76). possibly encodes a nitrogen regulatory protein homologous to PII proteins, but the biochemical functions of the and gene products in have not been reported. The operon is highly expressed during nitrogen-limited growth. More recently, Siewe et al. (61) characterized the first reported prokaryotic NH4+ transporter gene (gene in which complemented a mutant with less than 10% of the parental CH3NH3+ uptake activity. The complete sequence was published by Fabiny et al. (19). An analysis of the deduced amino acid sequence of the product of gene corresponded to gene in K-12. The gene product is homologous to transmembrane NH4+ transporters, but its functional characterization has not yet been reported. The gene is cotranscribed with encodes a second PII-like protein (6, 70). The gene product and the gene product (PII) are known to play a role in the reversible adenylylation of glutamine synthetase (GS) in response to the nitrogen status of the cells. In addition, PII stimulates the kinase-phosphatase enzyme, NtrB, to dephosphorylate the phosphorylated transcriptional activator NtrC. Phosphorylated NtrC is necessary to activate transcription from several RpoN-dependent promoters (reviewed in reference 65; 41, 46). Two PII homologs have been identified in (14, 15). is part of the nitrogen-regulated, but NtrC-independent, operon, and its product is required for nitrogen fixation. In contrast to what is found for other species, PII (gene product) is not involved in the ammonium control of GS activity by adenylylation. The level of expression is, however, lower in mutant strains than in the wild-type strain (14, 15). The second PII-like protein of have been characterized (40, 48). NtrC is involved in nitrate utilization (40) and (methyl)ammonium uptake (69). Notably, the mutant has a pleiotropic effect: nitrogen fixation, nitrate assimilation, ammonium uptake, and flagellar biosynthesis are impaired (48). We report here the isolation and characterization of a nitrogen-regulated (methyl)ammonium transporter gene from and strains used are listed in Table ?Table1.1. Plasmids mentioned in the text are also described in this table. Sequencing constructs and intermediate constructs are not given. A genomic library of Sp7 was constructed by ligation of fragments generated by partial HB101, and selected for isolation of tetracycline-resistant colonies. strains were grown in Luria-Bertani (LB) medium (57) at 37C. was grown in LB medium supplemented with 2.5 mM CaCl2 and 2.5 mM MgSO4 (LB* medium) at 30C. For solid media, 15 g of agar per liter was added. Conjugal transfers of recombinant plasmids, derived either from pLAFR1 or pLAFR3, from to were performed on D plates (containing 8 g of Bacto nutrient broth [Difco], 0.25 g of MgSO4 7H2O, 1.0 g of KCl, and 0.01 g of MnCl2 per liter). After conjugation, MMAB minimal medium (71) with 0.5% malate as the C source was used for selection of transconjugants. MMAB medium was also used in growth experiments and in [14C]methylammonium uptake, nitrogenase activity, and -glucuronidase assays. Growth rates CD8B in liquid minimal medium supplemented with 20 mM NH4+, 2 mM NH4+, 8 mM nitrate, or 10 mM aspartate as the nitrogen source were measured by.