Under nitrogen limitation, the intracellular glutamine levels are low and the bifunctional enzyme GlnD covalently links a UMP group to each monomer of PII. Conversely, when fixed nitrogen is abundant, GlnD binds glutamine, switching its enzymatic activity to perform PII deuridylylation (Jiang et al., 1998). The ability of PII proteins to sense carbon and energy levels is mediated by noncovalent binding of key metabolites such as 2-oxoglutarate and ATP and ADP (Jiang & Ninfa, 2007). The binding of these molecules to each

PII trimer regulates its interaction with different protein targets. Herbaspirillum seropedicae encodes two PII proteins, GlnB and GlnK (Benelli et al., 1997; Noindorf et al., 2006). For the vast majority I-BET-762 chemical structure of bacteria studied so far, the glnK gene is cotranscribed with the ammonium transporter amtB (Thomas et al., 2000). In H. seropedicae, amtB and glnK are coexpressed with a third gene, orf1, and expression of the orf1amtBglnK operon is induced under nitrogen limitation (Noindorf et al., 2006). The H. seropedicae glnB gene is apparently monocistronic and expressed constitutively

(Benelli et al., 1997). Although the PII proteins have been historically described as cytosolic proteins, recent data from several bacteria species and from Archea indicated that under certain conditions the PII proteins can be found in association with the cytoplasmic membrane (Tremblay & Hallenbeck, 2008). This association LDK378 mw is due to the

formation of a complex between PII proteins and the ammonium transporter AmtB. In Proteobacteria, the AmtB–PII complex formation is regulated by the availability of ammonium in the medium (Coutts et al., 2002). When ammonium-starved cells receive an ammonium shock, the PII proteins are deuridylylated and bind to AmtB in the cell membrane. Complex formation blocks the ammonia channel of AmtB (Conroy et al., 2007; Gruswitz et al., 2007) and significantly reduces the availability of PII protein in Cell press the cytoplasm (Javelle et al., 2004). Recently, it was observed that the AmtB–PII complex can direct other PII targets, namely the transcriptional regulator TnrA in Bacillus subtilis (Heinrich et al., 2006) and the DraG enzyme in Azospirillum brasilense (Huergo et al., 2006, 2007) to the cell membrane, thereby potentially regulating their activities. To determine whether membrane association of PII proteins might also play a role in the regulation of the nitrogen metabolism in H. seropedicae, we investigated the dynamics of membrane-associated proteins according to the ammonium levels using two-dimensional (2D) gel electrophoresis and MALDI-TOF-TOF MS analysis. Herbaspirillum seropedicae wild-type or amtB mutant strains (Noindorf et al., 2006) were cultivated in NFbHP-malate medium (Klassen et al., 1997) containing 5 mM glutamate (low-nitrogen, −N) or 20 mM NH4Cl (high-nitrogen, +N) as nitrogen source. Cells were grown at 30 °C in a shaker (120 r.p.m.