A global transcription study of the regulatory effects of potassium, glutamate and c-di-AMP in Bacillus subtilis

Since c-di-AMP has been implicated in the interplay of potassium and glutamate homeostasis, we analysed, using strand-specific tiling arrays (tiling step of 22 nucleotides), global gene expression in the wild type strain B. subtilis 168 at low (0.1 mM) and high (5 mM) potassium concentrations and in the presence of ammonium and glutamate as the nitrogen source as well as in the c-di-AMP free strain (∆disA, ∆cdaA, ∆cdaS) GP2222 and its isogenic suppressor mutant GP2223 that is able to grow at 5 mM potassium. In addition to strongly reduced expression of genes involved in fermentation and respiration (regulated by the NADH-responsive transcription factor Rex), genes of the SigO regulon involved in acid stress response and several competence genes was severely reduced in the mutant strain GP2222. In the wild type strain, the ktrAB and kimA genes were most strongly repressed by potassium. These genes encoding high affinity potassium uptake systems are controlled by a c-di-AMP sensitive riboswitch.Taken together, the analysis confirmed that c-di-AMP is involved in multiple cellular functions including genetic competence and revealed a particular role of genes involved in controlling NADH homeostasis. Cells were grown aerobically at 37 °C in a modified Spizizen's minimal medium (KH2PO4 was replaced by NaH2PO4, named as MSSM) with ammonium or glutamate as the nitrogen source and with 0.1 mM or 5 mM potassium. Cells were harvested in the mid exponential phase at an optical density at 600 nm of 0.4-0.6. Hybridizations of strand-specific tiling arrays were performed with RNA from two biological replicates. cDNA samples were labeled with Cy3.