Array Team
With global target analysis, which were performed in collaboration with the iGEM Groningen Team, we were interested in identifying novel c-di-AMP regulatory elements that control gene expression in Bacillus subtilis.
Our goal was to look for genes, whose expression level is affected by the level of c-di-AMP. Therefore we compared the wildtype with a hyperactive strain of Bacillus, which produces more c-di-AMP (Fig. 1).
By doing so, we are able to: first, find other compartments which also respond to c-di-AMP and therefore can be used to construct our reporter system, second we can shed light on the signaling web of c-di-AMP.
Fig.1: C-di-AMP concentrations determined via liquid chromatography-coupled tandem mass spectrometry method by our collaboration partner from the Hannover Medical School. In blue the c-di-AMP amount in low phosphate medium is shown, in red the c-di-AMP amount in high phosphate medium. We compared the wildtype and a hyperactive strain (1344) of Bacillus.
The microarray analysis in Groningen gave us a first glimpse on all the genes, which are affected by the c-di-AMP level. We were especially interested in ydaO. From a poster of another workgroup we knew that ydaO is connected to an upstream c-di-AMP sensing riboswitch. This means that in the presence of c-di-AMP the riboswitch will assemble and prohibit the expression of ydaO. Without c-di-AMP the riboswitch cannot be established and ydaO will be expressed. Therefore, ydaO was a perfect candidate for us to build another reporter around it. As the microarray analysis revealed, the expression of ydaO was indeed affected by c-di-AMP levels. To further analyse these results, we did some qRT-PCR analysis back in Göttingen (Fig. 2).
Fig.2: qRT-PCR analyses of c-di-AMP affected ydaO and a of control gene. The analyses revealed that in comparison to the wild type ydaO is upregulated with low c-di-AMP levels.
Our qRT-PCR analyses showed that ydaO is upregulated with low c-di-AMP levels. This means that the expression of ydaO is supposedly linked to the c-di-AMP levels inside the cell.
References
1. Mehne et al. (2013) Cyclic di-AMP homeostasis in Bacillus subtilis: both lack and high level accumulation of the nucleotide are detrimental for cell growth. J. Biol. Chem. 288:2004-2017.
2. Witte et al. (2008) Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. Mol. Cell. 30:167-178.
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