Team:Goettingen/Team/Array

Array Team

The genome of the Gram-positive model bacterium Bacillus subtilis contains the disA, cdaA, and cdaS genes encoding the three enzymatically active adenylate cyclases DisA, CdaA, and CdaS (Mehne et al. 2013). At least one of the three enzymes is needed for growth of the bacteria. By using the microarray technique we would like to analyze how many genes are regulated by c-di-AMP in B. subtilis. We are not only interested in the impact of c-di-AMP on the transcriptome, we also hope to identify novel regulatory elements (i.e. riboswitches) and also transcription factors that bind to the essential signaling molecule. In our experiments we use three different B. subtilis strains. The wild-type strain contains all three diadenylate cyclases. The second strain is a disA mutant strain lacking DisA, a protein that was shown to be involved in DNA metabolism (Witte et al. 2008). This strain should produce less c-di-AMP than the isogenic parent strain. The third strain synthesizes a hyperactive CdaS mutant variant, which produces a lot of c-di-AMP (Mehne et al. 2013). In our microarray experiments we will compare the transcriptomes of each mutant strain with that of the wild-type strain. Moreover, we want to generate a triple knock-out mutant (ΔdisA, ΔcdaA, ΔcdaS) lacking all diadenylate cyclases. We try to construct this mutant strain by feeding the cells with exogenous c-di-AMP that is commercially available.

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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