Opportunistic for c-di-AMP synthesis, as well as phosphodiesterases

Opportunistic human
pathogens need to resist osmotic stress. To adapt to increasing osmolarity,
they control their potassium ion uptake. The signaling nucleotide cyclic
diadenosine monophosphate (c-di-AMP) inhibits proteins responsible for
potassium uptake (Schuster et al. 2016) and serves as a second messenger for
various signals (Römling 2008). Generally, bacteria can use signaling
nucleotides to react to changing conditions, as they serve as molecules to
respond rapidly to environmental changes (Schuster et al. 2016).

Bacteria
synthesising
c-di-AMP contain diadenylate cyclases (DAC) with a conserved catalytic domain
for c-di-AMP synthesis, as well as phosphodiesterases for the degradation of
cyclic dinucleotides (Mehne et al.
2013). Another protein containing a diadenylate cyclase activity is the DNA
integrity scanning protein (DisA), the activity of which is repressed when it
binds to branched DNA substrates, built during DNA replication through
double-strand breaks (Römling 2008). Although diadenylate cyclase activity was
first studied in Bacillus subtilis,
it is now clear that archaea, Gram- negative as well as Gram-positive bacteria
produce proteins containing a DAC domain, showing the importance of c-di-AMP
for cell stability (Mehne et al.
2013).

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For
Gram-positive bacteria, the amount of c-di-AMP in the cell is crucial for cell
proliferation. In Listeria monocytogenes,
in the absence of c-di-AMP an accumulation of the toxic compound (p)ppGpp
has been observed, which leads to cell growth arrest (Schuster et al. 2016), by inhibition of GTP
synthesis and transcriptional control of mRNAs. (Mehne et al. 2013)

Bacillus subtilis contains
three DACs, the cyclic di-AMP synthase S (CdaS)
for sporulation, the DNA- binding DisA protein and the membrane bound cyclic
di-AMP synthase A (CdaA), the only DAC known 
in Listeria monocytogenes
(Mehne et al. 2013) (Commichau et al. 2017).

In
Listeria monocytogenes as well as in
other firmicutes, the CdaA gene is
encoded through an evolutionarily conserved operon, together with its regulator
protein CdaR and the phosphoglucosamine mutase GlmM, which is involved in the
biosynthesis of the cell wall (Commichau et al. 2017). Previous
literature suggest that CdaR negatively controls c-di-AMP formation by
interacting with integral membrane protein CdaA. The study suggests interaction
between the signal peptide of CdaR and the transmembrane domain of CdaA while
the YbbR motifs of CdaR presumably act in the cytoplasm (Rismondo et al., 2016)

In
our study, we wanted to get a broader understanding concerning the
influence of CdaA on cell proliferation and potassium uptake. Therefore, we
continued to work on plate assays, as this method
offers the possibility  to observe an immediate
impact of different potassium concentrations on cell proliferation in interdependency of CdaA expression.
Additionally, we used a phosphatase and ß-glucosidase assay to investigate the CdaR
membrane topology to get further insights into how CdaR might sense and process
signals from the environment.

For the screening systems E.
coli cells were used, as its wildtype does not produce c-di-AMP in contrast
to many Grampositive bacteria. Controlling CdaA and therefore the c-di-AMP
levels might work as a target for new antibiotics, which would work against
pathogens like Staphylococcus aureus.
This is especially important, concerning growing resistance against established
antibiotics.