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Firmicutes such as S. aureus and Bacillus subtilis,
alarmones don’t directly bind to RNAP and there is no DksA co-factor (Krasny et al., 2004).
 Instead, many genes are indirectly
regulated through the lowering
of GTP pool that accompanies alarmone production. In B. subtilis, ribosomal promoters initiate with GTP
which is the transcription-initiating nucleotide (iNTP)
and therefore, Low GTP pool that occurs in stringent response leads to
inhibition rRNA promoters ( Krásný et al., 2004) (Krásný et al., 2008).
 Also in S. aureus,
ribosomal genes such as rpsL (encoding
for ribosomal protein S2) are repressed under stringent conditions due to low
GTP pool (Geiger et
al., 2010).

alarmones can indirectly affect gene expression through CodY repressor.
In B.
subtilis, CodY
is activated by GTP under normal conditions and represses genes having CodY
conserved motif through inhibition of RNAP (Handke et al., 2008).
Under stringent conditions, CodY is not activated since there is a decrease in
the level of GTP. Therefore, it does not bind to its conserved motif on DNA
leading to de-repression (activation) of CodY-dependent genes (Shivers
et al., 2004). In S.
aureus, CodY repressor is activated by GTP in addition to branched chain
amino-acids leading to repression of genes such as virulence genes and genes
involved in transport and metabolism of amino acids and nitrogen. In stringent
response, low GTP pool and the descent of level branched chain amino-acids
leads to activation of CodY regulated genes in S. aureus (Pohl et al., 2009)
 as illustrated in Figure (1.5).

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Figure (1.5) Scheme of the role of CodY repressor in S. aureus under nutrient rich
conditions (left) and nutrient limitation (right).
Under nutrient rich conditions, there are high levels of GTP and BCAA which
bind and activate CodY repressor. CodY binds to its conserved CodY box on
DNA and inhibits RNAP leading to repression of genes.
Under nutrient limitation, there is low level of BCAA. In addition, RSH is
activated and synthesizes (p)ppGpp
leading to a decrease in the level of GTP. CodY does not bind to its motif
leading to activation of genes required for stringent response (Geiger et
al., 2014).














When S. aureus is treated with
Mupirocin, (p)ppGpp are synthesized with the induction of transcriptional
changes similar to those occurring in isoleucine depletion (Geiger et al., 2010).
Mupirocin is a potent antibiotic which mimics isoleucine starvation through
inhibition of bacterial isoleucyl tRNA synthetase. Mupirocin is a
structural analogue of isoleucyl-adenylate (Ile-AMP) where it competes with Ile-AMP
on binding sites of isoleucyl tRNA synthetases. Therefore, mupirocin leads to
the accumulation of uncharged isoleucyl –tRNAs which is a signal of amino-acid
depletion in the cell that activates the stringent response (Nakama et al., 2001) (Reiß et al., 2011).


1.4 Protein-Protein Interactions

Interactions seem to play an important role for the correct function
of RSH. It was shown that in E. coli, interactions
are crucial for regulating the activity of RelA where it is active in a monomeric
state and inactive in an oligomeric state. In addition, it was shown that the
C-terminal domain is the part involved in RelA-RelA interactions (Gropp et al., 2005). Furthermore, RelMtb which is a
bifunctional RelA/SpoT homologue in Mycobacterium tuberculosis was
reported to form trimers and the removal of the
C-terminal part results in a monomeric state indicating its essentiality for
multimerization. It was also shown that the trimer state is less active than
monomer state indicating that interactions affect the enzyme functionality (Avarbock et al., 2005). However, protein-protein interactions between RSH
from S. aureus have not been tested before.



1.5  S. aureus
resistance to oxidative stress

Bacteria can encounter oxidative stress endogenously (inside the bacteria)
or exogenously (from an outside source). Endogenous oxidative stress can take
place during bacterial aerobic respiration and intracellular redox reactions.
In aerobic respiration, oxygen is the final electron acceptor where it is
completely reduced forming H2O. Sometimes, incomplete reduction of
oxygen takes place when oxygen interacts with reduced FAD cofactor leading to
generation of reactive oxygen species (ROS) such as superoxide anion (O2?)
and hydrogen peroxide (H2O2) (Massey et al., 1969)
(Korshunov et al., 2010).

In addition, iron in ferrous form
can react with H2O2 generating hydroxyl radicals (HO•) in a
reaction called Fenton reaction (Imlay et al., 1988)
(Repine et al., 1981).
In addition to endogenous oxidative stress, S. aureus encounters exogenous oxidative
stress through host innate immune cells as macrophages and neutrophils. These
immune cells have NADPH oxidase enzyme (NOX) which reduces oxygen into O2?
in oxidative burst. Dismutation of O2?can
take place generating H2O2 which can be further converted
by myeloperoxidase enzyme into cytotoxic hypochlorite ion (OCl-) (Panday
et al., 2014)
(Harrison et
al., 1976).

Moreover, immune cells can produce
Nitric oxide (•NO) which is a cytotoxic reactive
oxidant. •NO can further react with O2? producing
peroxynitrite (OONO?)
which is a highly reactive bactericidal compound (Huie
and Padmaja, 1993). ROS and nitrosative
stressors damage bacterial DNA and proteins. An illustration of the oxidative
stressors to S. aureus is shown in figure

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