Biology Reference
In-Depth Information
An up-to-date approach set up by
Coppola et al. (2013)
to establish the
participation of
Ph
-2/2HbO in RNS detoxification, tested the influence of
heterologous expression of the
PSHAa0030
gene
in vivo
on protection from
NO toxicity in a NO-sensitive
E. coli
strain (
E. coli hmp
, defective in the
FHb) (see
Section 4.1
).
The growth properties and O
2
uptake of
E. coli hmp
having the
PSHAa0030
gene was analysed in an attempt to demonstrate that
Ph
-2/
2HbOoffers resistance to nitrosative stress.Wild-type
E. coli
and a
hmp
mutant,
carrying the
PSHAa0030
gene or not, were grown at 25
C under aerobic
conditions and treated with either the NO-releaser DETA-NONOate or
the nitrosating agent GSNO. As expected, exposure to these sources of NO
has no effect on the growth of wild-type
E. coli
or the expression of Hmp from
the complemented plasmid, and a comparable level of resistance is evident in
cells expressing
Ph
-2/2HbO. In contrast, in the absence of
Ph
-2/2HbO,
expression results in severe growth inhibition (
Coppola et al., 2013
).
Moreover,
Coppola et al. (2013)
demonstrated that upon addition of
NO,
E. coli hmp
not expressing
Ph
-2/2HbO shows prolonged inhibition
of O
2
uptake (
Fig. 8.8
A) until the NO level falls steadily. In contrast, in
the mutant strain carrying
hmp
þ
, the addition of NO does not inhibit O
2
uptake (
Fig. 8.8
B), confirming that Hmp is able to detoxify NO, as reported
previously (
Membrillo-Hern
´
ndez et al., 1999; Mills, Sedelnikova, Søballe,
Hughes, & Poole, 2001; Stevanin et al., 2000
). Upon addition of NO to the
E. coli
mutant carrying
Ph
-2/2HbO, only very short periods of inhibition of
respiration are observed and, again, the disappearance of NO is very fast
(
Fig. 8.8
C). When NO reaches negligible levels, the O
2
uptake is brought
back to a rate similar to that occurring before NO addition, unlike in the
cells bearing the empty vector (
Fig. 8.8
A). Following exhaustion of O
2
, fur-
ther addition of NO results in a larger signal and a slower rate of consump-
tion (
Coppola et al., 2013
).
Under aerobic conditions, over-expression of
Ph
-2/2HbO provides sig-
nificant resistance to NO and nitrosating agents and distinct NO consump-
tion ability to the NO-sensitive
E. coli hmp
mutant. In contrast, growth
curves and cellular respiration are strongly inhibited in
E. coli hmp
not
expressing the Antarctic globin gene. These results are clear evidence of a
very important physiological role of
Ph
-2/2HbO in
Ph
TAC125.
In vitro
kinetic studies of peroxynitrite isomerisation by the ferric protein
support the NO and O
2
detoxification activity as a possible functional
role of the cold-adapted globin, thus confirming the involvement of
Ph
-2/
2HbO in the protection from nitrosative stress. The high reactivity of
