Biology Reference
In-Depth Information
M. smegmatis
showed NO uptake activity of 27.82 nmol NO haem
1
s
1
,
but when the protein with the pre-A region deleted was subjected to the
same tests, rates of only 3.43 nmol NO haem
1
s
1
were observed (
Lama
et al., 2009
). Conversely, adding the pre-A region onto the
M. smegmatis
trHbN increased its NO detoxification abilities: expression of wild-type
trHbN on a plasmid gave rates of 4.9 nmol NO haem
1
s
1
, with the mod-
ified protein showing an NO consumption of 11.8 nmol NO haem
1
s
1
(
Lama et al., 2009
). These results confirm the authors' idea that the pre-A
region is a requirement for efficient NO detoxification.
As NO is a respiratory inhibitor, the ability of trHbN to protect respi-
ration of cells exposed to NO has been investigated. In
M. bovis
, respiration
was initially inhibited by 1 and 2
m
M NO and then recovered; however,
when
glbN
was knocked out, cells lost the ability to detoxify NO which
showed that the trHbN protein is responsible for the NO uptake activity
in wild-type cells (
Ouellet et al., 2002
). Furthermore, complementation
of the mutant and therefore probable over-expression of trHbN leads to cells
that are more resistant to respiratory inhibition by NO than wild-type cells.
Under high O
2
concentrations, the
glbN
mutant recovered somewhat and
there was less inhibition of respiration, suggesting that ambient O
2
concen-
trations are an important factor in protection against NO toxicity (
Ouellet
et al., 2002
).When Mtb trHbN was expressed in both
E. coli hmp
and
M. smegmatis
,O
2
uptake was protected from inhibition by NO; for example,
in
E. coli hmp
when 5
m
M NO was added to cells, O
2
uptake was
0.2
mol min
1
10
8
cells
1
, whereas expression of trHbN increased this
to 6.8
m
mol min
1
10
8
cells
1
; control values without addition of NO
were 7.3 and 7.5
m
mol min
1
10
8
cells
1
, respectively (
Pathania, Navani,
Gardner, et al., 2002
). Although the authors do not specify where on the
O
2
concentration curves the rates were calculated from, these data clearly
show that the expression of Mt
m
trHbN can protect respiration from
NO inhibition.
Expression of trHbN in
M. tuberculosis
and
M. bovis
is higher in late expo-
nential and early stationary phases than in early exponential phase (
Pathania,
Navani, Gardner, et al., 2002
), and
glbN
shows an early response to
nitrosative stress (
Joseph et al., 2012
), reflecting the point at which these spe-
cies may require NO detoxification activities during infection. Haem
staining did not show any detectable amount of Hb-like protein in
M. smegmatis
, although we know it possesses a trHbN homologue. The pres-
ence of Mtb trHbN in an
E. coli hmp
-
mutant protected against NO toxicity
under aerobic conditions. Importantly, this protection was sustained over