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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
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