Biology Reference
In-Depth Information
the genetic organisation is different in the non-pathogenic bacteria; for
example, in
M. tuberculosis
, a short-chain alcohol dehydrogenase is tran-
scribed in the opposite direction to
glbN
, whereas in
M. smegmatis
, the gene
in this position is
lprL
(
Pawaria et al., 2008
). In
M. leprae
, this region of the
genome is occupied by a pseudogene (
Pawaria et al., 2008
). In both
M. tuberculosis and M. bovis
,
lprI
is positioned directly adjacent to
glbN
(
Pawaria et al., 2008
); it is co-transcribed in
M. bovis
but has not been impli-
cated in trHbN activity. Western Blot analysis was used to prove when Mb
trHbN is expressed in
M. bovis
BCG growing in aerobic cultures; expression
is low both at the beginning of growth and in exponential phase, peaking
when cells are in stationary phase (
Couture et al., 1999
). This suggests that
in
M. bovis
, and probably in other pathogenic species of Mycobacteria,
trHbN is expressed when the bacteria is entering a period of growth where,
for example, nutrients are scarce and O
2
levels are low, corresponding to the
environment encountered in low O
2
granulomas. Less is known about the
genetic regulation of
glbO
, the gene encoding for trHbO. The region
around the
glbO
gene is conserved across pathogenic and non-pathogenic
Mycobacteria (
Pawaria et al., 2008
).
In order to probe the genetic regulation of both
glbN
and
glbO
from
M. tuberculosis
, potential promoter regions containing the -10 and -35 sites
and Shine-Dalgarno sequences were cloned in front of a promoter-less
gfp
gene and expressed in
M. smegmatis.
In agreement with data obtained in
M. bovis
(
Ouellet et al., 2002
),
glbN
was up-regulated in late exponential
and stationary phases of growth; its expression was also increased in these
phases when
M. smegmatis
was exposed to nitrite and SNP (
Pawaria et al.,
2008
). In contrast,
glbO
appeared to be more consistently expressed over
growth phases, and showed increased expression in response to nitrite in
all growth phases but SNP only in stationary phase, probably reflecting
the fact that trHbO is not thought to be involved in the detoxification of
NO (
Pawaria et al., 2008
). The same experiments were performed in
M. tuberculosis
H37Ra and the authors comment that both
glbN
and
glbO
promoters behaved in the same way as in
M. smegmatis
(
Pawaria et al.,
2008
). This strain of
M. tuberculosis
H37Ra was used to infect PMA-activated
THP-1 cells, which mimic human alveolar macrophages, to determine the
promoter activities
in vivo
. The results follow those obtained
in vitro
: FACS
analysis showed fluorescence in cells infected with
glbN
and
glbO
, although
this experiment was only done once (
Pawaria et al., 2007
). This suggests that
both promoters are active during macrophage infection, although more
work needs to be presented to further analyse this activity.
