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by Herrero
et al
. (2004). This is further confi rmed by the
ccbP
mRNA levels in the wild-type that
remained fairly constant during the fi rst 3 h of nitrogen step-down and NtcA deletion mutant which
showed 50% increase of
ccbP
mRNA levels during the same period. Interestingly, 2-OG promoted
interaction of NtcA with its binding sequence in the promoter of
ccbP
(Shi
et al
., 2006).
ii) 2-OG
:
The fi rst indication that cyanobacteria perceive nitrogen status by sensing intracellular 2-OG
levels came from the studies of Muro-Pastor
et al
. (2001) who monitored the levels of 19 different
amino acids and 2-OG in mutants of
Synechocystis
sp. strain PCC 6803 with altered levels of GS-
activity. Irrespective of the levels of glutamine and glutamate, the two key amino acids synthesized
during the ammonia assimilation (GS-GOGAT) pathway, it is the levels of 2-OG that are perceived
by cyanobacteria to sense combined nitrogen limitation. Another observation that lends support
to 2-OG as the signalling molecule is the increased
in vitro
binding affi nity of NtcA to the promoter
region for
S. elongatus
PCC 7942
glnA
in presence of 2-OG as well as its transcription rate (Vazquez-
Bermudez
et al
., 2002; Tanigawa
et al
., 2002). Another important protein that regulates nitrogen
metabolism in unicellular cyanobacteria is PII, a product of
glnB
. (There are three sub-families of
PII proteins. NifI
1
and NifI
2
constitute two sub-families while the third sub-family comprises of
all other PII proteins including products of
glnB
and
glnK
). The binding of 2-OG to PII not only
stimulates phosphorylation of PII (at Ser49) in
S. elongatus
PCC 7942 (Forchhammer and Tandeau
de Marsac, 1994, 1995) but also inhibits its dephosphorylation reaction (Irmler
et al
., 1997; Ruppert
et al
., 2002). Under nitrogen-limiting conditions, NtcA also regulates
glnB
at the transcriptional and
post-translational levels (Lee
et al
., 1999; Sauer
et al
., 1999). In turn it has also been observed that PII
stimulates and inhibits NtcA activity under nitrogen deprivation and in presence of nitrate enriched
medium, respectively (Aldehni
et al
., 2003; Paz-Yepes
et al
., 2003; Aldehni and Forchhammer, 2006).
The discovery of a transcriptional activator, PipX in
S. elongatus
PCC 7942 provides a connecting link
between NtcA and PII which are the two known regulators of nitrogen metabolism in cyanobacteria.
2-OG favours interaction between NtcA and PipX under nitrogen deprivation but impairs binding
to PII, the deciding factor being the intracellular concentration of 2-OG (Espinosa
et al
., 2006). But all
the above studies conducted mostly on unicellular cyanobacteria clearly emphasize that in perceiving
nitrogen status the most important interactions occur between NtcA, PII and 2-OG. However, it may
be noted that PII has not been well characterized in the fi lamentous heterocystous cyanobacteria.
In order to study the effect of enhanced intracellular levels of 2-OG, Li
et al.
(2003) constructed a
recombinant strain of
Anabaena
sp. strain PCC 7120 with
E
.
coli
ketoglutarate permease gene (
kgtP
)
under the infl uence of
petE
promoter that is expressed in presence of copper. The
kgtP
gene construct,
carried by a shuttle vector (pRL25c), was transferred through conjugation and the exconjugants
(KGTP strains) were selected in presence of 50 µg Nm ml
-1
. Uptake kinetics of [1-
14
C]2-OG by the
KGTP strain showed that in presence of copper the
kgtP
gene was expressed and signifi cant amounts
of 2-OG were taken up by the cells. In nitrate medium, the addition of 2-OG (1.0 mM) resulted in
heterocyst differentiation with a normal pattern. When PatS inactivated strain (AMC451; isolated by
Yoon and Golden, 1998) was grown in presence of 2-OG (25 mM), the interval between two heterocysts
was reduced to four cells (from the interval of 8 cells observed in the absence of 2-OG in the
patS
mutant). Another signifi cant feature is the occurrence of heterocysts in pairs that constituted 71%
of all the heterocysts when compared to 59% paired heterocysts in the absence of 2-OG. Ammonia
repressed heterocyst differentiation in the KGTP strain but in presence of 2-OG the time taken for
heterocyst differentiation was considerably reduced to 6 h after which ammonia could no longer
repress heterocyst differentiation. This signifi es that the presence high intracellular concentrations
of 2-OG in the cells, the commitment of cells for heterocyst differentiation is reached very early than