Biology Reference
In-Depth Information
and cold shock, exhibited a transient burst in [Ca
2+
]
i
in a recombinant strain of
Anabaena
sp. strain PCC
7120 that expresses constitutively apoaequorin (a calcium-binding sensitive luminescent protein). The
luminescence of the cells, measured in a luminometer after treatment with coelenterazine, depended
on the concentration of [Ca
2+
]
i
(Torrecilla
et al
., 2000). These studies paved the way for the identifi cation
of a specifi c calcium signature during heterocyst differentiation in the recombinant strain of
Anabaena
sp. strain PCC 7120 for apoaequorin and a
hetR
mutant strain expressing apoaequorin after a
nitrogen step-down. The fl uctuation in the calcium signature (increase, decrease or suppression)
due to the addition of compound A23187 (5 µM), trifl uoperazine (an inhibitor of eukaryotic
calmodulin, 5 µM) and BAPTA-AM (an intracellular calcium chelator; 1,2-bis(2 aminophenoxy)
ethane N,N,N',N' tetraacetic acid tetrakis(acetoxy-methyl ester at 300 µM) also prevented heterocyst
differentiation. The generation of calcium signature in
hetR
mutant strain signifi es that the calcium
signal during heterocyst differentiation may be an early step preceding expression of
hetR
gene
(Torrecilla
et al
., 2004).
A cyanobacterial calcium-binding protein (CcbP), encoded by the gene
alr1010
of
Anabaena
sp. strain PCC 7120, overexpressed in
E
.
coli
has molecular mass of 21 kDa with 126 amino acid
residues and no known motifs or domains to bind Ca
2+
. An insertion mutant of CcbP (CCBP-M) and
a transformant with P
petE
-ccbP
that had
ccbP
gene of
Anabaena
sp. strain PCC 7120 under the control of
petE
promoter (CCBP-IE) have been subjected to nitrogen step-down experiments to follow heterocyst
differentiation. CCBP-M showed Mch-phenotype with an increase in heterocyst frequency than that
of the wild-type. On the other hand, CCBP-IE formed low frequency of heterocysts in the absence of
copper (no expression of
ccbP
gene) but in presence of copper the expression of
ccbP
led to complete
suppression of heterocyst differentiation. These results thus show that CcbP negatively regulates
heterocyst differentiation. It has been further confi rmed by the expression of rat CaM gene (
cam
) in
the wild-type and CCBP-M of
Anabaena
sp. strain PCC7120 under the regulation of
petE
promoter.
The failure of wild-type carrying
cam
gene to differentiate heterocysts and the suppression of Mch-
phenotype in CCBP-M carrying
cam
gene in presence of copper confi rmed that CcbP negatively
regulates heterocyst differentiation. Additionally,
A
.
variabilis
ATCC 29413 transformed with a plasmid
P
petE
-cam
did not differentiate heterocysts after nitrogen step-down in presence of copper. Since
patS
deletion mutant exhibited Mch-phenotype, its transformation with plasmid P
petE
-ccbP (eryR)
resulted
in suppression of heterocysts when induced with copper. Likewise, the
hetR
overexpression strain
when transformed with plasmid P
petE
-ccbP(eryR)
, instead of Mch-phenotype heterocyst differentiation was
suppressed completely in presence of copper. The expression of
ccbP
gene in wild-type strain along
with GFP as reporter gene (P
ccbP
-gfp
) resulted in strong fl uorescence in vegetative cells and a weak
fl uorescence in the heterocysts. It is expected that due to down-regulation of
ccbP
in the heterocysts
the [Ca
2+
]
i
in the heterocysts is higher than in the vegetative cells (Zhao
et al
., 2005).The increase of
[Ca
2+
]
i
and down-regulation of
ccbP
in the developing heterocysts and mature heterocysts have been
linked to HetR and NtcA mediated reactions, respectively. The increase of [Ca
2+
]
i
has been shown to
be due to the degradation of CcbP by the proteolytic activity of HetR that requires Ca
2+
. During the
degradation process, two Ca
2+
held by each molecule of CcbP are released. The proteolytic activity of
HetR (besides the capability to autodegrade itself) seems to be specifi c for CcbP as it did not degrade
other proteins such as NtcA of
Anabaena
sp. strain PCC 7120 and bovine serum albumin. As HetR is
a serine-type protease, the active serine residues at 152 and 179 positions when mutated to Ala and
Asn respectively, the mutant forms of HetR did not bring about the degradation of CcbP. However,
another mutant form, HetR
C48A
that cannot exist as a dimer but possesses proteolytic activity, could
digest CcbP. The down-regulation of
ccbP
brought about by NtcA is attributed to the presence of
potential NtcA-binding site (GTTCTGAGTGGTCACA) in the promoter region of
ccbP
as noted earlier
