Biology Reference
In-Depth Information
of Ca
2+
/H
+
antiporter from
Synechocystis
sp. strain PCC 6803 and
A
.
halophytica
revealed that the
antiporter proteins ApCAX and SynCAX are localized in the cytoplasmic membranes of these
cyanobacteria and catalyzed Ca
2+
/H
+
exchange reactions at alkaline pH. Mutant cells of
Synechocystis
disrupted in the
SynCAX
gene showed lower Ca
2+
effl ux activity with a salt-sensitive phenotype.
There was gradual degradation of pigments in mutant cells at high pH where chlorophyll,
phycocyanin and phycobiliprotein were 30%, 20% and 10% of the wild-type, respectively. Though
the cellular K
+
levels were the same in the mutant and wild-type, the Na
+
content of the mutant cells
was three times higher than the wild-type.
S. elongatus
PCC 7942 cells with
ApCAX
and
SynCAX
genes and their overexpression resulted in a salt-tolerant phenotype. Glu-74 and Glu-324 are the
two conserved amino acid residues present in the transmembrane domain of the antiporter protein
that is responsible for the Ca
2+
/H
+
antiporter activity and for salt tolerance (Waditee
et al
., 2004).
Homologues of the NapA-type of Na
+
/H
+
antiporters of
Synechocystis
sp. strain PCC 6803 have been
characterized from
A
.
halophytica
with novel ion specifi cities that are involved in salt tolerance at
alkaline pH. Two genes Ap-
napA1
-
1
and Ap-
napA1
-
2
that encode Ap-NapA1-1 and Ap-NapA1-2
polypeptides of the same size, respectively have been isolated and these antiporters complemented
the salt-sensitive phenotype
E. coli
. The antiporter activities of Ap-NapA1-2 were signifi cantly lower
than the others but complemented K
+
uptake defi cient mutant of
E
.
coli
. Amino acid residues Glu129,
Asp225 and Asp226 of the transmembrane segment of the antiporter protein and Glu142 in the loop
region are responsible for the activity.
S. elongatus
PCC 7942 exhibited enhanced salt tolerance due
to overexpression of the gene
Ap-
napA1
-
1
especially at the alkaline pH. Thus these two antiporters
of
A
.
halophytica
exhibit different ion specifi cities (Wutipraditkul
et al
., 2005). Furthermore, they
identifi ed the pH dependence of Ap-NapA1-1 and Ap-NapA1-2. However, AP-NhaP1 exhibited
a high Na
+
/H
+
exchange activities at pH 6.0 to 9.0 whereas Ap-NapA1-1 had no Na
+
/H
+
exchange
activity at pH 6.0 but its activity increased with increasing pH with an optimum at pH 8.5. Likewise,
pH dependence for Li
+
/H
+
exchange activities by AP-NapA1-1 was demonstrated where as Ap-
NhaP1 showed very little or no Li
+
/H
+
exchange activity at all pHs examined. Genes encoding H
+
-
ATPase and Na
+
-ATPase have been identifi ed in
A
.
halophytica
. In case of the latter nine genes are
organized into an operon. Na
+
-ATPase conferred salt tolerance in a DKS mutant of
E
.
coli
defi cient
in ATPase.
S. elongatus
PCC 7942 acquired tolerance to salt stress up on the expression of
ApNa
+
-atp
operon and showed the localizationof Na
+
-ATPase in its cytoplasmic membrane (Soontharapirakkul
et al
., 2011).
The role of the genes
nhaS1
to
nhaS5
in governing the production of NhaS1 to NhaS5 in response
to salt stress and the regulation of internal pH in the cells of
Synechocystis
sp. strain PCC 6803 has
been identifi ed by the generation of mutants. Single, double and triple mutants impaired in the
respective genes of antiporters were generated by interposing genes conferring kanamycin resistance
gene (from pUCΔk), chloramphenicol resistance gene (from pACyC184) or streptomycin resistance
gene (from pBSL130) in ORFs cloned into
E
.
coli
vectors. Such plasmid DNA isolated from
E.
coli
was
used to transform the
Synechocystis
cells. Transformants for the respective antibiotics were selected.
Completely segregated mutants revealed the inactivated genes for the respective antiporter genes.
Double and triple mutants were generated by adding the required antiporter gene to the completely
segregated inactivated genes with the Sm
r
gene. The single, double and triple mutants of
Synechocystis
for
nhaS1
,
nhaS4
and
nhaS5
resembled wild-type cells in maintaining the same Na
+
and K
+
gradients
at high salt concentrations suggesting that they are not required for adaptation of
Synechocystis
for
salt stress. The activity of antiporter encoded by
nhaS3
seems to be suffi cient for giving protection
against salt stress (Elanskaya
et al
., 2002).