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was achieved, whereas the wild-type protein did not interact with streptactin-affi nity resin. Such
purifi ed sHsp16.6 subunits formed oligomers containing 20 or 22 subunits
in vitro
. The addition of
C-terminal tag did not cause any increase in the size of the oligomers and heat denatured luciferase
used as substrate was reactivated
in vitro
by the interactions of sHsps and other molecular chaperones.
In addition, strep-tagged Hsp18.1 from pea also exhibited similar
in vitro
interactions except that
the strep-tagged and wild-type Hsp18.1 could associate to form dodecameric oligomers that were
slightly larger than the wild-type Hsp18.1. Their interaction with the substrate molecules
in vitro
conform to the behaviour of their wild-type counterparts
in vivo
.
Balogi
et al
. (2005) identified a “heat-shock lipid” in the thylakoids that helps in the
acclimatization of cyanobacteria during heat/light stress. This lipid, known as highly saturated
monoglucosyldiacylglycerol (MGlcDG), increased in its level during stress.
In vitro
experiments
carried out with thylakoid MGlcDG liposomes showed them to be quite stable even at extremely
high temperatures. Moreover, of the fi ve polar lipids tested, only MGlcDG strongly interacted with
the sHsp (Hsp17) from
Synechocystis
sp. strain PCC 6803. Thus it was concluded that MGlcDG and
Hsp17 play an important role in preserving functional integrity of thylakoid membranes and help
in the development of acquired thermotolerance in light- or heat-stressed cyanobacteria.
S
.
vulcanus
, a thermophilic cyanobacterium, constitutively expresses a sHsp that showed high
homology to Hsp16.6 from the mesophilic cyanobacterium
Synechocystis
sp. strain PCC 6803.
The gene
hspA
encoding this protein was cloned (Roy and Nakamoto, 1998). The gene product
HspA showed homology to the α-crystallin-related sHsps from other organisms. HspA formed
a large homo-oligomer consisting of 24 subunits that prevented thermal aggregation of model
substrates such as porcine malic dehydrogenase at 45°C and 50°C and citrate synthase at 50°C
(Roy
et al
., 1999). In order to understand the role of sHsp in conferring cellular thermotolerance,
Nakamoto
et al
. (2000) cloned
hspA
from
S. vulcanus
and used it to transform
S
.
elongatus
PCC 7942
with an expression vector p-ECT under the control of
tac
promoter. The transformant (ECT-16-1)
constitutively expressed 16.6 kDa protein that accumulated in soluble and insoluble fractions.
Viability of ECT-16-1 cells when treated at 50°C for 15 min was enhanced when compared to
wild-type cells. Heat-adapted cells (45°C for 1 hour) when exposed to a temperature of 50°C for
60 min resulted in a 1000 and 10,000 fold increase in the survival at 50°C in case of wild-type and
ECT-16-1 cells, respectively. HspA plays a role in preserving the integrity of thylakoid membranes
under heat stress and its constitutive expression did not affect the expression of other Hsps such as
GroEL and DnaK. However,
hspA
deletion mutant of
Synechocystis
sp. strain PCC 6803 constitutively
expressed higher levels of GroESL1 and GroEL2 proteins under normal conditions (Asadulghani
et
al
., 2004). Nitta
et al
. (2005) showed that cultures of ECT-16-1 when subjected to heat shock (shifted
to 45°C or 50°C for 15 min) or exposed to high light (1000 µE.m
-2
s
-1
at 30°C), released HspA from
thylakoids. This suggested that under normal conditions thylakoid membranes act as the reservoir
for the HspA molecules. Under stressed conditions, the HspA protein molecules get dissociated
from the thylakoids and interact with the non-native proteins as well as proteins from thylakoids
and prevent them from aggregation. Immunocytochemical evidences confi rmed the localization
of the HspA protein molecules in the cytosol under stress conditions. This probably leads to the
induction of GroES/GroEL and DnaK/DnaJ/GrpE chaperone networks. Additional evidences in
favour of the sHsps to confer stress tolerance by protecting the integrity of thylakoid membranes
were presented by Sakthivel
et al
. (2009). ECT-16-1 cells not only exhibited thermotolerance but also
survived oxidative stress by exhibiting much better growth and viability in H
2
O
2
than the wild-type
strain. Moreover, all the pigment molecules (chlorophylls, carotenoids and phycocyanins) appeared
normal in ECT-16-1 cells when compared to a continuous reduction in their content in the wild-