STRESS RESPONSES - Cyanobacteria - page 658

DnaK protein acting as the direct thermometer for sensing heat (McCarty and Walker, 1991). The
ribosome sensing hypothesis envisages regulation of stress genes at the level of translation process.
The implication of Hsps in the acquisition of thermotolerance has been well established (Lehel
et al., 1993; Glatz et al ., 1997, 1999; Vigh et al ., 1998, 2007). Thus the Hsps protect and recover the
stress damaged proteins in the cell. In E . coli , IbpA/B (small Hsps), GroEL, DnaK, ClpB constitute
a sophisticated network of “holding” and “folding” chaperones that prevent protein aggregation
during heat stress. They not only promote protein disaggregation during heat stress but also help
in refolding the damaged proteins after the stress (Buchner, 1996; Mogk et al ., 1999, 2003). Initially
it is the small Hsps that are bound to the denatured polypeptides which hand them over to DnaK
and GroEL chaperones for refolding (Veinger et al ., 1998). Likewise, denatured protein aggregates
are successfully resolubilized and refolded by the sequential action of ClpB and DnaK (Singer and
Lindquist, 1998b; Goloubinoff et al ., 1999; Mogk et al ., 1999; Diamant et al ., 2000). Motohashi et al .
(1999) reported the functional co-operation between ClpB and DnaK. The existence of DnaK and
DnaJ as a stable trigonal ring complex in Thermus thermophilus (TDnaK.J complex) and the dnaK
gene cluster also contains ClpB gene. Substrate proteins could be protected from aggregation and
heat denaturation by TDnaK.J complex is dependent on ATP and TGrpE. Subsequent addition
of ClpB at moderate temperature resulted in an effi cient reactivation of the proteins. This study
emphasizes the cooperation of TDnaK.J complex, TGrpE, TClpB and ATP for complete reactivation
and TGroE/ES could not substitute TClpB. Diamant et al . (2001) demonstrated the regulation of
molecular chaperones by the chemical chaperones in vitro as well as in vivo when E . coli cells were
subjected to salt and heat stress simultaneously. Accordingly, the effects of four osmolytes (glycine
betaine, glycerol, proline and trehalose) on the folding activities of individual and combinations of
chaperones (GroEL, DnaK and ClpB) have been studied. Except trehalose, the low physiological
concentrations of the rest of three osmolytes activated molecular chaperones in their local folding
functions. Especially glycine betaine prevented protein aggregation during heat stress as well as
provided maximum levels of stimulation for the chaperone-mediated protein folding. On the other
hand, trehalose strongly inhibited DnaK-dependent chaperone networks such as DnaK+ GroEL
and DnaK+ClpB probably due to high viscocity levels. Thus the levels of activity of molecular
chaperones much depends on the level and nature of osmolytes that accumulate when the cells are
simultaneously subjected to a salt and heat stress.
i) Synthesis of Hsps in response to heat stress : All cyanobacterial genomes sequenced so far
possess two distinct hsp60 genes, a groEL gene as a part of groESL operon and a solitary cpn60 gene.
Synechococcus sp. strain PCC 6301 cells grown at 39°C when subjected to short-term exposure to
47°C synthesized a number of polypeptides that varied in their molecular weights (ranging from 91
kDa to 11.4 kDa) (Borbély et al. , 1985). Molecular cloning of genes cpn60 and dnaK from Synechocystis
sp. strain PCC 6803 revealed the existence of these genes in single copies and the transcripts were
monocistronic. The transcript levels of cpn60 and dnaK increased 30 and 25 fold, respectively after
90 min heat shock at 42°C. But after 2 h, the level of the transcripts of the two genes reached the pre-
stress levels. Oxidative stress and UV-exposure also triggered the accumulation of the transcripts of
these two genes. The deduced amino acid sequences of Cpn60 brings it closer to bacterial and plant
Cpn60 proteins whereas DnaK protein sequence is homologous to its bacterial counterpart than
to eukaryotic ones. Another siginifi cant feature is the presence of ORF60-5 that encodes a protein
belonging to the peripheral membrane proteins involved in metabolite transport in bacteria (Chitnis
and Nelson, 1991). When Synechocystis sp. strain PCC 6803 cells grown at 30°C were exposed to
heat stress at 42°C, the induction of Hsps of different molecular sizes (79, 64, 15 and 14 kDa) was