STRESS RESPONSES - Cyanobacteria - page 662

protected the E . coli null mutants (for dnaK ) from heat stress by conferring thermotolerance but DnaK1
from A . halophytica could do so to much lower levels. By contrast, dnaK homologues of Synechocystis
sp. strain PCC 6803 did not complement dnaK in E . coli dnaK -defective mutants. Rupprecht et al . (2007)
demonstrated that all three homologues of dnaK are functional in Synechocystis sp. strain PCC 6803
and the three DnaK proteins share a high degree of homology to each other. However, they differ in
their molecular masses, i.e. DnaK1, DnaK2 and DnaK3 possess 75 kDa, 67 kDa and 86 kDa molecular
weights, respectively and share the common DnaK domain. All the three proteins showed C-terminal
extensions and the observed differences are due to this. The expression of the three DnaK proteins
in Synechocystis sp. strain PCC 6803 was demonstrated by Western blot analysis using antibodies
specifi c to each one of the DnaK proteins. Specifi c physiological functions have been identifi ed by
the isolation of dnaK gene disruptant mutants by introducing kanamycin resistance marker. They
confi rmed the essentiality of dnaK2 and dnaK3 for survival of Synechocystis sp. strain PCC 6803 while
deletion of dnaK1 gene did not affect the survival as reported earlier in case of S . elongatus PCC 7942
(Nimura et al ., 2001). PCR and restriction analysis of the genomes of dnaK2 and dnaK3 disruptant
mutants revealed the presence of wild-type genes suggesting that all the copies of the two genes
could not be completely inactivated. DnaK1 protein was no longer produced in the dnaK1 disruptant
mutant but the levels of DnaK2 and DnaK3 in dnaK2 and dnaK3 disruptant mutants were almost
comparable to the levels of these proteins in the wild-type. Deletion of dnaK3 gene could only be
possible when the dnaK3 gene was expressed from a neutral site in the genome of Synechocystis sp.
strain PCC 6803. The C-terminal region proved to be essential for proper functioning of DnaK3
protein, as a truncated dnaK3 gene (without the C-terminal) region could not complement dnaK3
when expressed from a different site on the chromosome of Synechocystis . By using an expression
plasmid pMal-C2, all the three dnaK homologues have been individually cloned and E . coli dnaK756
mutant was transformed. In none of the transformants, dnaK homologues of Synechocystis sp. strain
PCC 6803 could complement the dnaK gene functions of E . coli . Although the three proteins increased
in their levels after a heat shock, only the levels of DnaK2 protein were up-regulated consistent with
the earlier observations on the same organsim (after a heat shock and other stresses like UV light,
ethanol, salt and high light; Chitnis and Nelson, 1991; Varvasovszki et al ., 2003; Mary et al ., 2004;
Fulda et al ., 2006) and on S. elongatus PCC 7942 (Nimura et al ., 2001).
A highly conserved N-terminal ATP-binding domain and a less conserved C-terminal peptide-
binding domain are characteristically present in the DnaK proteins. It is through the peptide
binding domain that DnaK3 is shown to be quantitatively associated with the cytosolic side of the
thylakoid membranes in S . elongatus PCC 7942. It means that due to the absence of a thylakoid-
targeting sequence, it was surmised that DnaK3 might require a receptor protein for its interaction
with thylakoid membrane (Nimura et al ., 1996). Zhu et al . (1996) showed that the substrate-binding
portions constitute β-sheet regions together with adjacent α-helical regions. In order to identify
the role of DnaK3 proteins and their binding interactions with thylaloid membranes, Katano et al .
(2006) isolated dnaK3 mutants and their suppressors from S. elongatus PCC 7942. The mapping of
the dnaK3 regions led to the identifi cation of T to C conversion in one of the mutants (NBC001) at
436 th codon from TTG (Leu) to TCG (Ser) in the β-sheet region. The growth and O 2 evolved upon a
temperature shift-up (from 30°C to 43°C) decreased in case of NBC001. Suppressor mutants were
isolated by incubating NBC001 at the non-permissive temperature (43°C). Of the three suppressor
mutants isolated, one of it mapped in rpll24 (ribosomal protein large; syc1876 ) that encodes 50S
ribosomal protein L24 whereas in the rest of the two suppressors the substrate-binding capacity was
restored suggesting that these two were intragenic suppressors. Further, DnaK1 and DnaK2 proteins
have been shown to be localized in the cytosol and partly in the membrane fractions in the