STRESS RESPONSES - Cyanobacteria - page 661

of dnaK gene is under circadian control (Aoki et al ., 1995). Isolation and characterization of dnaK
locus from A . halophytica led to the identifi cation of grpE and dnaJ genes in the order grpE - dnaK1 - dnaJ .
The mRNA levels for dnaK1 gene increased upon subjecting the cells to heat stress. The functional
aspect of the dnaK1 locus has been tested by its co-expression with a plant plastocyanin gene in
E . coli cells due to which there was a dramatic increase in the solubility of the plastocyanin protein.
This signifi es that the dnaK1 produces a functional protein and it performs the functions assigned
to DnaK/Hsp70 (Lee et al ., 1997).
Nimura et al . (2001) conducted gene disruption experiments in S. elongatus PCC 7942 by
introducing kanamycin resistance marker in dnaK1 , dnaK2 and dnaK3 . As S. elongatus PCC 7942
is known to possess multiple copies of the genome a successful transformation can lead to the
disruption of all copies of the particular dnaK gene. Thus DnaK2 and DnaK3 were found to be
essential as disruption of all copies of dnaK2 and dnaK3 was not possible but only in case of dnaK1
successful transformants were obtained suggesting that all copies of this gene could be disrupted
and the organism could lead a normal growth even in its absence. The expression of the three dnaK
homologues in response to heat shock (a shift up from 30°C to 45°C) showed that only DnaK2 and
GroEL proteins exhibited a typical heat shock response in the sense that the proteins increased in
their level during the fi rst 30 min and thereafter increased level was maintained for the entire period
under study. The effects of overproduction of the three gene products were studied in E . coli by using
an inducible expression system separately for each gene. Overproduction of DnaK1 and DnaK2
resulted in defective septation leading to the formation of long fi lamentous cells. Overproduction
of DnaK3 resulted in cells that were swollen and twisted. As deletion of dnaK in E . coli causes a
temperature-sensitive phenotype, a dnaK756 deletion mutant of E . coli was tested for its ability to
grow at non-permissive temperature when the three dnaK homologues of S. elongatus PCC 7942
were expressed. dnaK2 could suppress growth defi ciency at the non-permissive temperature while
dnaK1 and dnaK3 could not suppress this phenotype. Overproduction of DnaK1 or DnaK3 in mutant
dnaK756 of E . coli resulted in growth inhibition at the permissive temperature. Furthermore, the levels
of expression of DnaK2 and DnaK3 and GroEL proteins after heat shock in wild-type and dnaK1
disruptant mutant cells (designated as DK 1KM) were found to be the same as revealed by Western
blotting. The studies of Varvasovszki et al . (2003) further confi rmed that in Synechocystis sp. strain
PCC 6803, of the three dnaK homologues only dnaK2 is transcriptionally active. The co-chaperones
dnaJ and grpE were not inducible. Gene inactivation experiments with dnaK2 yielded partial mutants
that exhibited a temperature-sensitive phenotype with lower inducibility of GroEL and Hsp17.
Although DnaK1 of A . halophytica (Lee et al ., 1997) and DnaK1 of S . elongatus PCC 7942 (Nimura
et al ., 2001) showed extensive sequence similarity and comparable in vitro chaperone capabilities,
functionally they differed under in vivo conditions. Blanco-Rivero et al . (2005) compared the functional
capabilities of DnaK1 from A . halophytica and S . elongatus PCC 7942 in vivo by expressing them in
E . coli mutants defective in DnaK ( dnaK756 ) or a null mutant. DnaK1 from S . elongatus PCC 7942
effectively replaced and complemented E . coli DnaK by performing all the major functions recognized
for this chaperone. Some of the important functional differences in the two DnaK1 proteins are: (i)
production of fi lamentous cells of E . coli due to overproduction of DnaK1 from Synechococcus and
its absence due to overproduction of DnaK1 from A . halophytica ; (ii) reduced levels of production of
GroEL in E . coli cells when its own DnaK or Synechococcus DnaK1 are overproduced whereas there
was no signifi cant reduction in the levels of GroEL in E . coli when DnaK1 from A . halophytica was
overproduced; (iii) Overproduction of DnaK1 from Synechococcus prevented aggregation of misfolded
RuBisCO units in E . coli mutants while DnaK1 from A . halophytica could not prevent aggregation
of RuBisCO subunits; and (iv) most importantly, DnaK from E . coli or DnaK1 from Synechococcus