STRESS RESPONSES - Cyanobacteria - page 659

noted. Though the cells initially accumulated high levels of Hsp70 and Hsp15, the synthesis of
Hsp64 and Hsp14 continued through 2 h. The Hsp70 and Hsp64 have been identifi ed to be similar
to DnaK and GroEL of E . coli (Lehel et al ., 1992). The cloning and characterization of the groEL gene
of Synechocystis sp. strain PCC 6803 was performed by using groEL gene of S. elongatus PCC 7942 as
a probe. A fragment of 3.25 kbp from the genome of Synechocystis sp. strain PCC 6803 revealed three
ORFs. On the basis of sequencing of the three ORFs and deduced amino acid sequences, two of them
corresponded to GroEL and GroES proteins (Lehel et al ., 1993). The third gene (ORF51), located from
460 to 612 bp of the sequence encodes a putative polypeptide of 51 amino acids, that did not bear any
signifi cant homology to any known sequence in the database. The GroES of Synechocystis sp. strain
PCC 6803 showed 80% and 62-72% homology to GroES proteins of S. elongatus PCC 7942 and other
bacterial species, respectively. In addition to groESL operon, Synechocystis genome also possesses
two copies of groEL analogous genes. The transcript of the groESL operon was barely detectable at
30°C but after a heat shock at 42°C for 15 min the level of the transcript was enhanced by 100-fold,
reaching a maximum in 14 h. The sequence of Cpn60 of Synechocystis sp. PCC 6803 (Chitnis and
Nelson, 1991) showed same degree of similarity to the sequence of GroEL described by Lehel et al .
(1993). However, the degree of homology of the sequence of GroEL of Synechocystis sp. PCC 6803 is
higher to Cpn60 of Bacillus subtils than to its own sequence of Cpn60.
The role of HtpG, a prokaryotic homologue of Hsp90, in thermal stress management of
cyanobacteria has been investigated. A single copy of the htpG gene sequence (ORF, sll0430 ) has
been cloned from S . elongatus PCC 7942 and a comparison with the sequences in the database
suggested that the deduced amino acid sequence bears closest homology (64% overall identity) to
that of HtpG from Synechocystis sp. strain PCC 6803. Two unique features of HtpG are that a portion
between 210 and 224 (numbered according to HtpG of E . coli ) amino acid residues did not exist and
residues at 14 and 33 in homologues from S. elongatus PCC 7942 and Synechocystis sp. strain PCC
6803, respectively were inserted between position numbers 409 and 410. Mutants of S . elongatus PCC
7942 in which htpG was inactivated by targeted mutagenesis exhibited similar growth pattern as
that of wild-type at 30°C and 42°C but the cultures turned yellow. At 45°C, the htpG mutant grew
with a lag period while the wild-type grew normally. After a heat shock (cells exposed to 50°C for
20 min) the survival of wild-type was 20% whereas the htpG mutant surived to 0.01 % only. Wild-
type cells exhibited the ability of acquired thermotolerance (a pre-treatment for 60 min at 42°C
before giving heat shock at 50°C) but the htpG mutant lost this ability. These results emphasize the
essential nature of htpG gene in thermal stress management in cyanobacteria (Tanaka and Nakamoto,
1999). A htpG null mutant was generated by inserting a chloramphenicol resistance cassette in the
htpG coding sequence of Synechocystis sp. strain PCC 6803. A comparison of htpG mutant with a
mutant of hsp16.6 and the double mutant ( htpG - : hsp16.6 - ) for their growth, cell survival rate and
rates of O 2 evolution revealed that all the three of them were sensitive to heat stress. Basal level of
thermotolerance and acquired thermotolerance of the double mutant was lowest among the three
when compared to wild-type (Fang and Barnum, 2003). Apart from the essential nature of HtpG
during heat (Tanaka and Nakamoto, 1999), cold (Hossain and Nakamoto, 2002), salt (Huang et al .,
2002) and oxidative (Hossain and Nakamoto, 2003) stresses, other studies have indicated that htpG
transcripts and HtpG protein accumulation occurred under high light intensity as well (Hihara et
al ., 2001; Mary et al ., 2004).
Cloning and characterization of groEL gene and its contribution to thermotolerance of
Anabaena sp. strain L-31 has been reported (Rajaram et al ., 2001; Rajaram and Apte, 2003). Cloning
and characterization of cpn60 gene from Anabaena sp. strain L-31 and its expression due to heat
shock have been compared with that of groEL in response to nitrogen status. Severe inhibition of