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sp. strain PCC 7120 the former formed heterocysts like the wild-type but the latter did not form
heterocysts after nitrogen step-down. However, the mutant synthesized the HetR Ser152Ala protein after
a nitrogen step-down very much in a similar manner to the mutant 216 that synthesized HetR Ser179Asn
even in the absence of heterocyst differentiation. When compared to wild-type which supported
maximum levels of HetR synthesis during early hours (within 6 h), the two mutants continued to
synthesize mutant proteins (HetR Ser152Ala and HetR Ser179Asn ) for 24 h after nitrogen step-down.
Buikema and Haselkorn (2001) turned their attention to studies on expression of hetR in
Anabaena sp. strain PCC 7120. The presence of four tsps in the promoter region of hetR located at
-184, -271, -696 and -728 positions relative to the start codon of HetR has been demonstrated. In
the hetR mutant 216, the 1.4 kb and 1.9 kb transcripts were produced at considerably lower levels
with apparently no transcripts of 1.5 kb corresponding to the tsp located at -271. HetR produced
from -271 tsp appears to be responsible for autoregulation of hetR in Anabaena sp. strain PCC 7120.
They have chosen the petE gene that encodes a copper protein, plastocyanin in S. elongatus PCC
7942 under inducible conditions in presence of copper for the expression of hetR (Ghassemian et al .,
1994). Introduction of hetR gene under the regulation of petE promoter on a multicopy plasmid into
Anabaena resulted in the production of Mch-phenotype in presence of copper (due to induction of
petE gene) but in absence of copper (due to repression petE gene) the transformant produced normal
pattern of heterocyst differentiation. Since patA gene regulates the normal pattern of heterocysts and
its deletion caused the production of only terminal heterocysts, they introduced a hetR - gfp fusion
construct into wild-type and patA deletion mutant. After a nitrogen step-down, the GFP fl uorescence
appeared mostly in all cells but later appeared to be restricted to the proheterocysts and heterocysts
in the wild-type whereas it is restricted to only in the terminal heterocysts without any fl uorescence
being detected in vegetative cells in the patA deletion mutant. This shows that hetR expression is
primarily in the differentiating cells leading to heterocyst formation and for localization of HetR
protein in the proheterocysts or heterocysts it requires the presence of PatA.
Of the four tsps identifi ed for hetR , the tsps located at -728 and -271 were found to be under
the infl uence of NtcA, the regulator for nitrogen metabolism (Muro-Pastor et al ., 2002) It would be
worthwhile to recall here that the tsp located at -271 is also dependent on HetR, through which
autoregulation of hetR is reported to occur (Buikema and Haselkorn, 2001). Expression of NtcA
was found to be higher in cells subjected to nitrogen step-down than in cells grown in presence of
nitrate or ammonium. For this purpose, the tsps located at positions -180 and -49 bp in the promoter
region of ntcA are put to use. In ntcA (CSE2 reported by Frías et al ., 1994) or hetR (DR884a reported
by Black et al ., 1993) insertional mutants, the tsps at positions -180 and -49 of ntcA are not utilized.
Muro-Pastor et al (2002) thus concluded that there is a mutual dependence of the two regulatory
genes ntcA and hetR. This has been explained by the timing of expression of the two genes. In the
wild-type, hetR expression begins in just less than 3 h and assumes maximum levels by 4.5 to 9 h
whereas ntcA transcripts begin to appear only after 4.5 and 6 h after nitrogen deprivation. However,
in ntcA mutant the hetR transcripts did not increase in response to nitrogen deprivation. Since ntcA
expression continues even in organisms grown on fi xed nitrogen sources such as nitrate or ammonia,
the basal levels of NtcA present in cells after nitrogen step-down suffi ce the purpose for induction
of hetR gene. Higher levels of ntcA transcripts required for heterocyst differentiation are induced in
turn by hetR gene, a situation that is completely lacking in hetR mutant Thus the mutual dependence
of NtcA and HetR is confi rmed. Rajagopalan and Callahan (2010) analysed the utility of the four tsps
of hetR by separating the promoter region into fi ve segments and each of these having one or more
tsp fused to gfp was individually introduced into wild-type Anabaena sp. strain PCC 7120. Of the four
tsps, tsp located at -271 appeared to be tightly regulated and more widely used during early phases
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