Biology Reference
In-Depth Information
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 FrÃas
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