Biology Reference
In-Depth Information
HetF protein resembled a capsase-hemoglobinase type of protease (Aravind and Koonin, 2002). The
wild-type and
hetF
disruptant mutant (UCD416) have been transformed with a multicopy plasmid
(pSCR60) carrying
hetF
gene to generate strains UCD487 and UCD488, respectively. The presence
of the multicopy plasmid with functional
hetF
gene not only restored the ability of UCD416 to
differentiate heterocysts but also resulted in Mch-phenotype upon a nitrogen step-down. In this
respect, the effects of inactivation of
hetF
and the presence of
hetF
in multiple copies resembled
the effects of
hetR
. The absence of transcripts of
hetF
in UCD416 cells after nitrogen step-down
as evidenced by the unchanged luciferase activity when
luxAB
gene was used as a reporter gene
indicates that
hetF
is constitutively expressed. Moreover, the
hetR
gene expression, which is found
in the wild-type within 3 h of nitrogen step-down with a maxiumum at 12 h, has been found to be
delayed in UCD416 till 6 to 12 h and the maximum levels of
hetR
transcripts appeared only after
24 to 36 h. Further, transcriptional fusion of
hetR
with
gfp
showed GFP fl uorescence mainly in the
heterocysts after a nitrogen step-down in wild-type but in UCD416 when
hetR
-
gfp
was expressed
on a multicopy plasmid, the HetR-GFP fusion protein appeared in all cells under nitrogen enriched
medium. These results signify that
hetF
cooperates with
hetR
in a positive regulatory pathway of
heterocyst differentiation in
N
.
punctiforme
strain ATCC 29133 (Wong and Meeks, 2001). The necessity
of
hetF
homologue in
Anabaena
sp. strain PCC 7120 for nitrogen fi xation has been demonstrated (Wolk
et al
., 2007). PatS controls the formation of intercalary heterocysts and so the pattern formation in
Anabaena
sp. strain PCC 7120. Since PatS also regulates the transcription and HetR activity much
in a similar manner as
hetF
, the combined effects of
hetF
and
patS
on the
hetR
transcription and
activity of HetR has been examined by Risser and Callahan (2008). The main fi ndings of their study
are summarized here: (i) the
hetF
gene (
all1730
) of
Anabaena
sp. strain PCC 7120 is similar to
hetF
from
N
.
punctiforme
ATCC 29133; (ii) deletion of
hetF
in
Anabaena
sp. strain ATCC 7120 resulted in
a Het
-
phenotype (UHM130); (iii) since HetF belongs to CHF class of cysteine proteases, the active
site His201 and Cys246 residues at the respective codons have been mutated to the codons of Tyr
and Ala, respectively and the mutated
hetF
gene sequences have been introduced into UHM130.
Due to failure of the two mutations to complement UHM130, the protease nature of HetF has been
confi rmed; (iv) HetR protein levels in
hetF
and
PatA
single and double mutants were found to be
higher than in the wild-type of
Anabaena
sp. strain PCC 7120; (v) the increased levels of HetR in
hetF
and
patA
deletion single and double mutants has been found to be a post-transcriptional event and
(vi) The necessity of
hetF
for transcription of
hetR
from -271 tsp indicates that
hetF
is required for
positive autoregulation of
hetR
and its localization in cells that differentiate into heterocysts. Risser
and Callahan (2008) confi rmed this by changing the expression profi le of
hetR
from its native promoter
into a transposition carried on a multicopy plasmid with two independent inducible promoters of
petE
(inducible in presence of copper) and
nirA
(inducible in presence of nitrate). The corresponding
constructs were then introduced into strains having either
hetF
,
patA
or
hetF
and
patA
deleted and
also
hetR
deleted strains. The levels of HetR in wild-type and
hetR
deletion mutant increased due
to expression under inductive conditions in presence of copper and nitrate and this in turn caused
Mch-phenotype as reported earlier (Buikema and Haselkorn, 2001). Likewise, the levels of HetR in
hetF
and/or
patA
deletion mutants also increased suddenly under both the inductive conditions.
Since transcription of
patS
and
hetR
is dependent on
hetR
, the effects of deletion of
hetF
on the
patterned induction of
patS
and
hetR
was examined by introducing transcriptive fusions of
gfp
with
the promoters of
patS
and
hetR
in
hetR
deletion,
hetR
mutant (Ser179Asn) and
hetF
deletion strains.
The appearance of green fl uorescence due to expression of P
hetR
-
gfp
fusion after nitrogen step-down
was restricted to only heterocysts in the wild-type whereas it was uniformly seen in all vegetative
cells in the other three strains. Likewise, P
patS
-
gfp
fusion was also expressed in a patterned manner that
