HETEROCYST - Cyanobacteria - page 220

of copper and the Mch-phenotype was suppressed and with only 1% heterocysts in nitrogen-free
medium and none in presence of combined nitrogen. A heterocyst suppression signal by hetN has
been suggested to originate from the heterocysts as is evident by the localized fl uorescence of GFP
in the heterocysts. They further showed that hetN is not required for Hgl synthesis (Callahan and
Buikema, 2001). However, Li et al . (2002) suggested that HetN plays a role in fatty acid synthesis.
Overexpression of hetN from Anabaena sp. strain PCC 7120 in E . coli caused the accumulation of
HetN as inclusion bodies. Purifi ed recombinant protein was used to raise polyclonal antibodies. The
presence of HetN protein, in the heterocyst preparations of Anabaena sp. strain PCC 7120 subjected
to nitrogen step-down, was demonstrated by immunoblotting and the concentration of HetN was
0.5 to 1.0% of the total protein. The association of HetN in higher concentrations (3.5 times) on
the thylakoid membranes than that with the cytoplasmic membrane prompted them to conclude
that HetN participates in fatty acid synthesis. They confi rmed the observations of Callahan and
Buikema (2001) on the effect of overexpression of HetN on hetR . The C-terminal pentapeptide
RGSRG motif, that is characteristic of patS which suppressed heterocyst differentiation, has also
been found in the gene sequence of hetN . If the suppression of heterocyst differentiation by hetN
is due to the presence of this sequence, any mutation caused in these bases should not suppress
heterocyst differentiation. Mutant copies of hetN ( Arg132Lys , Gly134Ser , Ser135Asp and Arg136Leu )
when introduced into wild-type on a multicopy plasmid (pRL25C), also suppressed heterocyst
differentiation ruling out the possibility of the functional nature of the pentapeptide sequence (Li et
al ., 2002). The biochemical properties of HetN revealed that it could hydrolyze ATP or GTP in vitro
in presence of magnesium ions and the ketoacyl reductase activity has been predicted to reside in
the conserved triad (Ser142-Tyr155-Lys159) of amino acids. Purifi cation of recombinant proteins
of HetN produced in E . coli has been feasible with the introduction of an eight amino acid fusion
tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) at the C-terminus, known as Strep Tag II. Mutations in the
conserved triad ( hetNSer142Asp , hetNTyr155Val and hetNLys159Glu ) did not affect the activity of
ATP hydrolysis indicating there by that the catalytic mechanism of ATP/GTP hydrolysis of HetN
is different from that of the reductase activity. The heterocyst suppressing activity of HetN has been
tested by introducing mutant alleles of hetN , hetNSer142Asp , hetNTyr155Val and hetNLys159Glu on
a multicopy plasmid (pRL25T) into Anabaena sp. strain PCC 7120. Mutant alleles, hetNSer142Asp
and hetNTyr155Val suppressed heterocyst differentiation whereas mutant hetNLys159Glu did not
suppress heterocyst differentiation. This is suggestive of the fact that the heterocyst suppression
activity of hetN resides in Lys159 (Liu and Chen, 2009).
iv) patS : Originally identifi ed as a missense mutation in a fragment of DNA (3.3 kb), carried by the
conjugal plasmid and when introduced into Anabeana sp. strain PCC 7120, it completely suppressed
the heterocyst formation. Yoon and Golden (1998) identifi ed a short ORF of 54 bp from the above
fragment that encodes a small peptide of 17 amino acids with its N-terminal part containing fi ve
hydrophobic amino acids and 5 to 8 amino acids at the C-terminal position that are important for
its activity. The ORF has been named as patS (for pattern suppression). Overexpression studies
were conducted by using patS gene under the control of glnA promoter and it resulted in complete
suppression of heterocysts. When patS was put under the infl uence of promoter of petE , in the absence
of copper since there was no expression of patS , the organism exhibited a normal pattern of heterocyst
differentiation but in presence of copper due to induction of patS a decrease in heterocyst frequency
was noted. Deletion of patS gene (strain AMC451) resulted in Mch-phenotype. The fl uorescence of
GFP has been restricted to proheterocysts during fi rst 6 h after nitrogen step-down when P patS - gfp
was used as a reporter gene. A synthetic pentapeptide PatS-5 (RGSGR), of the C-terminal amino