HETEROCYST - Cyanobacteria - page 190

of heterocyst differentiation. This is evident by the expression of -271 tsp-gfp fusion construct in the
wild-type during the fi rst 4 h in spatially patterned cells destined to differentiate into heterocysts after
a nitrogen step-down. The wild-type strain bearing the -728 / -696 tsp - gfp fusion constructs showed
signifi cant levels of fl uorescence in all cells in nitrogen-replete and -starved conditions. Similarly, -184
tsp - gfp fusion construct exhibited fl uorescence uniformly in all cells. In a patA deletion mutant the
expression of 271-lacZ fusion construct was down-regulated which correlated with reduced heterocyst
frequency where only terminal heterocysts differentiated but the transcripts from -271 tsp of hetR
were completely lacking in strains overexpressing HetN and PatS, the two negative regulators of
heterocyst differentiation. Important features of heterocyst differentiation such as commitment of cells
to differentiate, the number of cells that undergo differentiation and the timing of these all depended
on -271 tsp of hetR as its deletion caused a delay in all these parameters.
Structural studies on HetR of Anabaena sp. strain PCC 7120 showed that it exists in vivo as a
homodimer by the formation of a disulphide bond between Cys48 residues of the two monomers. A
mutant of hetR with a change in the Cys48 to Ala48, isolated after site-directed mutagenesis, lost the
ability for HetR dimerization as well as heterocyst differentiation. So HetR dimerization is required
for heterocyst differentiation. The mutant protein HetR Cys48Ala retained its active Ser residue and so its
proteolytic activity. In order to show the importance of HetR homodimer formation for heterocyst
differentiation, Huang et al . (2004) introduced the mutant hetR Cys48Ala gene into a hetR - mutant 884a of
Anabaena sp. strain PCC 7120 reported earlier by Black et al . (1993) to generate a mutant C48. Mutant
C48 produced HetR Cys48Ala protein that did not form a dimer in vivo . The isolated HetR Cys48Ala protein
monomers did not interact with each other as well as with wild-type HetR monomers in vitro . Upon
nitrogen step-down, mutant C48 failed to form heterocysts. When the hetR Cys48Ala gene construct was
introduced into wild-type on a multi-copy plasmid, the resulting strain differentiated heterocysts
with a normal pattern suggesting that the expression of mutant gene did not interfere with normal
HetR function. Huang et al . (2004) also adduced evidences to show that HetR homodimer is a DNA-
binding protein and regulates the activity of hetR , hepA and patS by binding to their promoter regions
during early phases of heterocyst differentiation. The up-regulation in the activity of hetR required
for heterocyst differentiation is prevented by the C-terminal pentapeptide (RGSGR for Arg-Gly-Ser-
Gly-Arg) sequence of PatS due to its inhibition of the DNA-binding activity of HetR. This is how PatS
or the synthetic RGSGR pentapeptide (at 1 µM) inhibits the formation of heterocysts in Anabaena sp.
strain PCC 7120 as reported by Yoon and Golden (1998). For example, patS deletion causes an aberrant
pattern with Mch-phenotype after nitrogen step-down (Yoon and Golden, 1998). Like PatS, another
negative regulator of heterocyst differentiation is HetN. When hetN expression is switched off due
to non-expression of P petE - hetN in the absence of copper (a situation resembling hetN deletion) it caused
the formation of fi rst a normal pattern followed by Mch-phenotype during the subsequent rounds
of heterocyst differentiation (Callahan and Buikema, 2001). Both PatS and HetN act independently
but have complementary function and help in the establishment and maintenance of the pattern.
Their non-expression leads to an aberrant pattern as well as Mch-phenotype. In this background, the
characterization of a mutant S2-45, isolated upon Tn5-1058 insertion in Anabaena sp. strain PCC 7120
genome, showed that it formed very long, irregularly spaced strings of heterocysts. Since attempts
for the reconstruction of the mutant by introducing the Tn5 inserted region of S2-45 into wild-type
failed, Khudyakov and Golden (2004) cured the plasmid from S2-45 by growing it in the absence of
the antibiotics and the resulting strain (AMC1285) showed the original Mch-phenotype. In order to
make conjugative transfer possible through different pDU-1-based plasmids carrying Nm r marker,
the original Nm-Bm-Sm resistance genes in the chromosome of S2-45 mutant have been replaced by
Sp-Sm resistance cassette. The resulting strain AMC1286 exhibited Mch-phenotype. Since extra copies