HETEROCYST - Cyanobacteria - page 192

required for mutual regulation of ntcA and hetR . Although prpJ2 is nonessential for diazotrophic
growth, a double mutant for prpJ1prpJ2 was unable to grow diazotrophically and resembled in its
properties with the S20 mutant of prpJ1 . The double mutant showed decreased transcripts for ntcA
and hetR and accordingly the proteins NtcA and HetR when compared to the wild-type after a
nitrogen step-down. A consensus NtcA-binding site (GTAGCCACAGGTAC) upstream of tsp in the
promoter region of prpJ2 has been recognized and a change in this sequence to CCGGCCACAGGCA
resulted in a loss of NtcA-binding. This emphasizes the role of NtcA in regulating the expression of
prpJ2 . Another signifi cant feature is the absence of induction of prpJ2 in a hetR mutant. A nitrogen
responsive regulator, NrrA encoded by nrrA (ORF all4312 ) has been described by Ehira and Ohmori
(2006a). The expression of nrrA in the prpJ1prpJ2 double mutant was unaffected so also the expression
of prpJ2 remained unaffected in a nrrA mutant. Taking these into consideration, Jang et al . (2009)
concluded that for interaction of NtcA and HetR, the presence of PrpJ1 and PrpJ2 is required.
Direct regulation of heterocyst differentiation by HetR in Anabaena sp. strain PCC 7120 has
been also determined by an epistasis analysis conducted by Orozco et al . (2006). They created a hetR
null mutant (UHM103) that showed a Het - phenotype under all conditions tested and UHM103 is
identical to the previously described hetR - mutants (Buikema and Haselkorn, 1991b; Black et al .,
1993; Dong et al ., 2000). By utilizing UHM103, they produced double mutants, i.e. hetR - patA - , hetR -
hetN - and hetR - patS - which also showed a Het - phenotype identical to UHM103. This confi rms that
HetR directly regulates heterocyst differentiation and inactivation of hetR leads to epistatic effect on
the genes tested. Risser and Callahan (2007) employed error-prone PCR amplifi cation procedure for
introducing mutations in the promoter and coding sequence of hetR of Anabaena sp. strain PCC 7120
and introduced the mutated hetR gene sequences into UHM103 in a het - background by conjugation.
The exconjugants selected were further characterized for their growth and heterocyst differentiation
in a nitrogen-defi cient medium. Thirty-three mutants having amino acid substitutions from residue
33 to 150 at N-terminus of the HetR were unable to survive nitrogen deprivation and differed in
heterocyst frequencies to less than 2%. Out of three amino acid substitutions in hetR gene, i.e hetR Cys48Ala ,
hetR Ser152Ala and hetR Ser179Asn , Risser and Callahan (2007) showed that hetR Ser152Ala and hetR Cys48Ala gene
sequences on a plasmid could complement the mutant UHM103, restoring the heterocyst frequencies
almost at par with that of the wild-type. This shows that these two amino acid residues Cys48 and
Ser152 are not essential for heterocyst differentiation, in contrast to their essentiality reported earlier
(Dong et al ., 2000; Huang et al ., 2004). Similarly, mutant 216 of Anabaena sp. strain PCC 7120 that
had a mutation in hetR Ser179Asn (Dong et al ., 2000; Huang et al ., 2004) did not differentiate heterocysts
and in the experiments of Risser and Callahan (2007) hetR Ser179Asn did not complement UHM103.
Mutants with amino acid substitutions hetR Asp17Glu and hetR Gly36Ala caused a Het - phenotype whereas
hetR His69Tyr substitution resulted in heterocyst differentiation to less than 0.1% of cells. These three
amino acid substitutions were found to be essential for normal heterocyst differentiation and levels
of HetR turnover. The other important fi ndings are that (i) Residue Asp17 of HetR is important
for DNA-binding activity but not Cys48 of HetR as has been reported earlier for this activity by
Huang et al . (2004); (ii) the amino acid substitution in hetR Cya48Ala prevented the formation of HetR
homodimer formation, thus confi rming the earlier observations of Huang et al . (2004) and HetR
homodiner formation has been observed in the wild-type, and other HetR amino acid substitutions
such as HetR Asp17Glu , HetR Gly36Ala and HetR His69Tyr ; (iii) replacement of 2 bp in the hetR coding
region in the wild-type of Anabaena sp. strain PCC 7120 to cause a Cys48Ala substitution did
not affect heterocyst differentiation; (iv) the wild-type HetR protein and the recombinant proteins
produced (in respect of the amino acid substitutions in hetR mentioned in this study) in the
experiments of Risser and Callahan (2007) were all devoid of the protease activity and accordingly