HETEROCYST - Cyanobacteria - page 219

PCC 7120 genome, led to the identifi cation of a 1.6 kb ORF named as patB . The encoded protein is
predicted to contain a Fe 4 S 4 bacterial ferredoxin sequence at its N-terminus and a helix-turn-helix
at its C-terminus, characteristic of a transcriptional regulator. The essential nature of patB gene has
been demonstrated by Jones et al . (2003) who showed that patB deletion mutants were unable to
grow in a nitrogen-defi cient medium. Mutants of patB with defect in the N-terminal portion or the
original PAT-2 grew slowly and revealed Mch-phenotype after nitrogen shift-down. Expression of
P patB - gfp gene construct showed green fl uorescence exclusively in heterocysts.
iii) hetN : From amongst the repertoire of mutants isolated by Ernst et al . (1992), mutant N10 with
a Het - phenotype has been further characterized by Black and Wolk (1994). During reconstruction
of a series of Tn5 insertion mutants at positions adjacent to the original site of insertion, one of the
mutants that exhibited Mch-phenotype had Tn5 insertion nearest to the 5'-end of an ORF. This
gene with 915 bp has been termed hetN whose product is similar to ketoacyl reductases. Two other
genes, one upstream of hetN (at a distance of 556 bp) designated as hetM (1,518 bp) and another
downstream of hetN , designated as hetI (711 bp) but present on the opposite strand, have been
discovered. The N-terminal end of HetM resembles an acyl-carrier protein with its central portion
similar to keto reductases whereas hetI encodes products similar to biosynthesis and export of cyclic
peptides. When hetN is introduced into Anabaena sp. strain PCC 7120 on a replicating plasmid it
completely suppressed heterocyst differentiation. But when hetN is introduced along with hetI on
a replicating plasmid heterocyst formation is not suppressed. Attempts to inactivate hetI failed as
fully segregated double recombinants could not be obtained. An intact copy of wild-type hetN gene
could not complement mutant N10 to regain the wild-type character. Inactivation of hetN resulted in
Mch-phenotype but on repeated subculturing the mutant changed to Het - phenotype. Coincidentally,
Bauer et al . (1997) also identifi ed similar sequence of genes in the order hetM ( hglB )- hetN - hetI . The
gene hetM has been redesignated by them as hglB responsible for Hgl synthesis. The role of hetN in
the maintenance of heterocyst pattern in Anabaena sp. strain PCC 7120 has further been investigated
by Callahan and Buikema (2001) who replaced chromosomal hetN promoter region with the petE
(that encodes plastocyanin) promoter (inducible in presence of copper) of Anabaena sp. strain
PCC 7120. The resultant strain 7120PN, showed expression of hetN in presence of copper that
suppressed heterocyst differentiation. This is in agreement with earlier observations (Black and Wolk,
1994; Bauer et al ., 1997). In the absence copper, as there was no expression of hetN (this situation
almost equals to creating a null mutant for hetN and also obviates the ill effects of transposon
mutagenesis), the organism showed a normal wild-type pattern of heterocyst differentiation up to
48 h but afterwards the Mch-phenotype prevailed. This has been attributed to the complete lack
of HetN protein. Since the presence of hetI along with hetN on a replicating plasmid did not cause
any inhibition in heterocyst differentiation and to rule out the possibility of any cis effects of hetI on
hetN (Black and Wolk, 1994), they introduced P petE - hetN on a replicating plasmid (pSMC15) into wild-
type. Complete suppression of heterocyst differentiation occurred as noted in case of 7120PN. hetN
also prevented the patterned expression of hetR , the master regulator. This has been confi rmed by
introducing a P hetR - gfp fusion construct in plasmid pSMC127 into wild-type and 7120PN. The wild-type
showed increased GFP fl uorescence characteristically in groups of cells and to a lower level in all
other cells in presence of copper but 7120PN exhibited decreased fl uorescence of hetR uniformly in
all cells. As it is reported earlier that ectopic overexpression of hetR leads to Mch-phenotype, P petE - hetR
translational fusion was introduced into wild-type. In presence of copper overexpression of hetR led to
Mch-phenotype in nitrogen-free medium as well as nitrate enriched medium. When a plasmid with
P petE-hetR was introduced into 7120PN strain, overexpression of both hetR and hetN took place in presence