HETEROCYST - Cyanobacteria - page 199

layer due to a defi ciency in any one of the three genes of devBCA operon. Ultrastructural studies
confi rmed the absence of heterocyst glycolipid laminated layer (Fiedler et al ., 1998). Fiedler et al .
(2001) found that the promoter regions of devBCA operon have specifi c NtcA-binding sites at tsp
positions located at -762 bp in the devBCA operon of A . variabilis ATCC 29413 and at -704 bp position
in the devBCA operon in case of Anabaena sp. strain PCC 7120 suggesting that the devBCA operon
is activated by NtcA. Another gene, devH , encoding a putative DNA-binding protein required for
heterocyst function, has been detected during the characterization of mutant AD239 of Anabaena
sp. strain PCC 7120. DevH protein bears resemblance to the cyanobacterial proteins of the cAMP
receptor (CRP) proteins to which NtcA, the global nitrogen regulator belongs. Although DevH lacks
the fi ve conserved glycine residues in the N-terminal region necessary for regulatory properties
of the proteins of the CRP family, it possesses the helix-turn-helix motif in the C-terminal region
implicated in DNA binding (Hebbar and Curtis, 2000). A Nm resistance cassette has been introduced
in the sequence of devH and its transfer via plasmid pBN1-239B into Anabaena sp. strain PCC 7120
through conjugation from E . coli resulted in inactivation of the devH gene. The disruption of devH
has been further confi rmed by Southern analysis. The devH mutant (A57) phenotypically is Fox - ,
differentiates heterocysts without a laminated layer and so is unable to fi x nitrogen aerobically. Two
transcripts of devH , the shorter (1.0 kb) more abundant one and the longer (1.25kb) less abundant
one in a ratio of 5:1 have been induced in the wild-type after 24 and 48 h after nitrogen step-down,
respectively and these are absent in A57 (Hebbar and Curtis, 2000). The requirement of devH for
the synthesis of heterocyst glycolipid layer has further been demonstrated by Ramírez et al . (2005).
E . coli BL21 (DE3) was transformed with plasmid pET23930a carrying devH gene sequence and the
recombinant DevH protein has been utilized for production of polyclonal antibodies. The presence
of DevH protein (29 kDa) in the cell-free extracts of Anabaena sp. strain PCC 7120, after nitrogen
step-down, has been demonstrated by cross reaction with the antiserum of recombinant DevH.
Mutant A57 though produced DevH protein it has been non-functional due to the presence of extra
58 amino acid residues in between the helix-turn-helix and the C-terminal portion. The mutant
A57 also showed rearrangements in the nifHDK operon but the corresponding transcripts are not
detectable in the extracts grown under aerobic conditions. But under anoxic conditions mutant A57
exhibited Fix + phenotype signifying the synthesis of nitrogenase. Importantly, the expression of
genes specifi c for heterocyst-specifi c glycolipid biosynthesis, i.e. hglC and hglE1 is greatly reduced
while those of others ( hglB , hglD and hglE2 ) are not detectable in mutant A57. All these genes are of
course expressed in the wild-type (Ramírez et al ., 2005).
A devR gene, essential for the development of mature heterocysts in N . punctiforme ATCC
29133, a symbiotic cyanobacterium of A . punctatus has been discovered by Campbell et al . (1996).
The discovery of devR can be traced to a transposon-induced mutant UCD311 which is classifi ed
as a Fox - mutant. The identity of DevR with the receiver domain of a response regulator of the
two-component regulatory system emphasizes its role in the development of mature heterocysts.
Though the heterocyst-specifi c glycolipids are synthesized in the cells they are not transported
and deposited in the devR -disruptant mutant. The participation of DevR in a phosphorelay signal
transduction pathway has been confi rmed by the biochemical and genetic studies on DevR protein
of N . punctiforme ATCC 29133 (Hagen and Meeks, 1999). A recombinant His-tagged protein of
DevR, produced in E. coli BL21 (DE3), has been phosphorylated in vitro by a histidine kinase, EnvZ
(of E . coli involved in osmoregulation). The phosphotransferase role for DevR has been confi rmed
by the isolation of mutants defective in phosphotransferase activity. Since aspartate (D) residue at
position 53 constitutes the phosphorylation site, a change in codon 53 for aspartate GAT to CAA (for
glutamine, Q) and GAA for glutamate (E) resulted in mutants UCD425 and UCD436, respectively.