HETEROCYST - Cyanobacteria - page 179

the application of DNA microarray techniques that a wealth of information on gene expression profi les
has been generated. Ehira et al (2003) elegantly demonstrated that the large-scale expression of genes
during heterocyst differentiation in Anabaena sp. strain PCC 7120 is physically clustered in such a
manner that they could be termed as 'expression islands'. They fi rst synthesized cDNA in presence
of Cy3-dUTP label or Cy5-dUTP label by reverse transcriptase PCR (RT-PCR) from the different
mRNAs isolated at early (1-3 h), middle (8 h) and late stages (24 h) of heterocyst differentiation. The
respective single-stranded cDNAs were pooled. The double-stranded fl uorescently-labelled cDNA
so synthesized was denatured at 95ºC for 2 min, cooled and then applied to a DNA microarray on
a glass slide consisting of 2407 clones of genomic sequences of 3 kbp each with 10 ORFs. There was
suffi cient overlapping of the ORFs in the selected clones so as to allow hybridization a very successful
event. A procedure of normalization of fl uorescence intensity was followed to determine the level of
expression of genes. A comparison of such microarray from Anabaena grown on ammonium nitrogen
was made with the one that has been subjected to nitrogen step-down. Transient gene expression
was noted at very early stages of 1 to 3 h and middle stage (8 h). A co-ordinated up-regulation of
continuous genomic regions, termed as expression of genomic islands, took place. The expression
of hetR at 3rd h with a maximum of its accumulation by 8th h coincided with expression of ntcA
which is consistent with the expression of hetR and ntcA as previously reported by Muro-Pastor et
al . (2002). The expression islands pertained to 21 ORFs of carbohydrate metabolism probably related
to heterocyst envelope polysaccharide, genes related to nitrogen fi xation (expressed as operons nifB -
fdxN - nifS - nifU , nifHDK , hesAB-fdxH and nifENX after the excision of necessary DNA elements) and
heterocyst glycolipid synthetase genes ( hglE , hglD , and hglC ).
Another such a gene expression profi le pertaining nitrogen fi xation and heterocyst differentiation
by a Nostoc strain 0102 symbiotic to Gunnera megallanica and G . manicata was constructed along a
symbiotic profi le from apex to mature parts of the leaf glands. The transcripts for hetR , ntcA , glnB
and nifH were isolated and the expression patterns were determined as one-step RT-PCR along
eight different positions in the leaf gland. The use of hetR and glnB as signalling molecules indicated
an up-regulation of the former with a concomitant increase in the frequency of heterocysts and a
down-regulation of the latter along the symbiotic profi le. These results were corroborated with the
respective proteins isolated and confi rmed through Western blot analysis. The expression of nif H
was quite high at the growing apices. Soon after infection, the heterocyst frequency increased to
about 10-20% at the 3-4 mm from apex, and a little farther away (5-6 mm from apex) the heterocyst
frequency reached 20-30% and 11-12 mm away from apex the heterocyst frequency attained a
maximum of 50-60% (Wang et al ., 2004). Global gene expression profi les for N . punctiforme , another
symbiotic cyanobacterium, were determined from steady-state cultures undergoing heterocyst
differentiation. Three developmental states of hormogonium, heterocysts and akinetes were
subjected for this analysis after a nitrogen step-down and compared with those of the ammonium-
grown cultures. As many as 495 genes were expressed in the heterocyst stage, of which 373 were
up-regulated (Campbell et al ., 2007). A signifi cant discovery is the synthesis in large amounts of a
nitrogen-stress induced non-coding RNA, designated as NsiR1 of 60 nucleotides long, in Anabaena
sp. strain PCC 7120 that triggers heterocyst differentiation leading to nitrogen fi xation. That is NsiR1
acts as a switch from nitrogen-replete conditions to nitrogen-limited cultures. Thus its expression is
restricted to developing heterocysts and requires the participation of HetR and NtcA. The location
of the gene for NsiR1 upstream of hetF and individual transcripts with cell-type specifi c promoter
and a Rho-dependent terminator make it a unique non-coding RNA transcribed in high amounts
after a nitrogen step-down (Ionescu et al ., 2010).