HETEROCYST - Cyanobacteria - page 182

of heterocysts is not exclusively under environmental control but a certain degree of development
must precede the synthesis of nitrogenase. The different phenotypic features and their combinations
in different mutants of Anabaena sp. strain PCC 7120 isolated after Tn5 insertional mutagenesis have
been presented. They are as follows: (i) Fox - mutants are those that are incapable of fi xing nitrogen
in the presence of O 2 ; (ii) Fix - mutants are those Fox - mutants that expressed very low C 2 H 2 reducing
activity under any experimental condition; (iii) Het - mutants are unable to differentiate heterocysts
even after prolonged nitrogen deprivation, of these some showed fragmenting phenotype and others
non-fragmenting phenotype; (iv) Hen - mutants are those that are unable to synthesize heterocyst
envelope; (v) Hgl - represents the inability to synthesize Hgls and (vi) Dab - phenotypic feature is
denoted by the inability to oxidize diaminobenzidine (DAB) at the polar nodules that represents
a defi ciency in the respiratory enzymes and the fully developed normal heterocysts oxidize DAB
as fi rst reported by Murry and Wolk (1989). Mutants with a combination of the above phenotypic
features are classifi ed into different classes: (i) Fox - Het - Fix - fragmenting; (ii) Fox - Het - Fix - non-
fragmenting; (iii) Fox - Het + Dab - Hen - Fix - ; (vi) Fox - Het + Dab + Hen - Fix + ; (v) Fox - Het + Dab + Hen + Fix + ;
(vi) Fox - Het + Dab + Hen + Fix - and (vii) Hen - Hgl - Hep - . The properties of the above classes of mutants
point out to the existence of different developmental stages through which the differentiation of
heterocyst proceeds with a concomitant expression of the concerned genes or clusters of genes.
The discovery of triparental conjugation and the use of Tn5 to obtain gene disruptant mutants
of cyanobacteria paved the way for the elucidation of gene expression profi les in association
with specifi c reporter genes. The specifi c molecular tools designed for this purpose involved
localization of transcription using fusions with lac , lux , and gfp as reporter genes (Wolk, 1996). In
E . coli lactose is hydrolysed by the activity of β-galactosidase (β-gal) encoded by the lacZ gene.
Besides lactose, β-gal can also hydrolyze other chromogen substrates such as O-nitrophenol β-D-
galactopyranoside (DNPG), 5-bromo-4-chloro-3-indolyl-β-D galactopyranoside (X gal) and 3,4 cyc
lohexenoesculetin-β-D-galactopyranoside (S-gal) which produce yellow, blue and black products/
precipitates, respectively. There are two major limitations for using lacZ as the reporter gene.
Firstly, it involves the usage of very costly chemicals and secondly the cells have to be lysed for
the assay of β-gal activity. A number of vector systems such as multicopy (plasmid) and sigle copy
(bacteriophage) vectors have been used to construct lacZ gene fusions by either in vivo or in vitro
manipulations (Silhavy and Beckwith, 1985). Insertion of an RNaseIII cleavage site in between a
polylinker cloning site and the promoterless lacZ gene facilitated the generation of an unchanging
5'-end for the lacZ mRNA. This led to the independent translation of lacZ mRNA and the β-gal
level refl ects the expression of gene in question (Linn and Pierre, 1990). Lammers et al . (1986) used
lacZ as a reporter for fi nding out the region in 11-kb element of nifD responsible for excision during
heterocyst differentiation in Anabaena sp. strain PCC 7120. This was based on an assay developed in
E . coli by transposing a mini-Mu-lac gene fusion in nifD element. The excision of nifD element also
resulted in the loss of lacZ gene and the colonies could be easily recognized by the absence of blue
colour on X-gal plates. The fi refl y luciferase gene ( lux ) has been cloned (de Wet et al ., 1985) and it is
a reporter with high sensitivity and could be easily identifi ed by its luminescence that requires no
post-translational modifi cations and is active immediately after translation. Luciferin, the substrate
required for monitoring the assay of fi refl y luciferase is very costly and the use of tetracycline (an
antibiotic that is photoinactivated) as a selectable marker and the necessity to permeabilize the
cells during assay of the luminescence are the other disadvantages. So luxABCDE operon from the
bacterium Vibrio fi scheri has been generally used as reporter gene. The bacterial luciferase encoded
by luxAB genes requires no addition of substrates but the gene products of luxC , D and E serve as
substrates in vivo . In addition, bacterial luciferase oxidizes n -decanal, an aldehyde in presence of