HETEROCYST - Cyanobacteria - page 181

Anabaena sp. strain PCC 7120 possesses three restriction enzymes Asp7120I, Asp7120II and
Asp7120III (corresponding to AvaI, AvaII and AvaIII of A . variabilis ) that act as isoschizomers on the
corresponding restriction sites at Asp7120I , Asp7120II (Duyvesteyn et al ., 1983) and Asp7120III (Elhai et
al. , 1997) on the plasmids introduced into its cells. In order to overcome the activity of isoschizomers
in vivo , the restriction sites on the plasmids are removed. In this way it has been possible to develop
shuttle vectors capable of getting transferred in between E . coli and Anabaena sp. strain PCC 7120
(Wolk et al ., 1984). Wolk et al . (1988) for the fi rst time utilized molecular tools for the identifi cation
of genes governing the synthesis of heterocyst envelope polysaccharides of Anabaena sp. strain
PCC 7120. In doing so, they took advantage of the RP-4 (as helper plasmid)-mediated conjugative
transfer of pBR322-based shuttle plasmids from E . coli to Anabaena sp. strain PCC 7120 (Wolk et al .,
1984). A pRL25C cosmid vector that is equipped with the capacity to replicate in E . coli as well as
Anabaena sp. strain PCC 7120 was used to generate a library of 1,054 cosmid clones consisting of
size-fractionated (about 40 kb) genomic DNA of Anabaena sp. strain PCC 7120 and RP-4 was
transferred to this library by conjugation. A number of mutants unable to grow aerobically in a
nitrogen-free medium but required fi xed nitrogen sources for their survival were isolated after UV-
mutagenesis. Among these, EF116 which showed defects in heterocyst envelope polysaccharide
(which lost cohesiveness when compared to the wild-type) was chosen and a complementation of the
mutant EF116 by transfer of genomic DNA fom the cosmid library through triparental conjugation
enabled them to identify a 2.8 kb fragment of DNA derived fom one of the cosmids. Another mutant
EF113 was complemented by a single cosmid bearing a 4.8 kb genomic DNA. Other mutants EF114
and EF122 are unique for the lacking the Hgl layer whereas EF104 showed the absence of nitrogen
fi xation even under anaerobic conditions. This technique of conjugative transfer of genes into
Anabaena sp. strain PCC 7120 has been followed invariably by most of the investigators all world
over and has become the focal point for transfer of reporter genes to identify various functions during
heteocyst differentiation (Elhai and Wolk, 1988). Elhai et al . (1997) suggested that the success of
triparental conjugation very much depended on the number of restriction sites ( Asp7120I , Asp7120II
or Asp7120III ) carried on the plasmids intended to be transferred into Anabaena cells and the number
of corresponding restriction enzymes present in Anabaena cells. Plasmids with their restriction sites
( Asp7120I , Asp7120II or Ava7120III ) modifi ed by methylation in E . coli have a greater effi ciency of
being transferred as the restriction sites are protected against the activity of restriction enzymes.
Another technique that has become handy is the transposon Tn5-mediated mutagenesis. Tn5 has four
AvaI restriction sites and when transferred through pBR322bla::Tn5 construct into Anabaena cells, it
might be cleaved by the Asp7120I restriction enzyme. In order to protect Tn5 from being cleaved, in
the fi rst instance pBR322bla::Tn5 construct is used to transform E . coli HB101 cells containing AvaI
and AvaII methylase activity. That is AvaI restriction sites on Tn5 get methylated so that upon entry
into Anabaena cells the Asp7120I restriction enzyme is unable to act on these sites. The pBR322::Tn5
is then transferred into Anabena sp. strain PCC 7120 cells through triparental conjugation. Tn5 has
been found to get transposed into a number of sites on the genome of Anabaena sp. strain PCC 7120.
Such cells with Tn5 can be screened and selected by the antibiotic resistance cassette present in Tn5
sequence. Out of a total of 400 colonies isolated from four conjugation plates, one isolate T-123 was
unable to grow in a medium without combined nitrogen. That is it is unable to fi x nitrogen (Fix - )
although it formed the heterocysts that resemble in every respect with those of the wild-type strain
(Borthakur and Haselkorn, 1989).
A comprehensive account on the diversity of mutants related to the structure and function of
heterocysts was published by Ernst et al . (1992). Their study is most signifi cant for two reasons. The
fi rst is that they provided a classifi cation of the mutants and the second is that the differentiation