Biology Reference
In-Depth Information
nifD
elements examined. The upstream region of
xisA
(129 bp),
xisA
itself
and a small ORF (of ≈500
nucleotides) are well conserved regions in the
nifD
elements (Henson
et al
., 2005). The importance of
a 6-bp region within the 11-bp direct repeats in excision and recombination has been stressed by the
studies conducted on the ability of XisA to excise substrate plasmids with the
nifD
fl anking regions
in
E
.
coli
. Any change in the nucleotide sequence from a pyrimidine to a purine by mutation in the
11-bp repeats affected recombination (Henson
et al
., 2008).
Brusca
et al
. (1989) demonstrated that the 11-kb element present in
A
.
variabilis
had a sequence
similarity of 96% with the 11-kb element of
Anabaena
sp. strain PCC 7120 and it was also excised
during heterocyst differentiation very much in a similar manner as the 11-kb element of
Anabaena
sp.
strain PCC 7120 is excised. Though the two cyanobacteria are different in their diazotrophic growth
potential, the organization of the 11-kb element, the presence of 11-bp direct repeats at the two ends
of the 11-kb element, its excision during heterocyst differentiation, the presence of
xisA
gene encoding
the site-specifi c recombinase and the ability of
xisA
gene of
A
.
variabilis
to complement the
xisA
gene
of
Anabaena
sp. strain PCC 7120 all point out to the similarities in the genome rearrangement during
heterocyst differentiation. The 5'-regulatory region of
xisA
gene has been shown to be important
for excision of 11-kb element as its deletion blocked the expression of
xisA
in vegetative cells of
Anabaena
sp. strain PCC 7120 but not in
E
.
coli
cells. Mutants of
Anabaena
sp. strain PCC 7120 lacking
the
nifD
element grew normally in presence of combined nitrogen and differentiated heterocysts
in a nitrogen-free medium (Brusca
et al
. 1990). A vegetative cell DNA-binding factor (VF1) has
been reported to govern the expression of
xisA
in cells undergoing heterocyst differentiation in
Anabaena
sp. strain PCC 7120. The 5'-upstream region of
xisA
has a 66-bp region consisting of three
binding sites for VF1 adjacent to one another with consensus repeat sequences (ACATT) probably
required for VF1-DNA interaction. This region seems to be the same as the regulatory region of
xisA
described by Brusca
et al
. (1990). Step-by-step deletion of 16 and 18 bp from the 66-bp region
resulted in decreased VF1-binding and the deletion of a further 12 bp caused complete elimination
of VF1-binding. Certain other genes whose upstream promoter regions bear an affi nity for binding
by VF1 are
rbcL
and
glnA
(Chastain
et al
., 1990).
The excision of 11-kb element and the transcription of genes of the
nifHDK
operon seem to be
independent of one another. A
xisA
mutant of
Anabaena
sp. strain PCC 7120 (DW12-2.2), which could
not excise the 11-kb element and fi x nitrogen, exhibited transcription of
nifH
and
nifD
genes without
gene rearrangement. The absence of
nifK
transcription in DW12-2.2 suggests that its expression is
dependent on the
nifH
promoter. Another mutant of
Anabaena
sp. strain PCC 7120 (LW1), with a
deletion of 1.68-kb DNA fragment consisting of the promoter region of
nifH
, formed heterocysts but
could not grow diazotrophically due to lack of transcription through
nifD
. However, LW1 showed
normal excision of the 11-kb element
from the
nifHDK
operon (Golden
et al
., 1991).
The transposition of
mini
-
Mu
-
Kan-lac
gene construct in the 11-kb element and the fragment
carrying the
nifD
gene was utilized in the transformation of
E
.
coli
. Such transformants of
E
.
coli
showed excision of the 11-kb element from the
nifD
gene and along with this the loss of
lac
gene
facilitated the recognition of the white colonies (with a frequency of 3%) amongst the vast majority
of the blue-colony formers on X-gal plates (Lammers
et al
. 1986). The sequencing of the
xisA
gene
revealed that it runs counter to the direction of
nif
genes beginning 240 bp from the recombination
site. The cloning of the 11-kb element into a plasmid vector and its propagation in
E
.
coli
resulted
in a decreased frequency of the rearrangement. A second rearrangement of genes involving the
excision of an interrupting sequence in
nifS
gene takes place at the same time of the excision of the
nifD
element but mediated by a site-specifi c recombinase other than XisA protein (Golden
et al
.,
1987). Golden
et al
. (1988) reported the excision of the third DNA element from the
fdxN
gene of
