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In-Depth Information
down-regulation after 3 days in akinete differentiation medium and after
24 h within hormogonia-inducing media (
Campbell et al., 2007;
Christman, Campbell, & Meeks, 2011
). The down-regulation of
NpR1005 was further verified by subsequent experimentation on hormo-
gonia induction using both a hormogonia-inducing factor (HIF) as well as
nitrogen stress induction (
Campbell, Christman, & Meeks, 2008
). The
up-regulation of NpR0416 follows the same pattern as the nitrogen fixing
(
nif
) genes, which can be correlated to the fact that the
glbN
gene, coding for
the NpR0416 protein, is found within the
nif
gene cluster (
Christman
et al., 2011
).
A recent gene expression study in
N. punctiforme
(
Soule, Gao, Stout, &
Garcia-Pichel, 2013
) assessed stress from exposure to ultraviolet light and
found that exposure to UVA radiation causes an up-regulation of the
NpR1005 TrHb1-2 protein. Ultraviolet light is known to cause significant
damage to the cells and, although not investigated in this study,
up-regulation of the TrHb may in fact be linked to minimizing this
radiation damage.
4.3. Synechococcus sp. strain PCC 7002
The cyanobacterium
Synechococcus
sp. strain PCC 7002 contains a TrHb1-1
protein referred to as GlbN and coded for by the
glbN
gene (
Scott et al.,
2002
). The GlbN protein has been produced heterologously in
E. coli
and the recombinant GlbN protein studied
in vitro
(See
Section 5
). How-
ever,
Synechococcus
sp. PCC 7002 is also the first of the cyanobacterial species
in which physiological evidence exists for the function of the GlbN protein
in vivo
(
Scott et al., 2010
). As a first step, the
glbN
gene was deleted from the
Synechococcus
strain by introducing an antibiotic resistance gene (the
aadA
gene for the aminoglycoside adenyl transferase protein), which confers resis-
tance to the antibiotic spectinomycin (
Goldschmidt-Clermont, 1991
), into
the genome in place of the native
glbN
gene. In brief, the antibiotic resistance
gene was part of an engineered recombinant plasmid, situated between two
fragments of genomic DNA from
Synechococcus
sp. PCC 7002. The two frag-
ments were located directly upstream and downstream of the native
glbN
gene. When the recombinant DNA was delivered into the
Synechococcus
sp. PCC 7002 strain, homologous recombination between the plasmid
and the host genome inserted the antibiotic resistance gene in place of
the
glbN
gene, thereby removing the native gene from the cell's genome
and creating the novel
D
glbN
strain. Subsequent PCR analysis verified that
