Biology Reference
In-Depth Information
glbN
strain is a homologous deletion mutant. This strain allows side-
by-side comparison with the wild-type strain to determine the physiological
effects resulting from the complete loss of the
glbN
gene.
Under standard growth conditions, no obvious differences exist between
D
the
the
glbN
and wild-type strains when grown in replete medium. However,
when medium is limited, either in carbon dioxide or iron, the growth rate of
D
D
glbN
noticeably slows relative to wild type. Transcriptional analysis sug-
gests the
glbN
gene expression is constitutive, so altered growth rates would
indicate that the loss of
glbN
inhibits growth under these nutrient
limitations. Low-temperature chlorophyll fluorescence measurements of
both strains demonstrate that the
glbN
strain alters its chlorophyll compo-
sition when starved of these nutrients, limiting the amount of photosystem
I present in this strain. Both the fluorescence emission change and altered
growth rates suggest that the
D
glbN
strain experiences more stress under
nutrient-limiting conditions than does the wild-type strain (
Scott et al.,
2010
). Further testing using spermine NONOate (which decomposes to
release nitric oxide) demonstrates that the stress in
D
glbN
is consistent with
nitrosative damage to the cell. Quantitation of this damage was estimated
using 5- (and 6-) chloromethyl-2
0
,7
0
- dichlorodihydrofluorescein diacetate,
acetyl ester (CM-H
2
DCFDA) to measure ROS/RNS content in CO
2
/Fe-
limited
D
glbN
strain to have a
reactive molecule content over sixfold higher than in the control cells.
Thus, with reservations associated with the interpretation of data obtained
with fluorescent probes (
Wardman, 2007
), it appears that the GlbN
protein confers protection from oxidative damage to the
Synechococcus
sp.
PCC 7002 cells.
Both the wild-type strain and the
D
glbN
. CM-H
2
DCFDA experiments find the
D
glbN
strain can grow in media sup-
plemented with sodium nitrate; however, as the concentration of nitrate
increases to about 90 mM, the growth rate of the
D
D
glbN
strain slows signif-
icantly (
Scott et al., 2010
). The
glbN
strain cannot grow at media concen-
trations of 240 mM nitrate, and the ROS/RNS content of the
D
glbN
strain
is noticeably higher than the wild type with the highest ROS/RNS content
being seen when the
D
glbN
strain is placed under 240 mM nitrate and the
media is made micro-oxic by sparging with nitrogen and CO
2
. These results
are strengthened by the transformation of the
D
glbN
strain with a plasmid
containing the functional
glbN
gene, with the resulting rescued strain
regaining its ability to grow under the nutrient stress lethal to the
D
glbN
strain. The processing of nitrate appears to require the GlbN protein for
effective nitrogen metabolism, with excessive nitrosative stress resulting
D
