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genetic data and the recombinant structural results. But the simple verifica-
tion of the presence of these holoproteins within actively growing cultures
will definitively link the structural results to the physiology of the native
cells. This necessary bridge between the
in vivo
state of native cells and
the
in vitro
structural and mechanistic characterization of globins will lead
to a comprehensive understanding of the function of these proteins within
the cell.
6.1.2 Genetic manipulation and phenotypic analysis
One of the exciting aspects of cyanobacteria and green algae is the genetic
tractability offered by both of these systems. Within cyanobacteria, several
species, such as
Synechococcus
sp. PCC 7002 and
Synechocystis
sp. PCC
6803, are capable of genetic transformation. In green algae, as discussed
in
Section 4
,
C. reinhardtii
has an extensive molecular toolkit available for
genetic manipulation (
Harris, 2001
). This genetic malleability will be valu-
able as a method for determining the necessity of globin genes by the gen-
eration of 'knock-out' mutants, such as the
glbN
mutant created by for the
experiments involving
Synechococcus
sp. PCC 7002 (
Scott et al., 2010
).
An important result from this initial study using
Synechococcus
sp. PCC
7002 was the identification of a distinctive phenotype following the deletion
of the globin from the wild-type strain. Using variations to the conditions
under which this phenotype is displayed allows the activity of GlbN to be
investigated. This can now be built upon, using the well-characterized struc-
ture of GlbN to make predictions as to how alterations to the amino acid
sequence of the peptide would affect the function of the protein. The rescue
of the
D
glbN
strain with the native
glbN
gene also demonstrates the ability to
add back genetic information to the cell. By transforming cyanobacteria
with a plasmid containing an altered form of the
glbN
gene, it will be possible
to connect functional and physical properties.
The availability of
C. reinhardtii
is especially interesting because it is an
easily accessible eukaryotic model system. This has been shown by the recent
creation of a strain with reduced expression of
THB8
(
Hemschemeier et al.,
2013
). The genome of
C. reinhardtii
offers additional candidate genes for
investigation, as discussed in
Section 4
, with an initial review of trans-
criptomic data highlighting the expression of
THB1
and
THB4
for further
study. Both of these genes are candidates for knock-out or knock-down,
well-established procedures in
C. reinhardtii
. The challenge will not neces-
sarily be to generate novel strains of
C. reinhardtii
in which TrHb genes have
been deleted but rather to identify a definitive phenotype that results from
D
