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In order to gain a better understanding of the origin, function
and localization of the divinyl-chlorophyll a/b-binding antenna
complexes of Prochlorococcus species and how these properties relate
to the light niche to which different strains are adapted, we have
undertaken gene expression and structural studies on the moderate
low-light-adapted strain MIT 9313 for which the full genome sequence
is available (http://www.jgi.doe.gov/JGI_microbial/html/). This
strain contains two pcb genes: pcbA (PMT 1046) and pcbB (PMT 0496).
This contrasts with the very low-light-adapted strain SS120 that
contains eight pcb genes (pcbA to pcbH)—analysis of its genome
sequence (http://www.sb-roscoff.fr/Phyto/ProSS120/)
revealed the presence of one more pcb gene (pcbH) than previously
thought5—and the high-light-adapted strain MED4, which has
only a single pcb gene (pcbA)4,5.
Recently it was shown by electron microscopy and single-particle
analyses that SS120 contains a giant supercomplex consisting of
the PSI reaction centre trimer surrounded by a light-harvesting
antenna ring composed of 18 Pcb subunits9, similar to the 18-mer
IsiA–PSI supercomplex induced in cyanobacteria when deprived
of iron6,7. However studies on MIT 9313 grown under similar conditions
to SS120 did not reveal the presence of an 18-mer Pcb–PSI supercomplex
but only ‘naked’ trimeric PSI complexes, which matched
with the cyanobacterial X-ray structure10 (Fig.
1a, b). Instead, electron microscopy (Figs
1c, d and 2a) indicated that Pcb proteins associate with the
dimeric reaction centre complex of PSII to form a Pcb–PSII
supercomplex having dimensions of approximately 210 x 290 Angstrom.
Our interpretation is that this Pcb–PSII supercomplex consists
of eight Pcb subunits with four distributed on each side of the
PSII dimer as shown in Fig. 1c.
This is emphasized by overlaying onto the projection map the published
X-ray-derived models of the PSII reaction centre dimer and CP43,
a PSII antenna protein structurally similar to Pcb9,11 (Fig.
1d). In some cases, the four Pcb subunits on one side of the
dimer were missing (Fig. 1e, f).
The ‘naked’ PSI trimers and Pcb–PSII complexes shown
in Fig. 1 were located in a chlorophyll-containing band (band 2
in Fig. 2b, insert +Fe) obtained
by sucrose density centrifugation after solubilizing isolated thylakoid
membranes with the detergent b-D-dodecyl maltoside. Also contained
in this band were some PSII reaction centre dimers free of Pcb proteins
(Fig. 1g, h).
Analysis of all discernible particles, taken from band 2 sample
micrographs, resulted in 1,192 particles assigned to PSI and PSII,
and gave a PSI:PSII ratio of about 2.We assume this to be indicative
of the ratio in the intact thylakoid membrane given that most PSI
and PSII particles were in band 2. Amino-terminal sequencing of
the Pcb protein in band 2 from +Fe conditions showed it to be the
product of the pcbA gene only, a result which was also found for
the free-Pcb proteins in band 1 and for Pcb protein in thylakoid
membranes (Fig. 3). The absence
of the PcbB protein and the 18-mer Pcb–PSI supercomplex in
iron-replete MIT 9313 cells spurred us to investigate the expression
of pcbA and pcbB genes in this strain.When the cells were grown
in medium supplemented with iron (áFe), we found that only
the pcbA gene was expressed (Table
1). However, when cells were transferred to culture medium without
added iron (-Fe), expression of the pcbB gene was activated, a surprising
result as pcb genes of Prochlorococcus were not known to be regulated
by iron as is the iron-stress-induced isiB gene (Table
1). The latter gene encodes flavodoxin12, which substitutes
for ferredoxin as an electron acceptor to PSI, and its expression
indicates that the cells had acclimatized to conditions of iron
depletion. On the other hand, the expression of the pcbA as well
as the psaA and psbA genes, encoding PSI and PSII reaction centre
proteins, respectively, were downregulated (Table
1).
The question arising from these expression studies is: where is
the PcbB protein targeted in cells exposed to low iron levels? As
before, thylakoid membranes were isolated, solubilized and subjected
to sucrose density gradient centrifugation. As shown in insert -Fe
of Fig. 2b, an additional chlorophyll-containing
band was observed compared with the iron-supplemented cells (band
3). Electron microscopy (Fig. 2b) revealed that this band contained
the 18-mer Pcb–PSI supercomplex (Fig.
1i, j), similar to that observed in Prochlorococcus SS120 (ref.
9). SDS–polyacrylamide gel electrophoresis (PAGE) and N-terminal
sequencing indicated that the Pcb protein of the PSI supercomplex
is derived from the pcbB gene. This gene product was readily observed
in SDS–PAGE profiles of thylakoid membranes isolated from cells
grown under iron deficiency and shown in Fig.
3 (white asterisk). It ran at a slightly higher apparent molecular
mass compared with the PcbA protein observed in both +Fe and -Fe
cells (black asterisk), consistent with the difference in their
predicted molecular masses of 38,511 Da (PcbA) and 40,737 Da (PcbB).
Notably, despite the downregulation of the
pcbA gene under iron-depleted conditions, a relatively high level
of the PcbA protein was detected by SDS–PAGE (see Fig.
3) and Pcb–PSII structures similar to those present in
band 2 of iron-supplemented (see Fig. 2, insert +Fe) cells were
observed by electron microscopy and image analysis in iron-deprived
cells. Intriguingly, although in cyanobacteria the induction of
the 18-mer IsiA–PSI supercomplex under iron deficiency seems
to compensate for the reduction in the level of PSI relative to
PSII and for a decrease in the synthesis of phycobiliproteins, these
reasons cannot apply to Prochlorococcus MIT 9313. Immunoblotting
analyses (Fig. 3) showed that the
PSI:PSII ratio, estimated to be about two, remained approximately
the same in +Fe and -Fe conditions despite the lowering of the expression
of psaA and psbA genes in iron-depleted cells (Table
1).
Phylogenetic analyses of the pcb/isiA/psbB/psbC gene superfamily5,13
indicate that the pcbA and pcbB genes of MIT 9313 partition into
two different clusters and that the latter is very closely related
to the pcbG gene of Prochlorococcus SS120, which is the gene
providing the Pcb protein of the 18-mer Pcb–PSI supercomplex
of this strain under iron-replete conditions, as indicated by SDS–PAGE
and N-terminal sequencing (data not shown). In the case of MED4
we have been unable to detect an 18-mer pcb–PSI supercomplex
either under iron-rich or iron-depleted conditions. Indeed in
this strain the expression of the single pcb gene (pcbA) was not
significantly effected by iron depletion (Table
1). In fact, using electron microscopy and associated image
analyses of single particles we found that the Pcb proteins of MED4
associated with PSII in a similar fashion to those of MIT 9313.
Therefore we conclude that the PcbA protein of MIT 9313, similar
to the PcbA protein of MED4, is targeted to PSII where they interact
with the reaction centre dimer and increase the lightharvesting
capacity of this photosystem. In contrast the PcbB protein of MIT
9313, similar to the PcbG protein of SS120, is targeted to PSI where
it forms an 18-mer light-harvesting antenna ring around the PSI
reaction centre trimer. Under iron depletion conditions, expression
of the pcbC gene of strain SS120 strongly increased while expression
of pcbG was downregulated (Table
1). It is therefore possible that PcbC replaces PcbG in Pcb–PSI
supercomplexes under these conditions. The observations reported
here give support to the hypothesis that the common ancestor of
Prochlorococcus and marine Synechococcus possessed
an isiA-like gene, similar to that found in other cyanobacteria12,
even if, surprisingly, no isiA is present in the only currently
available genome of a marine Synechococcus (strain WH8102;
http://www.jgi.doe.gov/JGI_microbial/html/).
The difference in Pcb structure between strains can be interpreted
in terms of their respective light niche. For SS120, having both
constitutive PSI and PSII antennae possibly confers to this strain
an adaptive advantage to grow under the very low irradiances found
at the bottom of the euphotic zone3. In the high-light-adapted strain
MED4, only a PSII-related pcb gene is required because in the upper,
well-illuminated layer of the ocean an additional antenna for PSI
may not be needed. Indeed, according to the recent X-ray structure10,
the ‘naked’ cyanobacterial PSI reaction centre already
binds almost 100 light-harvesting chlorophyll molecules. The intermediate
situation found in MIT 9313 is harder to interpret, as it appears
to have an antenna ring around PSI only under iron depletion. Perhaps
this suggests that there is a more complex inter-relationship between
light intensity and availability of iron in the oceanic environment
than hitherto considered.
The apparent requirement of at least some Pcb proteins to associate
with PSII in Prochlorococcus is in line with the relatively
low level of about 30 light-harvesting chlorophylls bound to PSII
(ref. 12). Therefore in all types of photosynthetic organisms the
light-harvesting capacity of PSII usually needs to be boosted by
additional antenna systems: chlorophyll a/b-binding Cab proteins
of plants and green algae8 and phycobiliproteins of cyanobacteria
and red algae14. In the case of Prochlorococcus, each Pcb subunit
adds an additional 13 chlorophylls assuming that they bind the same
level of this pigment as CP43 (ref. 12). Consequently it takes the
binding of just two Pcb subunits to approximately double the light-harvesting
capacity of PSII.
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