From the
The rod substructures of the Anabaena sp. PCC 7120
phycobilisome contain the light harvesting proteins C-phycocyanin and
phycoerythrocyanin (PEC). Even at low light intensities, PEC represents
no more than 5% of the phycobilisome protein. The
Phycobilisomes are macromolecular complexes of several million
daltons that serve as important light-harvesting components of the
photosynthetic apparatus of cyanobacteria and red algae. These
complexes are made up of several homologous chromoproteins, the
phycobiliproteins, whose assembly to form the phycobilisome is mediated
by a set of ``linker'' polypeptides
(1) . The major
phycobiliproteins fall into three classes: the allophycocyanins, the
phycocyanins, and the phycoerythrins. The fundamental building block of
all these phycobiliproteins is an
The in vitro uncatalyzed reaction of
phytochromobilin with apophytochrome leads to a chromoprotein
functionally indistinguishable from native
phytochrome
(9, 10) . The possibility of non-enzymatic
addition has been investigated by analysis of the in vitro addition of PCB and phycoerythrobilin to
apophycocyanin
(11, 12) , and to the apophycoerythrin
Studies of C-phycocyanin
Analysis of a second-site revertant showed that a single amino acid
substitution in the
In sum, the available information suggests
that bilin addition at each site requires a specific lyase. In the
organisms with phycobilisomes having the simplest phycobiliprotein
composition, this would require eight such lyases; in those with the
most complex phycobilisomes, 22 such enzymes might be
needed
(17) . However, this supposition rests solely on the
studies of C-phycocyanin
In addition to a single cpc operon that
inter alia includes the C-phycocyanin
SDS-PAGE
(34) was performed in a Bio-Rad Mini Protean II system using 14%
acrylamide in the separating gel and 10% in the stacking gel and a
monomer/bis ratio of 37.5:1. Two-dimensional gel electrophoresis was
performed with the Bio-Rad Mini two-dimensional system. The first
dimension was composed of 5% acrylamide (40% T, 5% C), 2% Triton X-100,
9.2 M urea, and 2% ampholytes (4:1 mixture of pH 5-7 and
3-10). The second dimension was a SDS-PAGE on a 14% separating
gel and a 10% stacking gel. One-dimensional separations by horizontal
isoelectric focusing were performed under the conditions given for
vertical isoelectric focusing. These gels were then stained with
Coomassie or 10 mM zinc acetate in 0.1 M glycine. The
Coomassie-stained gels were scanned with an imaging system, and the
intensity of the spots was calculated by integration with the program
Scan Analysis (BioSoft; Cambridge, UK). Zinc-stained gels were scanned
with a two-color, confocal fluorescence gel imaging system
(35) ,
and the images were processed with the NIH image program Image 1.54 and
IPLAB (Signal Analytics Corp; Vienna, VA). The fluorescence intensity
of the spots was calculated by integration with Scan Analysis.
Plasmids pRL278:PECE, pRL278:PECF, and pRL278:PEC
The growth
rates of the mutant strains were compared to that of wild-type. Under
limiting light conditions, the growth rate of the mutants was
30-50% lower than that of the wild-type, and the mutant strains
had an average of 20% less PCB relative to chlorophyll a than
wild-type (). The reduction in PCB content exceeds that
expected for the loss of PEC alone and suggests a modest decrease in
the mutants in the cell content of phycocyanin (and, possibly
allophycocyanin) as well. For comparison, the cpcE and
cpcF mutants of Synechococcus sp. PCC 7002 grew two
times slower than wild-type
(16) . Under high light, there was no
significant difference between the growth rates of wild-type and mutant
strains.
SDS-PAGE of the wild-type, PECE-3, PECF-6, and PEC
The subunits of
phycobiliproteins can be separated by HPLC on reverse-phase C-4
resin
(33) . When phycobiliproteins from Anabaena sp.PCC 7120 are analyzed by this method, the PEC
Anabaena sp.PCC 7120 pecE and pecF genes
are 47% identical at the amino acid sequence level to the cpcE and cpcF
genes of the same organism
(14) . Initial studies by Zhou et
al.(15) using interposon mutagenesis showed that the
homologous cpcE and cpcF genes from the cpc operon of Synechococcus sp. PCC 7002 are involved in the
attachment of PCB to the C-phycocyanin
Independently selected mutants in pecE (strains PECE-3 and PECE-5), or pecF (strains PECF-6 and
PECF-7), all show loss of PEC and decrease of the PEC-associated linker
polypeptide in phycobilisomes. The PEC
It is interesting to compare the consequences of
mutations in cpcE and cpcF with those in pecE and pecF. Synechococcus sp. PCC 7002 mutants in
cpcE and cpcF (or in both genes) show a much
decreased level of phycocyanin (to
A surprising
difference is seen with respect to the PEC
The average homology between
the corresponding subunits of different C-phycocyanins is 74% while
that between the corresponding
Phycocyanin
How is the near-absence of such
a PCB adduct of the PEC
subunits of
both proteins carry thioether-linked phycocyanobilin (PCB) at
-Cys-82 and
-Cys-155; however, C-phycocyanin has PCB at
-Cys-84 whereas PEC
subunit carries phycobiliviolin at this
position. The Anabaena sp. PCC 7120 pec operon is
made up of five genes. PecB and pecA encode the
and
subunits of PEC, pecC encodes a linker polypeptide
associated with PEC in the rod substructure, and pecE and
pecF are genes of unknown function that show a high degree of
homology to cpcE and cpcF, that encode a
C-phycocyanin
subunit PCB lyase (Fairchild, C. D., Zhao, J.,
Zhou, J., Colson, S. E., Bryant, D. A., and Glazer, A. N.(1992)
Proc. Natl. Acad. Sci. U. S. A. 89,
7017-7021). Insertional mutants in pecE and
pecF, and an interposon mutant in which a portion of both
pecE and pecF was deleted, were constructed. All
three types of mutants grew 1.3 times slower than wild-type under
limiting light conditions and showed a 20% reduction in the PCB content
of whole cells relative to chlorophyll a. Holo-PEC was missing
from the phycobilisomes of all three types of mutants and the level of
the PEC linker polypeptide was reduced relative to the wild-type.
However,
30% of the wild-type level of the PEC
subunit was
present in all of these phycobilisomes. In contrast, the PEC
subunit was barely detectable in the pecE and pecF mutants, but was present in the pecEF deletion mutant as
a PCB-adduct in a 1:1 ratio with the PEC
subunit. The identity of
this ``unnatural'' adduct was confirmed by isolation of the
subunit and amino-terminal sequencing. These biochemical results
support the inference that pecE and pecF encode a PEC
subunit phycobiliviolin lyase, and, in conjunction with earlier
findings, demonstrate that phycobiliprotein bilin lyases show high
selectivity (rather than absolute specificity) for both the bilin and
the polypeptide substrate.
heterodimer. Their ability
to absorb visible light is conferred by linear tetrapyrrole prosthetic
groups (bilins) attached through thioether linkages to specific
cysteinyl residues. Four isomeric bilins, differing only in the
arrangement of double bonds, are found on cyanobacterial and red algal
phycobiliproteins: phycocyanobilin (PCB),
(
)
phycobiliviolin, phycoerythrobilin, and
phycourobilin
(2) . The positions of the cysteinyl residues that
serve as the bilin attachment sites are conserved among the different
phycobiliproteins, e.g.
-Cys-84 carries a bilin in
allophycocyanins, phycocyanins, and phycoerythrins, but the total
number of bilin attachment sites is the major distinguishing feature of
phycobiliproteins belonging to different classes. Thus,
allophycocyanins carry one bilin on each subunit, phycocyanins one on
the
and two on the
subunit, and phycoerythrins carry either
two or three on the
and three on the
subunit
(2, 3) . The amino acid sequences of many
phycobiliproteins have been determined either directly or by sequencing
of the structural genes
(4) . The crystal structures of several
C-phycocyanins
(5, 6) , phycoerythrocyanin
(7) ,
and B-phycoerythrin
(8) have been determined at high resolution.
In spite of such a large base of structural information, the features
that lead to the attachment of particular bilins at specific sites are
not evident.
subunit
(13) . With these apophycobiliproteins, spontaneous
thioether bond formation does take place between the bilins and certain
of the cysteinyl residues normally involved in bilin attachment.
However, the products of in vitro addition differ from native
phycobiliproteins. The predominant products are bilins, with an
additional double-bond between the C-2 and C-3 carbons of ring
A
(11, 12, 13) . There is no discrimination in
the addition of PCB and phycoerythrobilin at particular sites. Finally,
addition takes place at some, but not all of the attachments sites.
These results argue strongly against spontaneous bilin addition to
apophycobiliproteins.
subunit
phycocyanobilin lyase provide powerful support for the existence of
multiple specific bilin lyases. The phycocyanin operon has been
sequenced in several cyanobacteria (for references, see Ref. 14). This
operon includes two genes, cpcE and cpcF. Insertional
inactivation of either cpcE or cpcF in
Synechococcus sp. PCC 7002 results in the same distinctive
phenotype. These mutants produce a much lower amount of phycocyanin
than the wild-type, and the phycocyanin that is produced lacks the PCB
chromophore at
-Cys-84 (15); however, the bilin-binding site is
competent to react with PCB in vitro(16) . A minor
fraction (
20%) of the phycocyanin from the mutants does carry
unnatural bilin adducts at the
-84 site (16). The predominant
adduct is mesobiliverdin (see Ref. 12). The two phycocyanin
subunit PCB prosthetic groups are apparently unaffected in the mutants
nor are the PCBs on allophycocyanin
(16) . Direct biochemical
studies of the catalytic activities of recombinant CpcE and CpcF
decisively confirmed that these polypeptides together function as a
specific phycocyanin
subunit PCB lyase
(17, 18) .
subunit of phycocyanin is sufficient to
partially suppress the defect in PCB addition caused by the
inactivation of cpcE(16) . Examination of the position
of this substitution,
-129-Tyr
Cys, in the crystal structure
of wild-type phycocyanin reveals that this residue is within 5 Å
of the thioether linkage of the PCB to
-Cys-84. This finding hints
that the recognition of bilin-binding sites by bilin lyases involves
three-dimensional features in the apopolypeptide and that the
-129-Tyr
Cys substitution may allow recognition of the
-84 bilin attachment site by a protein bilin lyase specific for
another site
(16) .
subunit PCB lyase referred to above.
This paucity of information prompted us to search for other protein
bilin lyases.
-subunit PCB lyase
genes cpcE and cpcF, Anabaena sp. PCC 7120
has an adjacent phycoerythrocyanin (pec) operon made up of
five genes
(19, 20, 21, 22) . PecB and pecA encode the
and
subunits of PEC,
pecC encodes a linker polypeptide that functions in the
assembly of PEC in the rod substructure, and pecE and pecF are genes of unknown function that show a high degree of homology
to cpcE and cpcF (21, 22). We describe here the
phenotypes and biochemical studies of pecE and pecF mutants. These studies support the inference that pecE and pecF encode a phycoerythrocyanin (PEC)
subunit
phycobiliviolin lyase.
Cultures and Strains
Anabaena sp. PCC
7120 was maintained in BG-11 medium
(23) at room temperature
with constant illumination at 40 µE m s
. Plates of BG-11 contained washed Difco agar
at 1%. One-liter cultures were constantly bubbled with 4%
CO
, 96% N
and maintained at 30 °C. DH5
cells were used for transformation and plasmid maintenance and grown in
Luria Broth medium with the appropriate antibiotics (kanamycin and
streptomycin at 25 µg ml
and ampicillin at 50
µg ml
).
DNA Manipulations
Miniprep plasmid was isolated by
the SDS-alkaline lysis method
(24) . Larger scale plasmid
preparations were prepared by a modified SDS-alkaline lysis method
which included polyethylene glycol (average molecular weight 6,000)
precipitation of the plasmid. Restriction endonucleases and other
modifying enzymes were from Life Technologies, Inc. or New England
Bio-Labs (Beverly, MA). DNA fragments were separated on agarose gels
and eluted with Geneclean (Bio-101, La Jolla, CA).
DH5
cells were transformed by the CaCl
method or using
the DNA transformation kit from Boehringer Mannheim. Southern analysis
was employed on total DNA isolated from 10-ml cultures of the
Anabaena strains as described by Cai and Wolk
(25) .
Southern blots were probed and visualized using the Luminescent
Detection Kit (Genius 7, Boehringer Mannheim). In this method, the
probe was made by random primed labeling of the DNA with a dNTP mixture
that included digoxigenin-11-dUTP. The blot was incubated with the
probe and then with anti-digoxigenin-alkaline phosphatase.
Visualization was with Lumigen PPD chemiluminescent alkaline
phosphatase substrate [4-methoxy-4-(3-phosphate-phenyl) spiro(1,
2-dioxetane-3, 2`-adamantane)].
Construction of Anabaena sp. PCC 7120 Mutant
Strains
Plasmid pF500
(21, 22) containing the
pec operon was digested with NheI and XhoI
in order to excise pecB, pecA, and part of
pecC. The ends were filled in with Klenow and the remaining
plasmid containing the pBluescript KS vector
(Stratagene, La Jolla, CA), part of pecC, pecE, and
pecF was ligated to form pF500C`EF. PecE was
interrupted with the C.S4 cassette
(26) as follows. The C.S4
cassette, which confers resistance to streptomycin and spectinomycin,
was excised with XbaI from pRL42
(26) . The
cassette was partially filled in with dCTP and dTTP, and then ligated
to a 4.5-kb fragment of pF500C`EF which had been partially digested
with HindIII and then partially filled in with dATP and dGTP.
A clone was isolated which contained the C.S4 cassette inserted into
the HindIII site in pecE (Fig. 1A).
The 4.5-kb fragment containing part of pecC, pecE interrupted with C.S4, and pecF was excised from
pBluescript KS
with XhoI and XbaI
and ligated into pRL278 digested with XhoI and SpeI.
The resulting plasmid was named pRL278:PECE. Plasmid pRL278 is a
shuttle vector for transferring genes to Anabaena sp. PCC 7120
which contains the sacB gene
(25, 27) . PecF was interrupted by the C.S4 cassette in a similar manner. C.S4
bounded by XbaI sites was ligated into pF500C`EF digested with
SpeI (Fig. 1B). The 4.5-kb fragment containing
part of pecC, pecE, and pecF interrupted
with C.S4 was excised from pBluescript KS
with XhoI and XbaI and the resulting 4.5-kb
fragment was ligated into pRL278 as described above and was named
pRL278:PECF. The pecE and pecF genes were interrupted
with the C.S4 cassette as follows. pF500C`EF was digested with
HindIII, and the HindIII sites were partially filled
in with dATP and dGTP. This deleted a 1.1-kb fragment
(Fig. 1C) and replaced it with C.S4 bounded by
XbaI sites and partially filled in with dCTP and dTTP. The
3.4-kb fragment containing part of pecC was excised with
XhoI and XbaI and ligated into pRL278 as described
above. This plasmid was designated as pRL278:PEC
EF
(Fig. 1C).
Figure 1:
Position of the interposon in the
pecE and pecF mutant strains of Anabaena sp.
PCC 7120. Shaded boxes indicate positions of the genes.
Arrows indicate the direction of transcription. Selected
restriction sites are indicated. A, the position of the
interposon, C.S4, which interrupts pecE in the PECE strains.
B, the position of the interposon, C.S4, which interrupts pecF
in the PECF strains. C, the position of the interposon, C.S4,
in strain PECEF-4 which replaces a portion of the pecE and pecF genes. The black boxes indicate
portions of pecE and pecF used as probes for Southern
analysis.
The interrupted gene(s) present in pRL278
were introduced into Anabaena sp.PCC 7120 by the
triparental mating technique of Elhai and Wolk
(28) . Strain
J53(Rp4) was the conjugal strain, and HB101 cells containing the helper
plasmid pRL528 were used in the mating. The cells were plated on
0.45-µm HATF filters (Millipore), placed on BG-11 agar plates for 2
days, and then transferred to plates containing spectinomycin and
streptomycin each at 2.5 µg ml. After 2 weeks
initial exconjugants appeared, several were grown in liquid BG-11 for 1
week and then plated on BG-11 plates containing 5% sucrose,
spectinomycin, and streptomycin to select for potential double
recombinants. After 2 weeks on this selective medium, colonies appeared
which exhibited resistance to spectinomycin, streptomycin, and sucrose.
Twenty of these colonies were replica-plated onto BG-11 plates
containing 5% sucrose, spectinomycin, streptinomycin, and neomycin to
confirm excision of pRL278 from the genome. Colonies unable to survive
on this combination were then screened by Southern analysis. The mutant
strains resulting from transformation with pRL278:PECE, pRL278:PECF,
and pRL278:PEC
EF are referred to as PECE, PECF, and PEC
EF,
respectively.
Whole Cell Difference Spectra
(29
Two 1-ml
aliquots of cells in mid to late log phase were taken. One sample was
heated in an Eppendorf tube at 58 °C for 10 min and then
immediately chilled on ice. The spectra of the untreated and
heat-treated samples were taken using the frosted side of a quartz
cuvette. To obtain the difference spectrum, the spectrum of the
heat-treated sample was subtracted from that of the untreated one.
Quantitation of Chlorophyll and
Phycocyanobilin
One ml of each strain at mid to late log phase
was pelleted by centrifugation in a microfuge at 3,000
g for 5 min. One ml of 90% methanol was then added to the
pellet, and the cells were sonicated two times for 5 min at 4 °C
and then incubated in the dark for at least 30 min at 4 °C. The
methanol extract was then centrifuged at full speed for 10 min in a
microcentrifuge at 4 °C. The methanol supernatant was collected,
and the reading at 663 nm was taken. The remaining pellet containing
the phycobiliproteins was then resuspended in 0.9 ml of 9 M
urea, pH 2.0, incubated for 15 min in the dark, at room temperature,
with shaking and then centrifuged at full speed in a microcentrifuge
for 10 min. The absorbance of the urea supernatants was measured at 665
nm. The concentration of chlorophyll was determined from the
relationship Chl (µg ml
) = OD
13.9
(30) ; an extinction coefficient of 35.4
mM
cm
at 665 nm was used
for protein-bound PCB
(31) .
Analysis of Phycobiliproteins from the
Phycobilisomes
Phycobilisomes were isolated by the procedure of
Yamanaka and Glazer
(32) . Absorbance spectra were obtained with
a Perkin-Elmer Lambda 6 UV/Vis spectrophotometer. Phycobiliprotein
subunits were separated by high pressure liquid chromatography (HPLC)
by the method of Swanson and Glazer
(33) with a Waters 600E
multisolvent delivery system and a Waters 991 photodiode array detector
for data acquisition. Samples were collected as indicated, concentrated
by evaporation with nitrogen gas, and aliquots subjected to
amino-terminal sequencing on a gas-liquid phase sequencer (Perkin Elmer
Applied Biosystems Division, Foster City, CA).
Interposon Mutagenesis of pecE and pecF
The
pecE and pecF genes were individually interrupted
with the C.S4 cassette at the sites shown in Fig. 1, A and B. A portion of the pecE and pecF genes was also deleted and replaced with the C.S4 cassette to
inactivate both genes simultaneously (Fig. 1C).
Initially, the ligation constructs were introduced into DH5 cells
by electroporation. However, approximately 70% of the resulting
transformants contained rearranged plasmids. Constructs in which the
intervening sequence between pecE and pecF was
deleted yielded a higher percentage (50%) of transformants with the
correct plasmid. To avoid such complications, the ligation constructs
were introduced by transformation of competent cells as this gave a
higher percentage of transformants with the correct plasmid. The
interrupted gene(s) were then ligated into the multiple cloning site in
pRL278. As described in the ``Experimental Procedures,''
pRL278 contains the sacB gene which confers sucrose
sensitivity as well as sequences which allow transfer of this plasmid
into Anabaena sp. PCC 7120. Thus, interrupted genes ligated
into pRL278 can be transferred to Anabaena sp. PCC 7120 for
site-directed inactivation of the genomic copy and double recombinants
are easily isolated by positive selection of sucrose-resistant clones.
EF were
introduced into Anabaena sp. PCC 7120 cells by the triple
mating procedure of Elhai and Wolk
(28) and double recombinants
were selected as described under ``Experimental Procedures.''
These recombinants were subjected to Southern analysis (Fig. 2)
in order to confirm the replacement of the genomic gene with the
interrupted one. Two probes from the coding regions of pecE and pecF were used (Fig. 1C). Digestion
with EcoRI yielded an 8 kb band from wild-type. Two bands from
PECE-3, PECF-6, and PEC
EF-4 were detected due to internal
EcoRI sites in the C.S4 cassette (Fig. 2A). A
probe which contained the noncoding region between pecE and
pecF was also used. Multiple bands were detected with this
probe, indicating that the intervening sequence is present in multiple
places in the genome (data not shown). The orientation of C.S4 was
determined by digestion with HindIII (Fig. 2B),
and mutant clones were selected in which the direction of transciption
of the cassette was parallel with that of the gene. The mutants
analyzed in this study are designated PECE-3, and PECE-5 for pecE mutants, PECF-6 and PECF-7 for pecF mutants, and
PEC
EF-4 for the mutant in both pecE and pecF.
Figure 2:
Southern blot of total DNA from wild-type
and mutant strains to confirm isolation of double recombinants.
Numbers to the left indicate the size of the DNA fragments in
kilobase pairs. A, total DNA digested with EcoRI and
probed with fragments from the coding regions of pecE and
pecF (Fig. 1C). These probes hybridized to an 8-kb
fragment in the wild-type, 4.5- and 3.3-kb fragments in strain PECE-3,
two 4.5 kb bands in strain PECF-6, and 3.9- and 3.3-kb fragments for
strain PECEF-4. B, total DNA digested with
HindIII to confirm the direction of transcription of the C.S4
cassette. Probes used are the same as in A. These probes
hybridized to 1.3- and 1.1-kb fragments in the wild-type, two 1.5-kb
fragments in strain PECE-3, 2.2- and 1.1-kb fragments in strain PECF-6,
and 3.7- and 1.5-kb fragments in strain
PEC
EF-4.
Whole Cell Absorption Spectrum of Mutant
Strains
The presence of PEC is revealed by a shoulder at 570 nm
(due to the phycobiliviolin attached to the PEC subunit) in the
wild-type absorption spectra of whole cells and
phycobilisomes
(36) . The contribution of the phycobiliproteins
to the absorption spectrum of whole cells can be determined from a
difference spectrum of untreated minus heated cells (29). Such
difference spectra for the wild-type and mutant strains showed that the
570 nm shoulder was absent in all the mutant strains.
Characterization of the Phycobiliprotein Content of the
Phycobilisomes
In phycobilisomes, PEC is present as a hexameric
disc associated with a 34.5-kDa linker polypeptide encoded by the
pecC gene
(37, 38) . Whole cell difference
spectra indicated a loss of PEC in strains PECE-3, PECF-6, and
PECEF-4. To examine this further, we analyzed the phycobiliprotein
composition and content in phycobilisomes from the mutant strains. The
phycobilisomes were isolated on sucrose step gradients
(32) and
three major fractions were found at the 0.25, 0.5, and 0.75 M
layers. Phycobilisomes isolated from wild-type and from strains PECE-3,
PECE-5, PECF-6, PECF-7, and PEC
EF-4 sedimented primarily into the
0.75 M sucrose layer, with wild-type phycobilisomes
sedimenting slightly further than those of the mutants. The shoulder at
570 nm that is present in wild-type phycobilisomes is absent in those
from strains PECE-3, PECF-6, and PEC
EF-4. This indicates a loss of
phycobiliviolin from the mutant phycobilisomes. When the absorption
spectra are normalized at 650 nm the mutant strains exhibit a
22-25% decrease in the absorption at 615 nm indicative of a loss
of C-phycocyanin as well as PEC, assuming that the content of
allophycocyanin is unaffected. This observation correlates with the 20%
decrease noted above in the PCB/chlorophyll a ratio in the
mutants as compared to wild-type. The absorption spectra for the other
two major fractions from the sucrose gradient were also examined. None
of the slower sedimenting fractions from PECE-3, PECE-5, PECF-6,
PECF-7, or PEC
EF-4 showed absorption spectra with a peak at 570
nm, which would indicate the presence of PEC or the PEC
subunit.
EF-4
phycobilisomes is presented in Fig. 3. In this system the
separation of the
and
subunits of PEC is incomplete, and
PEC is seen as a fuzzy band located between the
and
subunits of C-phycocyanin. Coomassie staining shows a distinct
reduction in the PEC polypeptides and the PEC linker polypeptide in
phycobilisomes from strains PECE-3, PECF-6, and PEC
EF-4.
Figure 3:
SDS-PAGE of the polypeptides of wild-type
and mutant phycobilisomes. The abbreviations used are: WT,
wild-type; PECE-3, pecE mutant; PECF-6,
pecF mutant; PECEF-4, pecE and pecF mutant. Each lane was loaded with 25 µg of phycocyanin.
Position of the PEC linker polypeptide (L
),
C-phycocyanin subunits (
and
),
and PEC are indicated at the left. Under these conditions the subunits
of PEC are not well resolved.
The
subunits of PEC are best resolved on two-dimensional gel separations,
with isoelectric focusing in urea in the first dimension and SDS-PAGE
in the second. Fig. 4A shows the separation of the
phycobiliprotein polypeptides from wild-type phycobilisomes. In the
PECEF-4 phycobilisomes, the quantities of the PEC
and
subunits were reduced relative to wild-type phycobilisomes
(Fig. 4, A and B). The PECE-3 and PECF-6
phycobilisomes showed a decrease in the PEC
subunit and absence
of the PEC
subunit (Fig. 4, C and D).
Thus, the PEC
subunit is still present in strain PEC
EF-4,
but is not detectable by eye after Coomassie staining in strains PECE-3
and PECF-6. Coomassie-stained gels were scanned, and the densities of
the PEC
and
subunits and the allophycocyanin
subunit
were obtained. The allophycocyanin
subunit was used as the
internal control and the densities of the PEC
and
subunits
were expressed as a fraction of this subunit. In strains PECE-3 and
PECF-6, 33 and 35% of the PEC
subunit was present and 9 and 0% of
the PEC
subunit as compared to wild-type (A). In
strain PEC
EF-4, the amounts of the PEC
and
subunits
were 45 and 40%, respectively, relative to wild-type (A).
Figure 4:
Two-dimensional separation of the
polypeptides of wild-type and mutant phycobilisomes. For each
phycobilisome sample, a load of 25 µg of phycocyanin was applied to
the gel. Selected subunits are labeled in each panel. The abbreviations
used are: ap, allophycocyanin
subunit;
cpc, C-phycocyanin
subunit;
cpc,
C-phycocyanin
subunit;
pec, PEC
subunit;
pec, PEC
subunit. Note the presence of a double
spot for the PEC
subunit in panel
A.
To quantitate the and
subunits on the basis of their
bilin content, a duplicate set of gels was soaked in zinc acetate.
Under these conditions polypeptides containing bilins fluoresce upon UV
excitation. These gels were scanned to quantitate the fluorescence
intensity of the polypeptide spots. For strains PECE-3 and PECF-6,
respectively, 30 and 27% of the PEC
subunit and 7 and 5% of the
PEC
subunit were present as compared to wild-type. In strain
PEC
EF-4, 32 and 30% of the PEC
and
subunits were
present relative to wild-type (see B).
subunit elutes just before the C-phycocyanin
subunit, but the
subunits of PEC and C-phycocyanin coelute
(33) . The
wild-type and mutant strain phycobilisomes were analyzed with this
chromatographic procedure. Chromatograms of these separations,
monitored at 560 and 640 nm, are shown in Fig. 5, A and
B. The wild-type phycobilisomes contain a major peak at 23.5
min which has a shoulder at 22.8 min (Fig. 5A).
Spectroscopic analysis shows that C-phycocyanin
subunit
represents the major peak, and the PEC
subunit gives rise to the
shoulder. (Note that the spectrum at the center of the shoulder
(Fig. 6A, 22.8 min) is characteristic of the PEC
subunit.) The absorption spectrum at 22.8 min for the chromatogram of
PECF-6 phycobilisomes showed that the phycobiliviolin-bearing subunit
was absent (Fig. 6A). The chromatogram for PECE-3
phycobilisomes was virtually identical to that of PECF-6. Surprisingly,
a new small peak was seen in the chromatogram monitored at 640 nm of
PEC
EF-4 phycobilisomes, which also eluted at 22.8 min
(Fig. 5, B and C) but had an absorption
spectrum identical to that of C-phycocyanin
subunit
(Fig. 6B). To characterize this minor 640-nm peak from
strain PEC
EF-4, samples from PECF-6, PEC
EF-4, and wild-type
chromatograms (shown in Fig. 5A) which eluted between
22.5-23 min were collected. All three samples were subjected to
amino-terminal sequencing. The sequences of the PEC
EF-4 and
wild-type samples matched the sequence of the PEC
subunit
(I). The sample from PECF-6 gave no detectable sequence.
Thus the 22.8 min peak from PEC
EF-4 has the absorption spectrum of
C-phycocyanin
subunit, but elutes at the position of the PEC
subunit.
Figure 5:
Separation of phycobiliprotein subunits
from wild-type and mutant phycobilisomes. A, chromatogram of
phycobilisomes from wild-type (-), PECF-6 (- - - -), and
PECEF-4 (
), monitored at 560 nm. The
phycobilisomes were separated by HPLC on a C-4 reverse-phase resin, and
1.5 mg of phycocyanin was loaded for each type of phycobilisome.
B, HPLC chromatogram monitored at 640 nm. Samples and
conditions are the same as in A. C, HPLC chromatogram
of wild-type and PEC
EF-4 phycobilisomes monitored at 640 nm.
Sample load was 0.5 mg of phycocyanin each. At this lower load the
minor peak containing the PCB-bearing PEC
subunit at 23.7 min is
resolved in the PEC
EF-4 phycobilisome sample. Note that at this
lower load the polypeptides elute about 1 min later relative to the
elution times seen in panel A.
Figure 6:
Absorption spectra of early eluting
components in the HPLC chromatograms of wild-type and mutant
phycobilisomes. Separation was as described for Fig. 5 and under
``Experimental Procedures.'' Spectra were obtained with a
diode array detector during subunit separation. A, the
wild-type spectrum (-) at this time point (22.8 min; Fig.
5A) corresponds to that of the PEC subunit. The
PEC
EF-4 spectrum (
), corresponds to that
of the C-phycocyanin
subunit. No spectrum was detected from the
PECF-6 (- - - -) sample. The sharp dip and spike at about 660 nm in the
spectra obtained with the diode array detector are a feature of normal
instrument operation. B, normalized absorption spectra
acquired for the C-phycocyanin
subunit (25 min; (-)) and
for the component eluting at 23.7 min (- - - -) in the HPLC
chromatogram of PEC
EF-4 (Fig. 5, panel
C).
A portion of these samples was also analyzed by
horizontal isoelectric focusing. A fluorescent band, which co-migrated
with purified PEC subunit, was detected in the wild-type sample.
Two bands were detected in the PEC
EF-4 sample, one which
co-migrated with C-phycocyanin
subunit and the other in between
the C-phycocyanin
and PEC
subunits (Fig. 7). Both
bands were also detected by Coomassie staining of the gel after it was
scanned for the determination of fluorescence intensity.
Figure 7:
Fluorescence scan of horizontal
isoelectric focusing pattern of samples collected from HPLC
chromatogram of wild-type and PECEF-4 phycobilisomes. PC,
phycocyanin (2 µg); PEC, phycoerythrocyanin (1 µg);
strain PEC
EF-4, component eluting from 22.5 to 23 min (Fig.
5B); and WT, wild-type, component eluting from 22.5
to 23 min (Fig. 5B).
subunit. The proof that
these genes encode a heterodimeric phycocyanin
subunit PCB lyase
was provided by subsequent extensive biochemical studies and
characterization of the CpcEF lyase
(17, 18) . The
function of the Anabaena sp.PCC 7120 pecE and pecF genes was investigated here by interposon
mutagenesis.
subunit was barely
detectable, and the PEC
subunit was reduced to approximately 30%
of wild-type level. Simultaneous deletion and interruption of the
pecE and pecF genes (strain PEC
EF-4) also
resulted in the loss of PEC and a reduction of the PEC linker
polypeptide in phycobilisomes from this mutant. As in the pecE and pecF mutants, the PEC
subunit was present at
33-40% of wild-type levels. In contrast, however, the PEC
subunit was also present at 32-45% of wild-type level, but as an
``unnatural'' bilin adduct; the PEC
subunit isolated
from strain PEC
EF-4 contained PCB rather than phycobiliviolin and
had a spectrum identical to that of C-phycocyanin
subunit
(Fig. 6B). In all three types of mutants, the spectra of
isolated phycocyanin and allophycocyanin appeared normal (data not
shown). Thus, the mutations in pecE and pecF had no
apparent pleiotropic effects on the structure of other
phycobiliproteins.
20% of wild-type), and this
phycocyanin is modified. Whereas the
subunit, with PCBs at
-82 and
-155, is normal, no wild-type phycocyanin holo-
subunit is detectable. Phycocyanin
subunit polypeptide in an
amount similar to that of the
is present in the mutants, but most
of this
subunit is present as the apopolypeptide, the rest
(
20%) carrying unnatural bilin adducts
(16) characteristic
of the in vitro addition of PCB to this
apopolypeptide
(11, 12) . A significant amount of the
altered phycocyanin is associated with phycobilisomes. A parallel
situation, but with some distinctive features, is seen in the
Anabaena sp. PCC 7120 pecE and pecF mutants.
Just as no wild-type C-phycocyanin is formed in the cpcE or
cpcF mutants, no PEC is formed in the corresponding pec mutants. The presence in the phycobilisomes from the mutants of
the PEC
subunit at levels of
30% of wild-type, in the near
absence of
subunit polypeptide, suggests that in the absence of
its normal partner, the PEC
subunit can form hybrid monomers with
the C-phycocyanin
subunit, and that such monomers can be
incorporated into phycobilisomes. This interpretation is plausible. The
amino acid sequences of Anabaena sp.PCC 7120
C-phycocyanin and phycoerythrocyanin are
60%
identical
(19, 20, 21) . The three-dimensional
structures of PEC and C-phycocyanin are likewise very similar as are
the contacts at the
/
interface
(5) . Glazer and Fang
(39) showed that the
and
subunits of different
C-phycocyanins, with primary structure divergence of up to 25%, formed
stable hybrid monomers. Finally, substantial amounts of the
PEC-associated linker polypeptide are present in the phycobilisomes
from the pecE and pecF mutants.
subunit in the
PEC
EF mutant versus the PECE and PECF mutants. In the
PEC
EF mutant, the
subunit is present at a level similar to
that of the PEC
subunit (33-40% of wild-type); moreover, it
carries the PCB chromophore. In the PECE and PECF mutants, the PEC
subunit is either absent or present at barely detectable levels.
What might account for this difference?
and
subunits of
C-phycocyanins and PECs is 58 and 63%, respectively
(14) . Hence,
it is plausible that in the absence of PecE and PecF, the endogenous
CpcEF lyase could attach PCB to the PEC apo-
subunit. This would
explain the high level of the PCB adduct of the PEC
subunit in
strain PEC
EF-4. It is unlikely that this PCB adduct is formed
nonenzymatically. In vitro experiments have shown that the
principal product of nonenzymatic addition of PCB to the C-phycocyanin
(12) or to the C-phycoerythrin
(13) apo-
subunits is
3`-cysteinylmesobiliverdin, which has an absorption peak in 10
mM trifluoroacetic acid at 686 nm
(12) . In 10
mM trifluoroacetic acid, the absorption spectrum of the PCB
adduct of the PEC
subunit peaks at 646 nm and is identical to
that of the C-phycocyanin
subunit. Consistent with the
interpretation offered here, it is noteworthy that CpcEF from
Synechococcus sp. PCC 7002 is able to use heterologous
C-phycocyanin
subunits as bilin donors in catalyzing the transfer
of bilin from holo-
subunit to apo-
subunits
(17) .
subunit PCB lyase can add either PCB or
phycoerythrobilin to the C-phycocyanin
subunit. However, CpcEF
has a 30-fold higher binding affinity for PCB and a turnover rate of 19
times that for PCB over phycoerythrobilin
(18) . Thus the ratio
of k
/K
PCB to
k
/K
phycoerythrobilin is 570.
This preference for PCB in binding affinity and in the rate of
catalysis is sufficient to account for the selective attachment of PCB
to the phycocyanin apo-
subunit in the presence of
phycoerythrobilin (18). The finding of a significant amount of PEC
subunit carrying PCB in the PEC
EF mutant suggests that this
is due to a similar selectivity (rather than absolute
specificity) with regard to the polypeptide substrate in the lyase
reaction. A testable hypothesis is that when the cognate lyase is
missing, the phycocyanin
subunit lyase ``rescues'' the
PEC apo-
subunit by adding PCB.
subunit in the PECE and PECF mutants
explained in the context of this hypothesis? When only PecE or PecF is
present, either of these components (or a hybrid PecE/CpcF or PecF/CpcE
lyase) may form a complex with the PEC apo-
subunit which is
thereby made unavailable to the CpcEF lyase. The formation of such a
non-productive complex may also permit proteolytic degradation of the
PEC apo-
subunit. These possibilities will be tested in
appropriately engineered Anabaena sp. PCC 7120 strains with
deletions in cpcE and cpcF genes and ultimately by
biochemical studies of the properties of recombinant PecE and PecF.
Table:
Characteristics of Anabaena sp. PCC 7120
wild-type and mutant strains
Table:
Quantitation of
the amounts of PEC and
subunits in phycobilisomes from
wild-type and mutant strains of Anabaena sp. PCC 7120
Table: 1819897957p4in
ND, not determined (no residue
could be identified at this step).(119)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.