(Received for publication, August 18, 1995; and in revised form, November 7, 1995)
From the
The light harvesting antenna 1 (LH1) complex of Rhodobacter
sphaeroides is intimately associated with the reaction center (RC)
as part of the reaction center RC-LH1 core complex. The pufA gene has been modified such that between 5 and 16 amino acid
residues were progressively deleted from the C terminus of the LH1
polypeptide. The two largest deletions produced strains which
were deficient in LH1. The remaining four deletion mutants exhibited
significant reductions in the average level of LH1 per reaction center.
Analysis of detergent-solubilized cores on sucrose gradients showed
that the mutant strains had a sizeable population of antenna-deficient
reaction centers in addition to core complexes with a reduced ratio of
LH1:RC. The decrease in the ratio of LH1:RC in core complexes of the
mutant strains was accompanied by a progressive blue shift of the
absorbance maximum of LH1, which we attribute to the reduced
aggregation state of LH1 in the smaller cores. The PufX polypeptide was
not required for photosynthetic growth in mutants with reduced core
sizes. We conclude that the level of LH1 in the bacterial membrane, and
the aggregation state of LH1 in core complexes, are both influenced by
the C terminus of the
polypeptide, and we discuss possible models
for the organization of the core complex in Rb. sphaeroides.
The photosynthetic bacterium Rhodobacter sphaeroides has a relatively simple membrane-bound photosystem comprising a
single type of reaction center (RC) ()and two types of light
harvesting complex. The peripheral light harvesting complex, LH2, is
present in variable amounts according to the incident light intensity
whereas the core light harvesting complex, LH1, is present in a fixed
stoichiometry to the RC(1, 2) . In the simple
``lake'' model (3) light energy captured by LH2
migrates via LH1 to a dimer of bacteriochlorophylls (Bchl) within the
RC, initiating the transfer of an electron through the RC from the
periplasmic side to the cytoplasmic side of the membrane. The products
of this transmembrane electron transfer, reduced ubiquinone and an
oxidized Bchl dimer, trigger a cycle of electron transfer reactions
involving the intramembrane pool of ubiquinone, the cytochrome bc
complex and a soluble cytochrome c that results in the translocation of protons from the cytoplasmic
side of the membrane to the periplasmic side, generating a proton
electrochemical gradient.
All bacterial light harvesting complexes
studied to date contain two small hydrophobic polypeptides designated
and
in a 1:1 ratio, both of which have a single
membrane-spanning helix(4) . Liganded to these polypeptides are
the light harvesting pigments which, in the case of Rb.
sphaeroides, are molecules of Bchl a. Carotenoids also
act as light harvesting pigments but have an additional photoprotective
role. In the LH2 complex of Rb. sphaeroides each pair of
and
subunits is associated with three molecules of Bchl, two of
which absorb at 850 nm and the third at 800 nm. Within the LH1 complex
there are only two Bchl molecules per pair of
and
subunits,
and these absorb maximally at 877 nm. Historically, the structure and
function of the bacterial antenna has been studied mainly through the
application of biochemical and spectroscopic techniques(5) .
However, the first prospects for carrying out an examination of the
relation between structure and mechanism based on detailed structural
information have recently appeared with the publication of a high
resolution structure for the LH2 complex from Rhodopseudomonas
acidophila strain 10050(6) . The complex consists of a
cylinder of 9
heterodimers, with the Bchl a molecules arranged in 18-member (850 nm Bchls) and 9-member (800
nm Bchls) rings. It is thought that this arrangement allows the excited
state of any Bchl a molecule to become rapidly delocalized
over the entire ring, allowing efficient transfer of energy to an
adjacent complex. Parallels with the LH2 structure have also been drawn
from low resolution structural information on the LH1 complex from Rhodospirillum rubrum(7) , which has been proposed to
have 16
units that are also arranged in a cylinder, with the
organization of the 880-nm Bchls being analogous to that of the 850-nm
Bchls of LH2. The exact spatial relationship of the LH2, LH1, and RC
complexes of the bacterial photosystem is still uncertain, although it
has been noted that there is sufficient space within the ring of the Rs. rubrum LH1 complex for a single RC complex (7) .
However, whether or not the RC resides within a ring of LH1
units remains to be proven.
A number of studies using a range of
bacterial species have shown that the N termini of both the and
polypeptides of the LH1 complex play essential roles in the
stable expression of the complex in the
membrane(8, 9, 10, 11) . In
contrast, the C terminus of the LH1 complex has not been studied in
detail. Gogel et al.(12) used dansyl chloride to
modify the Rs. rubrum LH1
K
residue
with no effect upon the absorbance of LH1; electron transport activity
in treated membranes was significantly reduced but due to the
nonspecific nature of dansyl chloride this could not be unambiguously
ascribed to the modified LH1
polypeptide. A more recent in
vitro study (11) using Rs. rubrum LH1
polypeptides demonstrated that an apparently wild type LH1 complex
could be formed from an intact
polypeptide plus an
polypeptide which lacked 10 amino acids from the C terminus, ending at
residue E
. This study raised questions concerning
the exact role of the C terminus of the LH1
polypeptide, since
its removal appeared to have little impact upon the stable assembly of
the LH1 complex in detergent solution. Comparison of a number of LH
polypeptides from a variety of species of photosynthetic bacteria had
previously shown that there was reduced homology at the C termini as
compared with the N termini or the membrane spanning
helices(13) , which supported the lack of any specific role in
formation or stability of the LH1 complex in vitro. However,
an examination of the structure of the Rps. acidophila LH2
complex reveals that the interaction between polypeptides in LH2 is in
part mediated by hydrogen bonds and hydrophobic interactions between
residues in the C terminus of the LH2
and
polypeptides and
between adjacent
polypeptide C termini (6) . Also, the
W
residue of the Rb. sphaeroides LH1
complex has been shown to form a hydrogen bond to the 2-acetyl carbonyl
group of one of the LH1 Bchls(14) .
In the present study, we
have examined the role of the C terminus of the polypeptide in
the assembly of the RC-LH1 core complex through the introduction of six
mutations into the 3` end of the pufA gene of Rb.
sphaeroides that alter specific codons to a stop codon, resulting
in the formation of
polypeptides that are truncated by between 5
and 16 amino acids at the C terminus. To simplify the spectroscopic
analysis of the altered complexes, the experiments were performed in an
LH2-deficient background by expressing a plasmid-borne copy of the puf operon harboring the mutated pufA gene in the
double-deletion strain DD13. This strain harbors genomic
deletion/insertion mutations in the pufQBALMX (RC+LH1)
and pucBAC (LH2) operons, and is devoid of pigment-protein
complexes. Our results demonstrate that truncation of the LH1
polypeptide has a major impact on the levels and aggregation states of
LH1 associated with RCs. They shed new light on the organization of the
core complex and on the influence of antenna oligomer size on the
spectroscopic properties of LH1 Bchls. We have also examined the effect
of the absence of PufX on the capacity for photosynthetic growth and on
the organization of the core unit in these truncation mutants.
The remaining five pufA mutants were constructed using the
polymerase chain reaction with the template in all cases being the
plasmid pSELBHX (see above). For all of the mutagenic changes, one of
the pair of PCR oligonucleotides was designed to be homologous to the
5` end of the pufA gene and encompassed the engineered HindIII site located between the pufB and pufA genes (see above). The second oligonucleotide for each PCR was
designed to have a common sequence at the 5` end that encompassed the
engineered NruI site immediately downstream of the pufA stop codon followed by tandem stop codons at a position
immediately 3` to this NruI site in the oligonucleotide
sequence. Appended to the 3` end of this common sequence were sequences
of 17 bases that were homologous to the appropriate region of the pufA gene. The 5` end of this homologous sequence started with
the codon of the amino acid that would form the C-terminal residue of
each mutated polypeptide. The HindIII-NruI
fragment encompassing the wild-type pufA gene in plasmid
pRKEK1H (18) was then replaced by the HindIII-NruI fragments produced by the mutagenic PCR,
and the resulting mutant constructs were shuttled as EcoRI-XbaI fragments into the expression vectors
pRKEH10 (20) and pRKEH10X
(15) to
form a series of expression plasmids for the mutated puf operon with and without the pufX gene, respectively (Table 1). The nomenclature for the mutant strains (see Fig. 1) follows the numbering system described in Loach et
al. (21) and Olsen and Hunter (13) , designating
the histidine that forms the axial ligand to the LH1 Bchl as residue
H
, with residues toward the C terminus having positive
numbers.
Figure 1:
Arrangement of the wild type puf operon and the nature of the truncation mutations. A, represents the intact pufQBALMX operon present on plasmid
pRKEH10 (20) and B, indicates the pufX-deficient version of this found in plasmid
pRKEH10X(15) . C shows the amino
acid sequence of the LH1
polypeptide as encoded by the pufA gene, and the sequences of the polypeptides that arise from the
six truncation mutations. The putative transmembrane helix is indicated
by the horizontal bar, and the histidine residue that is
thought to ligand the LH1 Bchls is shown by the arrow.
For the resequencing of pufA DNA from Rb. sphaeroides strains harboring mutant plasmids, 1 ml of a culture of the required strain was pelleted by centrifugation at 13,000 rpm for 1 min at room temperature in a microcentrifuge and the cell pellet was resuspended in 100 µl of water. This cell suspension was boiled for 2 min and cell debris was removed by centrifugation at 13,000 rpm for 5 min at room temperature. The region of plasmid DNA encompassing pufBA was then amplified from the supernatant by PCR using two oligonucleotides that were homologous to sequences immediately upstream of pufB and downstream of pufA. The latter of these two oligonucleotides was then used for sequencing of the PCR product.
Low temperature
(77 K) fluorescence emission spectra of membrane samples were obtained
using a FluoroMax spectrofluorometer (SPEX Industries Inc., NJ). The
sample absorbance at room temperature was adjusted to 0.1
cm at 885 nm in 60% glycerol (v/v), 10 mM Tris, pH 8.0. To promote accumulation of RCs in the photooxidized
state, potassium ferricyanide was added to a final concentration of 100
µM, and the sample was illuminated with a tungsten light
source through a RG 630 filter for 10 s immediately prior to cooling.
Fluorescence was detected at 90 °C to the incident light at a
resolution of 5 nm using 590 nm excitation.
The percentage of free RCs in detergent-solubilized membranes was estimated by dividing the total estimated amount of LH1 Bchl in a sucrose gradient by the mean core size for that gradient to give the number of RCs associated with LH1. This figure was then used together with the total estimated amount of RCs in the gradient to give the percentage of RCs not associated with LH1.
Fig. 2shows the room temperature absorbance spectra of
intracytoplasmic membranes from the mutant and control strains
normalized on the basis of RC concentration. Membranes from control
strain RCLH12 exhibited the characteristic wild-type LH1 absorbance at
877 nm and RC bands at 804 and 760 nm. In contrast, the spectrum of
strain RCO2 had only the latter two peaks, characteristic of
(photo)oxidized RCs, showing the complete absence of LH1. The 860-nm
band of the RC Bchl special pair was bleached since the Guided Wave
fiber optic spectrophotometer, used to acquire the spectra, irradiates
the sample with white light. For each of the mutant strains the
absorbance spectra showed reductions in the relative size of the 877-nm
LH1 band (Fig. 2), indicating that the truncations in the LH1
polypeptide had led to reductions in the number of LH1 Bchls per
RC, but that they had not significantly altered the pigment
environment. The extent of the apparent reduction in core size was
roughly correlated with the number of residues deleted from the C
terminus of the
polypeptide, while the two largest truncations
gave rise to spectra that were identical to that of the RC-only strain
RCO2.
Figure 2: Room temperature absorbance spectra of intracytoplasmic membranes of control and mutant Rb. sphaeroides strains. All spectra were normalized to a reaction center concentration of 0.5 µM, estimated as described under ``Experimental Procedures.''
The average number of LH1 Bchls per RC in each strain, denoted
here as MLH1/RC, was calculated as described under ``Experimental
Procedures.'' As shown in Table 2, the removal of the first
five amino acids from the C terminus of the subunit more than
halved the value of MLH1/RC, while the removal of between four and nine
additional residues led to a further decrease to about 20-30% of
wild-type levels. Deletion of the next residue (L
)
caused a complete loss of the LH1 complex. The estimates of MLH1/RC in Table 2took into account any changes in the extinction
coefficient for LH1, which did not vary for the mutant with the
smallest truncation (RCLH+21), but was reduced by between 10 and
20% in the remaining mutants.
The levels of the LH1 and
polypeptides in membranes from each strain were visualized by
SDS-polyacrylamide gel electrophoresis. Upon comparison with the LHO1,
RCLH12, and RCLH12X
controls the apparent molecular
weight of the LH1
polypeptide decreased in the order
RCLH+21, RCLH+17, RCLH+13, and RCLH+12 (Fig. 3) which correlated with the predicted decrease in size of
the
polypeptide in these strains. The levels of both the
and
polypeptides were also reduced as compared with RCLH12 and
correlated with the MLH1/RC ratios calculated above. For strains
RCLH+11 and RCLH+10 no
or
polypeptide was
detected (Fig. 3).
Figure 3:
SDS-polyacrylamide gel of intracytoplasmic
membranes from control and mutant Rb. sphaeroides strains.
Molecular weight markers are indicated on the left (M values shown
10
) and the positions of the wild-type LH1
and
polypeptides are marked on the right. 10 pmol of RCs
were loaded in each lane (except LHO1) and thus the amount of LH1
polypeptides is a reflection of the LH1:RC ratio in each membrane
sample.
In the strains harboring truncations of
the LH1 polypeptide, comparison of the PufX-deficient strains
with their PufX-containing counterparts revealed that, as for the
control strain, the removal of the pufX gene had led to a
small increase in MLH1/RC, with the exception of strain
RCLH+12X
(Table 2). This increase was
consistent with an average increase in the size of the mutant core
complexes of between one and two LH1 Bchls per RC. In those truncation
mutants which did not express an LH1 complex (strains RCLH+11 and
RCLH+10), removal of the PufX polypeptide did not restore an
assembled LH1 complex. PufX did not affect the extinction coefficient
for LH1 in the truncation mutants.
Visualization of the LH1
and
polypeptides by SDS-polyacrylamide gel electrophoresis of
intracytoplasmic membranes revealed no large differences in apparent
molecular weight or amount of either polypeptide when comparing
equivalent strains with and without the pufX gene (Fig. 3).
Figure 4: Profiles of detergent-solubilized pigment-protein complexes separated by centrifugation on sucrose gradients. Strains analyzed were (a) RCLH12, (b) RCLH+21, (c) RCLH+13, and (d) RCLH+10 (open triangles) and LHO1 (closed triangles). In panels a-c the arrow indicates the fractions used to obtain 77 K absorbance spectra.
Absorbance spectra of fractions from the sucrose
gradients of the detergent-solubilized pigment-protein complexes from
control strain RCLH12 indicated that the vast majority of RCs in this
strain co-migrated with LH1 complexes, with very few free RCs being
observed near the top of the gradient (Fig. 4a). In
contrast, sucrose gradients containing solubilized complexes from
strains RCLH+21 (Fig. 4b) and RCLH+13 (Fig. 4c) revealed a significant fraction of RCs that
were not associated with LH1, suggesting that there was a significant
population of free RCs in membranes from the truncation mutants. This
antenna-free population was estimated to be 50% of the total RCs
in strain RCLH+21,
25% in strain RCLH+13, and
2% in
the control strain RCLH12. The average size of the core complexes
(SLH1/RC) in the LH1-containing fractions from the sucrose gradient of
the control strain was estimated to be
29, close to the value for
MLH1/RC of
26 estimated on the basis of membrane spectra. In
strain RCLH+21, a value of
20 was estimated for SLH1/RC,
twice the value of MLH1/RC for this strain and consistent with the
level of free RCs seen in the gradient fractions. For strain
RCLH+13, SLH1/RC was
10, again consistent with the value of
7 estimated for MLH1/RC taking into account a 25% population of
free RCs.
To investigate whether the larger core complexes seen in
the sucrose gradients of strains RCLH+21 and RCLH+13 arose
from a detergent-induced redistribution of LH1 among the RC population,
77 K absorption spectra were recorded for the fractions indicated by
the arrows in Fig. 4and the of LH1
determined (Table 2). For the control strain RCLH12, SLH1/RC was
very similar to MLH1/RC and there were very few free RCs, suggesting
that no significant reorganization of the core complexes had taken
place. Therefore we attributed the observed 2-nm blue shift in
for sample RCLH12 to the detergent extraction of
the complexes. The
was also constant across the
RCLH12 gradient which suggested LH1 had a similar aggregation state
throughout the gradient. The solubilized core complexes from strains
RCLH+21 and RCLH+13 showed the detergent-induced 2-nm blue
shift, but despite the significant difference between SLH1/RC and
MLH1/RC in each of these strains, there were no further differences in
the
when 77 K spectra of gradient fractions and
membranes were compared (Table 2). This suggested that there had
been no large scale redistribution of LH1 among the RC population.
Detergent extraction of membranes from strains
RCLH12X, RCLH+21X
, and
RCLH+13X
was also performed to establish whether
a lack of PufX significantly altered the distribution of LH1 between
the RCs in the membranes of the mutant strains. No significant
differences were observed between the distribution profiles seen for
the three PufX-deficient strains (data not shown) and the profiles
shown in Fig. 4(a-c) for their PufX-containing
counterparts.
Figure 5:
Photosynthetic growth of the control and
mutant Rb. sphaeroides strains. Growth was monitored by the
absorbance of cultures at 680 nm. The strains containing pufX are shown by open circles and those lacking pufX by filled circles. Strains are (a)
RCLH12(X), (b)
RCLH+21(X
), (c)
RCLH+17(X
), (d)
RCLH+13(X
), (e)
RCLH+12(X
), (f)
RCLH+11(X
), (g)
RCLH+10(X
), and (h)
RCO2(X
).
With the sole exception of strain RCLH+11, all of the PufX-containing truncation mutants were capable of photosynthetic growth, but scrutiny of the growth curves for the mutant strains revealed a complex pattern. The mutant strains which expressed an LH1 complex, namely RCLH+21, RCLH+17, RCLH+13, and RCLH+12, all displayed a reproducible biphasic growth (Fig. 5, b-e). The initial phase occurred with no lag and proceeded for approximately 50 h whereupon a second phase became dominant and growth continued to an absorbance at 680 nm of between 2.0 and 2.5. The first phase of growth in the LH1-containing mutants was very similar to that seen during the first 50 h of growth of the RC-only strain RCO2 (Fig. 5h). The second phase of growth in the LH1-containing mutant strains, which became dominant at the end of the initial 50-h period, was similar to that seen after approximately 50 h in the control strain RCLH12, with in all cases the final absorbance of the culture rising to a value greater than 2.0. Thus the four LH1-containing truncation mutants appeared to display features of both ``RC-only growth'' and ``RC+LH1 growth.'' To determine whether this was due to segregation within the photosynthetic cultures into cells containing only the RC complex and cells containing both the RC and the mutant LH1 complex, aliquots of the photosynthetic cultures from early stationary phase were taken and appropriate dilutions were spread onto agar plates of M22+ medium containing kanamycin and tetracycline and then incubated under dark, aerobic conditions at 34 °C. Absorbance spectra taken of at least 20 individual colonies from each of the above four strains all displayed peaks for both RC and LH1 complexes (data not shown), showing that segregation had not occurred during photosynthetic growth, and making it unlikely that the biphasic growth patterns could be explained by two spectroscopically-distinct cell types within the cultures. The mutant strains which exhibited reproducible growth under photosynthetic conditions were also screened for the presence of supression mutations within the pufB and A genes. Cells grown under photosynthetic conditions to early stationary phase were harvested, the pufB and A genes were amplified by PCR and the DNA products sequenced as described under ``Experimental Procedures.'' In all cultures tested there were no secondary mutations within either the pufB or A gene.
Mutants RCLH+11 and RCLH+10 both had a RC-only absorbance spectrum (Fig. 2) but only strain RCLH+10 was capable of photosynthetic growth with kinetics similar to those displayed by the RC-only control strain RCO2. Mutant RCLH+11 also appeared to be capable of weak photosynthetic growth, but seemed to be impaired relative to strains RCO2 and RCLH+10. The reasons for this were not investigated further.
The pufX gene appeared not to
have any effect on the ability of the mutant strains to grow under
photosynthetic conditions, with the exception of strain
RCLH+21X (Fig. 5b) in which
there was a reproducible inhibitory effect due to the absence of the
PufX polypeptide. However, this inhibition was not as dramatic as that
seen for the control strain RCLH12X
which in the
absence of secondary suppression mutations was entirely
non-photosynthetic. In contrast, strain RCLH+21X
was still capable of biphasic photosynthetic growth, but the
second phase of growth became dominant after a longer period (75 h)
than was seen for the other LH1-containing truncation mutants (
50
h). Therefore there was some correlation between the number of LH1
Bchls per RC and the effect of PufX, since the only strains whose
growth was sensitive to the absence of PufX was
RCLH12X
, which had normal core complexes, and strain
RCLH+21X
, the truncation mutant with the largest
core complexes.
Although at present little is known about
the assembly pathway of the LH1 complex, topology studies have
demonstrated that the C-terminal regions of both the and the
polypeptides are located on the periplasmic side of the
membrane(25) . Thus it is conceivable that truncations of the C
terminus of the
polypeptide would affect the process by which it
is translocated across the bilayer, leading to a decrease in the level
of the
polypeptide in the membrane, accompanied by a decrease in
the stability of the
polypeptide. This was supported by
SDS-polyacrylamide gel electrophoresis of mutant intracytoplasmic
membranes in which the observed amounts of both the LH1
and
polypeptides correlated with the decreases in amounts of LH1 Bchl (Fig. 3). The RC-only phenotype exhibited by the RCLH+10
and RCLH+11 mutants may have arisen from a further destabilization
of the LH1 complex caused by disruption of the hydrogen bond that has
been shown to exist between the LH1
W
residue
and the 2-acetyl carbonyl group of one of the LH1 Bchls(14) .
While disruption of this hydrogen bond in an otherwise wild-type LH1
complex does not prevent assembly (14) the further
destabilization arising from breakage of this hydrogen bond may have
lead to a complete inhibition of LH1 assembly in mutants RCLH+10
and RCLH+11. Neither the
nor
polypeptide could be
detected in membranes from these strains (Fig. 3).
In a recent paper (15) we discussed the
possibility that the small increase in the aggregation state of LH1
observed in the absence of the PufX polypeptide
(RCLH12X) results in a physical blockage of the
Q
site of the RC, preventing ubiquinone/ubiquinol exchange
with the cytochrome bc
complex(28, 29) . From the results presented
here, RC-LH1 core complexes with a reduced LH1:RC stoichiometry remain
fully functional in the absence of PufX, thus supporting this
hypothesis. The threshold level of LH1 per RC at which the requirement
for PufX is lost is not known, but our results with strains
RCLH12X
and RCLH+21X
suggest
it lies somewhere between 29 and 20 Bchls per RC.