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J Bacteriol, February 1998, p. 642-646, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Demonstration of the Key Role Played by the PufX
Protein in the Functional and Structural Organization of Native and
Hybrid Bacterial Photosynthetic Core Complexes
Timothy K.
Fulcher,1
J. Thomas
Beatty,2 and
Michael R.
Jones1,*
Robert Hill Institute for Photosynthesis and
Krebs Institute for Biomolecular Research, University of Sheffield,
Sheffield, United Kingdom,1 and
Department of Microbiology and Immunology, University of
British Columbia, Vancouver, British Columbia,
Canada2
Received 18 July 1997/Accepted 21 November 1997
 |
ABSTRACT |
The role of a component of the bacterial photosystem, the PufX
protein, was examined by heterologous expression of the
pufX gene from Rhodobacter capsulatus in a
strain of R. sphaeroides that lacks the native
pufX gene. The strain of R. sphaeroides containing the R. capsulatus PufX protein was capable of
efficient transduction of light energy despite a low degree of sequence conservation between the PufX proteins from the two species. The organization of the hybrid reaction center/LH1 photosystem in strains
of R. sphaeroides containing the R. capsulatus
LH1 antenna complex was affected differently by the R. sphaeroides and R. capsulatus PufX proteins. We
discuss the implications of our findings for the role of the PufX
protein in organizing the bacterial photosystem for efficient
transduction of light energy.
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INTRODUCTION |
Photosynthetic organisms carry out
the conversion of light energy into electrochemical energy in
membrane-bound pigment-protein complexes termed reaction centers
(5, 6, 10, 27). The efficiency of the photosynthetic process
is greatly enhanced by the presence of light-harvesting pigment-protein
complexes, which funnel excitation energy to the reaction center. In
Rhodobacter sphaeroides, the reaction center is intimately
associated with the LH1 antenna in a characteristic stoichiometry,
forming the RC/LH1 core complex. Core complexes are surrounded and
interconnected by a variable amount of a peripheral LH2 antenna
(25). In Rhodopseudomonas acidophila, the LH2
complex has a ninefold symmetry, comprising a transmembrane cylinder of
nine 
polypeptides surrounded by a second cylinder of nine 
polypeptides (22). These concentric cylinders encase rings
of 18 B850 and 9 B800 bacteriochlorophylls (Bchls), as well as
carotenoid molecules. It has been proposed that LH1 antenna complexes
have a similar cylindrical structure and that the reaction center
occupies the space in the center of the LH1 cylinder (4, 15,
28).
In addition to the reaction center and LH1 complex, the core complex of
R. sphaeroides appears to contain one or more copies of a
polypeptide of 82 amino acids that is the product of the pufX gene (7). This gene is part of a
polycistronic message which includes both LH1 genes (pufAB),
as well as two of the reaction center genes (pufLM)
(18). The pufX gene has been identified unambiguously in only one other species of purple bacterium, namely, Rhodobacter capsulatus (29). These puf
operon genes are transcribed in the order BALMX in both
R. sphaeroides and R. capsulatus, and it is
thought that the structure and organization of the photosynthetic apparatus encoded by these genes are very similar in the two bacteria (11, 30). In support of this, sequence comparisons have
revealed a high degree of identity between the protein sequences of the reaction center L, M, and H subunits and the LH1 and polypeptides from the two species (~75%, on average). The role of
the PufX protein appears to be to facilitate photosynthetic growth in
strains of bacteria that have normal, or near to normal, levels of the LH1 antenna complex (9, 16, 20, 23).
Given the similarities between the photosystems of R. sphaeroides and R. capsulatus, it has been assumed
that the role played by the PufX protein is essentially the same in the
two species. However, the sequence identity in alignments between the
PufX proteins from the two species is very low (29%; see Discussion), with no clear regions of local high conservation. This study examined the functional properties of the PufX proteins from R. capsulatus and R. sphaeroides by heterologous
expression of the R. capsulatus protein in a strain of
R. sphaeroides that lacks the native PufX protein. The
functional similarity of the photosystems of these two species was
examined further by heterologous expression of the structural genes of
the R. capsulatus LH1 complex in R. sphaeroides and coexpression of these genes with the pufX gene from
R. capsulatus.
| |
MATERIALS AND METHODS |
Construction of plasmids for heterologous expression of the
pufX gene.
Replacement of the R. sphaeroides
pufX gene was achieved by modification of plasmid pRKEH10
(23). To avoid confusion, the pufX genes from
R. sphaeroides and R. capsulatus will henceforth be referred to as pufXs and
pufXc, respectively, and the corresponding gene
products will be called PufXs and PufXc. The
LH1 genes and complexes from the two species are described by a similar
nomenclature, i.e., pufAsBs,
pufAcBc, LH1s, and
LH1c. Plasmid pRKEH10 is based on broad-host-range vector
pRK415 and contains a 6.5-kb EcoRI-HindIII fragment of R. sphaeroides DNA encompassing the entire
puf operon. For subcloning, plasmid pSKBH was constructed;
this consisted of 3.2 kb of R. sphaeroides DNA from the
BamHI site located between pufM and
pufXs to the downstream HindIII
site cloned into plasmid pBlueSK+ (Stratagene). To facilitate excision
of the pufXs gene coding sequence, an
EcoRI site was introduced at a position 2 bp downstream of
the stop codon of the pufX gene by mismatch oligonucleotide mutagenesis. The template for mutagenesis was a 326-bp
BamHI-SmaI restriction fragment encompassing the
pufX gene cloned into plasmid pALTER-1 (Promega). After
successful introduction of the EcoRI site, the mutated
BamHI-SmaI restriction fragment was inserted into
plasmid pSKBH, replacing the equivalent native
BamHI-SmaI fragment and forming plasmid pSKBEH.
The EcoRI site downstream of pufXs
was then transferred to plasmid pRKEH10 as a 3.2-kb
BamHI-HindIII fragment to give plasmid
pRKEHXs. Replacement of the pufXs gene with the
pufXc gene was achieved by using plasmid pSKBEH.
The pufXc gene was isolated by PCR from a
pUC13-based plasmid containing the entire R. capsulatus puf
operon (pUC13::puf). The oligonucleotide primers used were
designed such that only the pufX coding sequence would be
changed in the final construct. The PCR product was digested with
BamHI and EcoRI and inserted into plasmid pSKBEH
in place of the 265-bp BamHI-EcoRI fragment that
encompasses the pufXs gene. Successful
replacement of the R. sphaeroides gene was confirmed by DNA
sequencing. The hybrid R. sphaeroides pufBALM-R. capsulatus pufX operon was then constructed by transfer of the
pufXc gene into plasmid pRKEHXs as a 3.2-kb
BamHI-HindIII fragment to give plasmid
pRKEHXc. The success of this procedure was confirmed by DNA
sequencing. The pufX deletion construct used in this study was plasmid pRKEH10X
(23).
Construction of plasmids for heterologous expression of the
R. capsulatus pufBA genes.
Replacement of the R. sphaeroides pufBsAs genes was achieved by
a strategy similar to that described above for
pufXs. The starting plasmid was pUCEBHNX, which
consists of a 1.6-kb EcoRI-XbaI restriction
fragment encompassing pufBsAs in
pUC19. This restriction fragment had an engineered BamHI
site located 1 bp upstream of the start codon of
pufBs, an engineered HindIII
restriction site located immediately upstream of the start codon for
pufAs, and an engineered NruI
restriction site located immediately downstream of the stop codon for
pufAs. These engineered restriction sites do not
affect expression of the R. sphaeroides puf operon
(26). The R. capsulatus pufBA genes were isolated
by PCR from plasmid pUC13::puf. The oligonucleotide primers
were constructed such that only the coding sequences for
pufBA, plus the 13-bp intervening region, would be changed
in the final construct. The PCR product was digested with
BamHI and NruI and inserted into plasmid pUCEBHNX in place of the 353-bp BamHI-NruI fragment that
encompasses the pufBsAs genes.
Successful replacement of the R. sphaeroides genes was
confirmed by loss of the R. sphaeroides HindIII
restriction site and subsequent DNA sequencing. The hybrid
R. sphaeroides-R. capsulatus construct was then shuttled
into plasmids pRKEHXs, pRKEHXc, and pRKEH10X as an
EcoRI-XbaI restriction fragment, generating
plasmids pRKEHLH1cXs, pRKEHLH1cXc, and
pRKEHLH1c10X, respectively.
Construction of mutant strains.
The six plasmids described
above were transferred to R. sphaeroides DD13
(13) by conjugation from Escherichia coli S17-1 (12). Transconjugant strains were selected on the basis of
growth of single colonies on M22+ agar plates supplemented with
tetracycline and kanamycin under semiaerobic conditions in the dark.
Bacterial growth and preparation of intracytoplasmic membranes.
R. sphaeroides strains were grown under dark,
semiaerobic conditions at 34°C in M22+ medium as previously described
(14). Photoheterotrophic growth was conducted in 15-ml
screw-top test tubes filled with M22+ medium and supplemented with
0.1% Casamino Acids and kanamycin at 20 µg/ml. The inoculum
consisted of cells harvested from a 70-ml culture grown under dark,
semiaerobic conditions to an optical density at 680 nm
(OD680) of approximately 1.5; harvested cells were added to
the 15-ml screw-top tubes to give an initial OD680 of
between 0.1 and 0.2. The tubes were incubated at 34°C in a
glass-sided water bath illuminated by three 500-W tungsten floodlamps,
giving an incident light intensity of approximately 40 W/m2. Growth was monitored at 680 nm with a Sherwood
colorimeter. Intracytoplasmic membranes were prepared from cells grown
under dark, semiaerobic conditions to late log phase as described
previously (14).
Spectroscopy.
Absorbance spectra of cells and
intracytoplasmic membranes were recorded in a Beckman DU640
spectrophotometer. Fluorescence emission spectra were recorded with a
FluoroMax spectrofluorimeter (SPEX Industries Inc.). Excitation was in
the Qx band of LH1 at 590 nm with 18-nm resolution.
Emission was monitored between 850 and 950 nm with a resolution of 9 nm. Samples were normalized to a concentration of 0.2 absorbance unit
cm1 at the maximum of the LH1 Qy band.
| |
RESULTS |
Heterologous expression of the R. capsulatus pufX gene
in R. sphaeroides.
Plasmids pRKEHXs, pRKEH10X,
and pRKEHXc (see Materials and Methods) were expressed in R. sphaeroides DD13 (13), which is devoid of RC/LH1
core complexes due to a deletion-insertion mutation in the genomic copy
of the puf operon and is devoid of LH2 antenna complexes due
to a similar mutation in the genomic copy of the puc operon.
Transconjugant strains were selected as described previously
(12) and were given the names RCLH1sXs, RCLH1sXd, and
RCLH1sXc, where the lowercase letters indicate the origins of the LH1
antenna and PufX protein (s, R. sphaeroides; c, R. capsulatus; d, deletion of complex). The strains had an
RC+ LH1+ LH2 phenotype.
To assess the effects of the PufXc protein on the
composition of the R. sphaeroides core complex,
intracytoplasmic membranes were prepared from cells grown under
semiaerobic conditions in the dark and examined by absorbance
spectroscopy (Fig. 1A). As seen in a
previous study (23, 24), deletion of the
pufXs gene resulted in an increase in the amount
of LH1 per reaction center (Fig. 1A; dotted and solid curves), seen as
an increase in the amplitude of the LH1 Qy band (at
approximately 875 nm) relative to the reaction center Qy
band (at approximately 802 nm). The position of the absorbance maximum
of the LH1 Qy band was not affected significantly by
removal of PufXs (23, 24). Experiments with
samples that had been normalized to contain approximately the same
concentration of LH1 showed that the amount of LH1 fluorescence from
membranes of strain RCLH1sXd was ~60% greater than that from membranes of control strain RCLH1sXs (Fig. 1B, cf. dotted and solid
curves). The emission maximum was at 895 nm for membranes from both
strains. These fluorescence data are in good agreement with
observations on R. capsulatus (21) which showed
that strains that lack the PufX polypeptide use harvested light energy
less effectively. For comparison, the amount of fluorescence emission was also measured for membranes from an LH1-only strain (i.e., a strain
which lacks the reaction center, LH2, and PufX). For samples normalized
to the same concentration of LH1 Bch1, emission was approximately
3.7-fold greater in membranes from the LH1-only strain than from strain
RCLH1sXs (data not shown).
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FIG. 1.
Room temperature absorbance (A and C) and fluorescence
emission (B and D) spectra of intracytoplasmic membranes. For
comparison, absorbance spectra were corrected for background scatter
between 650 and 950 nm and normalized to the same absorbance at 802 nm,
approximating the same concentration of reaction centers. For
fluorescence emission spectra, membranes were normalized to the same
absorbance (0.2 absorbance unit cm1) at the maximum of
the LH1 Qy band.
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|
Assembly of the core complex was not impaired by replacement of the
pufXs gene by the
pufXc
gene in strain RCLH1sXc. The level
of LH1 per reaction center seen in
the presence of the PufX
c protein
was essentially the same
as that seen in the presence of PufX
s and was quite
distinct from the elevated level of LH1 seen in
strain RCLH1sXd (Fig.
1A, dashed curve). The LH1 Q
y band was blue
shifted by 2 nm
relative to that seen in strains RCLH1sXs and
RCLH1sXd. Membranes of
the RCLH1sXc strain showed a level of LH1
fluorescence slightly below
that seen in membranes from the RCLH1sXs
strain and much lower than
that seen in the RCLH1sXd strain (Fig.
1B; dashed curve). This
indicated that the presence of the PufX
c protein improved
the effectiveness with which LH1 excitation energy
was used by the
reaction center, relative to core complexes which
lacked either PufX
protein. Taken together, therefore, the absorption
and fluorescence
data demonstrate a direct interaction between
the PufX
c
protein and the
R. sphaeroides core complex.
Photosynthetic growth of strain RCLH1sXc was similar to that exhibited
by control strain RCLH1sXs (Fig.
2A) in
that appreciable
photosynthetic growth was observed after a lag of 50 to 60 h.
This lag has been attributed to a period of adaptation
from semiaerobic,
dark growth conditions to photosynthetic growth that
is peculiar
to strains with an RC
+ LH1
+
LH2
phenotype and which have normal, or near to normal,
levels of
the LH1 antenna complex (
23,
24). Experiments
involving cycles
of semiaerobic, dark growth and photosynthetic growth
showed that
the growth exhibited by strains RCLH1sXs and RCLH1sXc after
the
60-h lag did not occur in response to suppressor mutations (data
not shown). Growth of strain RCLH1sXc was quite distinct from
that
exhibited by PufX-deficient strain RCLH1sXd, which only grew
after a
very long lag phase (if at all), presumably in response
to one or more
suppressor mutations (
1,
19). The PufX
c protein,
therefore, was competent in supporting the photosynthetic growth
of
R. sphaeroides.
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FIG. 2.
Photosynthetic growth of strains containing the
LH1s complex (A) and the LH1c complex (B).
Growth was monitored by measuring OD680 and is plotted on a
log10 scale. For each strain, the inoculum consisted of
cells taken from a culture that had been grown under semiaerobic
conditions in the dark. In B, the growth of antenna-deficient strain
RCO2 is shown for comparison.
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|
Heterologous expression of the pufBA genes from
R. capsulatus in R. sphaeroides.
The functional
identity between core complex proteins from the two species of purple
bacteria was investigated further by heterologous expression of the
pufBA genes from R. capsulatus in R. sphaeroides. The pufBcAc genes
were expressed in R. sphaeroides DD13 in tandem with the
pufXs gene (strain RCLH1cXs) and in the absence
of either pufX gene (strain RCLH1cXd).
Room temperature absorption spectra showed that the LH1
c
complex was successfully expressed in
R. sphaeroides but
that the
level of expression relative to that of the reaction center
was
50 to 60% of that of the native LH1
s complex,
regardless of the
presence or absence of the PufX
s protein
(Fig.
1C, solid and dotted
curves). This can be seen by comparing the
relative heights of
the LH1 Q
y band at 875 to 878 nm and
the reaction center Q
y band
at 802 nm in Fig.
1A and C. The
maximum of the LH1
c Q
y band in
strain RCLH1cXs
was located 2 nm to the red of the corresponding
band of native
LH1
s (877 versus 875 nm) and was red shifted by
a further 1 nm in strain RCLH1cXd. The amount of fluorescence
emission from
membranes of strain RCLH1cXd (Fig.
1D, dotted curve)
was similar to
that of strain RCLH1cXs (Fig.
1D, solid curve),
in contrast to the
significant difference shown in Fig.
1B for
the analogous strains
containing the LH1
s complex. The level of
fluorescence in
both strains was comparable to that seen for control
strain RCLH1sXs
(Fig.
1B, solid curve) and was therefore well
below the level expected
for RC-free LH1 (see above).
To examine whether the relatively low level of LH1
c
expression in
R. sphaeroides was attributable to the lack of
the PufX
c protein, plasmid pRKEHLH1cXc was constructed to
allow coexpression
of the
pufBcAc
genes with the
pufXc gene (see Materials and
Methods).
As can be seen in Fig.
1C (dashed curve), the absorption
spectrum
of membranes from the resulting strain (RCLH1cXc) was very
similar
to those of strains RCLH1cXd and RCLH1cXs, showing that the
relatively
low level of LH1
c in these membranes was not a
consequence of
the absence of the PufX
c protein.
Fluorescence emission from membranes
of strain RCLH1cXc was similar to
that shown by strains RCLH1cXs
and RCLH1cXd and control strain RCLH1sXs
(Fig.
1D).
All three of the LH1
c-containing strains were capable of
photosynthetic growth (Fig.
2B). Strain RCLH1cXs grew following a
60-h
lag (Fig.
2B, circles) in a manner similar to that of control
strain
RCLH1sXs (Fig.
2A, circles) and PufX
c-containing strain
RCLH1sXc (Fig.
2A, squares). In contrast, strain RCLH1cXc grew
without
the initial lag (Fig.
2B, squares) in a manner similar
to that of
antenna-deficient strain RCO2 (Fig.
2B, crosses), although
the final OD
reached was significantly greater than that achieved
by strain RCO2,
being comparable to that seen in the other strains
with an LH1 antenna.
Finally, strain RCLH1cXd showed both appreciable
growth with no lag and
a second phase of growth after ~70 h (Fig.
2B, triangles). It should
be noted that each of these patterns
of photosynthetic growth was
distinctive and highly reproducible,
despite the fact that the
absorbance and fluorescence properties
of the three strains were very
similar, over and above some minor
differences in the positions of
absorbance and fluorescence emission
maxima.
| |
DISCUSSION |
Sequence analysis of PufXs and PufXc and
implications for the role of PufX.
Alignments of the amino acid
sequences of PufXs and PufXc, such as that
shown in Fig. 3, reveal a maximum of 29%
identity between the two proteins. This low sequence identity is in
marked contrast to the high degree of identity seen in alignments
between the reaction center and LH1 polypeptides from R. sphaeroides and R. capsulatus (between 70 and 80%).
Part of this discrepancy may arise from the requirement of the LH1 and
reaction center complexes to bind carotenoid and Bchl pigments,
although such binding does not appear to require well-conserved and
extensive structural motifs. As can be seen from the alignment shown in
Fig. 3, such identity that exists between the two PufX proteins is not
focused on one or two areas of high conservation but rather is randomly distributed along the entire length of the two polypeptide chains. The
fact that the PufXc protein is very effective in
substituting for the PufXs protein suggests that the
primary sequence of a large part of the PufX protein is not
particularly important for function, raising the possibility that the
main requirement for PufX activity is a small number of residues,
perhaps widely distributed along the protein sequence. Alternatively,
the principal requirement could be the overall shape of the molecule or
some other structural property that can be provided by more than one
specific amino acid sequence. Hydrophobicity plots (Kyte-Doolittle
algorithm) (17) performed with the PufXs and
PufXc protein sequences suggested a stretch of hydrophobic
amino acids in both proteins that would, in principle, be sufficiently
long to span a lipid bilayer membrane as an alpha helix (Fig. 3).
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FIG. 3.
Alignment of the PufX polypeptide sequences from
R. sphaeroides (Rs) and R. capsulatus (Rc). The
alignment gives 29% identical residues and 49% similar residues.
Residue numbering is for R. sphaeroides. Identical residues
are indicated by bold type. Horizontal dashes indicate probable
transmembrane regions, as indicated by hydrophobicity plots. As
discussed in reference 8, the best alignment is
achieved with two gaps, one in each sequence.
|
|
Organization of the Rhodobacter photosystem by
PufX.
The data presented in this report show that the PufX protein
is a key factor in the proper functional organization of the LH1/RC
core complex in R. sphaeroides and R. capsulatus.
Two findings, in particular, point to this role. The first is the
observation that the restoration to photosynthetic competence seen on
expression of the pufXc gene in place of the
pufXs gene (in strain RCLH1sXc) was accompanied
by restoration of the LH1:RC ratio to the value seen in control strain
RCLH1sXs and restoration of the level of LH1 fluorescence to that seen
in the control strain. It has been proposed that the increase in the
number of LH1 Bchls per reaction center seen on removal of the PufX
polypeptide reflects an increase in the aggregation state of the LH1
complex surrounding the reaction center, the result of which is to
interfere with the rate of ubiquinone-ubiquinol exchange at the
QB site through steric constraints (2, 3, 20, 21, 23,
24). The effects of expression of the
pufXc gene in R. sphaeroides on the
optical properties of the core complex and the photosynthetic
competence of the bacterium revealed in this study lend further support
to this model, in which PufX defines the proper organization of LH1 in
the core complex.
The second finding is the distinctive patterns of photosynthetic growth
exhibited by the three LH1
c-containing strains, cells
and
membranes of which had very similar absorbance and fluorescence
properties. We believe that this observation shows that while
PufX is
not necessarily the main determinant of the relative levels
of reaction
center and LH1 in the membrane, it does affect the
organization of the
components of the core complex and that this,
in turn, determines the
ability of the bacterium to grow under
photosynthetic conditions. The
origin of the differences in membrane
organization that underlie the
markedly different patterns of
photosynthetic growth exhibited by
strains RCLH1cXs, RCLH1cXd,
and RCLH1cXc is the subject of ongoing
investigations.
| |
ACKNOWLEDGMENTS |
This work was supported by the Biotechnology and Biological
Sciences Research Council of the United Kingdom. M.R.J. is a BBSRC Advanced Research Fellow.
We thank C. N. Hunter, A. R. Crofts, and W. Westerhuis for
helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2UH, United Kingdom. Phone: 114-2224234. Fax: 114-2728697. E-mail: m.jones{at}sheffield.ac.uk.
| |
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J Bacteriol, February 1998, p. 642-646, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
