(Received for publication, May 10, 1995; and in revised form, June 28, 1995)
From the
One of the major problems in structural work on
membrane-spanning proteins is the identification of an expression
system which will allow the production of enough pure protein for
structural studies; an inadequate expression system can lead, for
example, to the formation of unwanted protein inclusion bodies. In the
present work we report the expression of genes encoding the
light-harvesting 2 (LH2) membrane-spanning proteins from a number of
species of purple bacteria in mutants of Rhodobacter sphaeroides that lack the native LH2 antenna. The LH2 structural genes (pucBA) from the photosynthetic bacteria Rhodopseudomonas
acidophila and Rubrivivax gelatinosus were amplified and
tailed by polymerase chain reaction, and cloned into an LH2 expression
vector, which was then introduced into three LH2-minus Rb.
sphaeroides mutants; DBC/G5 and DD13 (DD13/G1); the
resulting transconjugant strains synthesized LH2 complexes that were
examined using absorption and fluorescence spectroscopy, and Western
blotting. Thus, we have created a heterologous expression system which
supports the assembly of a functional ``foreign''
light-harvesting complex. This work opens up the possibility of
creating site-directed LH2 mutants from bacteria for which no genetic
system is available; this is particularly significant in the case of Rps. acidophila, since this bacterium has been the source of
the LH2 complex that has recently been structurally resolved to atomic
resolution.
Most of the light-absorbing pigments in photosynthesis are found
in the light-harvesting (LH) ()complexes, which funnel
absorbed solar radiation to the photochemical reaction centers, where
the primary redox reactions occur. An understanding of the molecular
details of the light-harvesting process requires a combination of
biochemical, structural, and spectroscopic information, and
photosynthetic bacteria provide excellent model systems in which to
unravel this photophysical process. In most purple photosynthetic
bacteria, energy migrates from a peripheral light-harvesting complex
(LH2) to an LH1-reaction center ``core'' complex (Hunter et al., 1989); both types of light-harvesting complex are
oligomers of two types of apoproteins (
and
) together with
bacteriochlorophylls and carotenoids. These pigments are not free
within the photosynthetic membranes but are liganded to specific
hydrophobic membrane-spanning apoproteins (Brunisholz and Zuber, 1992);
these apoproteins are small (molecular weight =
5,000) and
provide the structural scaffolding that serves to organize the pigments
so that energy transfer to the reaction center is both rapid and
efficient (Sundström and van Grondelle, 1990).
Moreover, these simple polypeptides apparently possess the information
that allows them to aggregate to form large assemblies (Brunisholz and
Zuber, 1992).
Some recent developments have provided more
information on these bacterial light-harvesting complexes. First, there
has been a breakthrough in the x-ray crystallography of the LH2 complex
from Rhodopseudomonas acidophila, which has built upon earlier
studies (Cogdell and Hawthornthwaite, 1993; Papiz et al.,
1989) and has progressed to the extent that a structure has been
determined to atomic resolution (McDermott et al., 1995). This
LH2 complex is composed of a ring of 18 bacteriochlorophyll molecules
sandwiched between an inner ring of nine membrane-spanning
subunits and an outer ring of nine membrane-spanning
subunits;
nine more bacteriochlorophylls also form a more widely spaced ring and
are located near the cytoplasmic face of the membrane (McDermott et
al., 1995). In order to examine the roles played by some of the
residues in this complex, it would be necessary to use site-directed
mutagenesis techniques. The second development is the availability of a
gene deletion and expression system for Rhodobacter sphaeroides (Burgess et al., 1989; Jones et al., 1992). It
has therefore been possible to introduce site-directed alterations into
the light-harvesting complexes in this species; a combination of
mutagenesis and spectroscopy has, for example, identified the
Tyr
and
Tyr
residues as
modulators of the bacteriochlorophyll Q
absorbance peak in
the LH2 complex from Rb. sphaeroides (Fowler et al.,
1992, 1994). Finally, it is possible to re-assemble bacterial LH
complexes from their constituent parts; this process allows the
investigator to exert control over the composition of the complex, for
example by using truncated polypeptides or bacteriochlorophyll
analogues (Loach and Parkes-Loach, 1994).
One of the major problems
in crystallization studies on membrane-spanning proteins is the
production of enough pure protein; the overproduction of protein in Escherichia coli, for example, can lead to the formation of
unwanted protein inclusion bodies or in some cases very low levels of
protein expression. The lack of availability of genetic systems for
many photosynthetic bacteria necessitates a different approach; the
expression of LH genes from other bacteria in the purple photosynthetic
bacterium Rb. sphaeroides reported here exploits the ability
of this bacterium to elaborate a large amount of intracytoplasmic
membrane (see, for example, Lommen and Takemoto(1978) and Drews and
Oelze(1981)). This could also be a valuable asset when designing
systems for overexpression of other membrane proteins. For the work
reported here, we have used LH genes from two photosynthetic bacteria, Rps. acidophila and Rubrivivax (formally Rhodocyclus) gelatinosus. Some of the LH2 genes of Rps. acidophila and Rv. gelatinosus (Fig. 1)
have been cloned and characterized elsewhere, and in general they
follow the pattern already established in Rb. sphaeroides and Rhodobacter capsulatus, and are organized in the order pucBA. ()One important difference, however, is the
presence of multiple copies of these genes, as also found for Rhodopseudomonas palustris (Tadros and Drews, 1990). This is
under further investigation, but it is already clear that some of these
are not expressed at detectable levels (see also Tadros et
al.(1993)). Similarly, it has been established that different Rps. acidophila light-harvesting proteins are synthesized at
different levels depending on the growth environment of the bacterium
(Hawthornthwaite and Cogdell, 1991; Gardiner et al., 1993).
The expression of the genes in Rb. sphaeroides would allow the
individual pucBA gene pairs to be studied in isolation from
the others, and would also provide a means of studying previously
``silent'' gene pairs.
Figure 1:
Sequences of Rb.
sphaeroides (RS), Rps. acidophila (RPA), and Rv. gelatinosus (RVG) LH2
polypeptides, aligned at the Bchl850 histidine ligand (shown in bold). The Rps. acidophila polypeptides are those
coded for by silent genes pucBA from strain 7050
(A. T. Gardiner, R. C. Mackenzie, S. J. Barratt, K. Kaizer, and R. J.
Cogdell, manuscript in preparation). The underlined residues
in the
-subunits have been shown to have a role in the regulation
of the spectral properties of the Bchl850 pigments (Fowler et
al., 1992, 1994).
In the present work we report the
expression of genes encoding the light-harvesting 2 (LH2)
membrane-spanning proteins from the purple bacteria Rps. acidophila and Rv. gelatinosus in mutants of Rb. sphaeroides lacking the native LH2 light-harvesting complex. The LH2
structural genes (pucBA) from these bacteria were amplified
and tailed by polymerase chain reaction, and cloned into an LH2
expression vector, which was then introduced into three LH2-minus Rb. sphaeroides mutants, DBC/G5 (LH2
,
LH1
, RC
), DD13 and DD13/G1
(LH2
LH1
RC
),
resulting in a series of LH2-containing transconjugant strains (see Table 1). The resulting foreign LH2 complexes were examined using
Western blotting and absorption and fluorescence spectroscopy. The
implications of this expression system for structural studies and for
the study of previously inaccessible and even silent gene pairs are
discussed.
Figure 2: Western blot analysis of LH2-only membranes prepared from transconjugant strains probed with Rps. acidophila LH2 antibodies (top) (strain 7750) and Rb. sphaeroides LH2 antibodies (bottom). Sample1, strain DD13(pRKGEL); 2, Rps. acidophila WT (strain 7050) as a control; 3, strain DD13(pRKACID); 4, strain DD13/G1(pRKACID); 5, Rb. sphaeroides WT, as another control. Each sample has been loaded into two adjacent gel lanes in amounts that differ 3-fold.
The
gene pairs shown in Fig. 1were thought to be good
candidates for use in the examination of Rb. sphaeroides as a
heterologous expression system, in that they are similar to the native
proteins. However, since we have observed that this system has been
less successful for the expression of LH genes from Rhodospirillum
rubrum and Rhodospirillum molischianum, (
)for
example, and that site-directed point mutations in the native genes can
abolish assembly of the complex (Bylina et al., 1988), (
)successful production of foreign LH2 complexes was not
assured at the outset.
Figure 3: Room temperature absorbance spectra of Rb. sphaeroides LH2-containing membranes from strains DD13(pRKCBC1) (a) and DD13/G1(pRKCBC1) (b); of Rps. acidophila LH2-containing membranes from strains DD13/G1(pRKACID) (c) and DD13(pRKACID) (d) and Rv. gelatinosus LH2-containing membranes from strain DD13/G1(pRKGEL) (e). Spectra have been scaled to reflect the levels of light-harvesting complex on the basis of the amount of complex per µg of membrane protein, as assessed by protein assay.
The absorbance spectra of
the membrane samples in Fig. 3clearly show that the assembly
and spectral properties of the Rps. acidophila LH2 complex are
sensitive to the type of carotenoid present. The Rb. sphaeroides recipients had either neurosporene (green) or
spheroidene/spheroidenone (orange/red) as the carotenoid background,
but neither of these pigments has a glucoside moiety, and yet the major
carotenoid in Rps. acidophila is rhodopin glucoside, a pigment
that is structurally different from either neurosporene or spheroidene
(see, for example, Gardiner et al.(1993)). Although some LH2
has been assembled in DD13/G1(pRKACID) and DD13(pRKACID), wild-type
levels of LH2 have not been reached; however, this could be explained
by the absence of the normal carotenoid for the Rps. acidophila complex. For example, we have recently shown that assembly of the Rb. sphaeroides LH2 complex is sensitive to the type of
carotenoid present (Lang et al., 1994). The Rps.
acidophila complex does tolerate both neurosporene and
spheroidenone, but to different degrees; the presence of spheroidenone
leads to marked reduction in the amount of complex, which is
accompanied by a marked attenuation of the B800 band (Fig. 3d). These types of shifts have been seen before
in LH2 complexes, but only in the context of site-directed mutagenesis.
For example, a complex that absorbs at 790-853 nm has been
observed in an LH2 mutant of Rb. sphaeroides in which the
Arg
(
Arg
) residue has been
changed to a Glu (Crielaard et al., 1994). Similarly, in a Rb. sphaeroides strain containing LH2 that has been
synthesized in a phytoene background (DD13/W1) of Rb. sphaeroides (that is, in a complete absence of colored carotenoids), an
absorption spectrum is observed in which the B800 peak is greatly
attenuated and in which the B850 is red-shifted by at least 10 nm. (
)It is interesting to note that in these last two cases,
and in the present work, either the carotenoid or the residue
Arg
exerts a strong effect on the binding of
B800: the recent determination of the structure of the Rps.
acidophila complex (McDermott et al., 1995) has shown
that these moieties are located close by the B800 pigment, which
provides an explanation of our results. Expression of the Rps.
acidophila genes in the neurosporene-containing strain of Rb.
sphaeroides results in relatively high levels of the complex with
absorbance peaks at 800 and 838 nm. In the LH2 structure it can be seen
that the carotenoid is closely associated with both the B800 and B850
pigments (McDermott et al., 1995), so it is possible for an
alteration in the carotenoid to have an effect on either pigment.
With respect to the altered spectral profile (800-838 nm) of
the Rps. acidophila LH2 complex in strain DD13/G1(pRKACID),
similar spectral properties can be seen with site-directed LH2 mutants
of Rb. sphaeroides (Tyr
Phe,
Tyr
Phe; Fowler et al.(1992)).
The results of Raman spectroscopy carried out on the Rb.
sphaeroides
Tyr
and
Tyr
mutants have led to the proposal that this type of shift is a
consequence of the breaking of a hydrogen bond with one of the B850
Bchls (Fowler et al., 1994). In both the Rb. sphaeroides LH2 mutant
Tyr
-Tyr
Phe-Tyr, and the Rps. acidophila genes studied in
the present work (which have the sequence
Phe
-Met
; see Fig. 1),
the aromatic residue at position
has been
altered (relative to the Rb. sphaeroides WT LH2 complex) to
Phe, an amino acid that has no potential to form a hydrogen bond. This
may help to explain in part why these two LH2 pigment-protein
complexes, each absorbing at 800 and 838 nm, have such similar spectral
properties, although the effect of altering the carotenoid background
in the Rps. acidophila LH2 complex from rhodopin glucoside to
neurosporene should not be underestimated, as mentioned above. More
needs to be done to investigate the requirements for binding different
carotenoid types, and the expression system described here provides a
way forward for this type of work.
Fig. 3also shows the
absorption spectrum ( = 800, 850 nm) from
strain DD13/G1(pRKGEL) containing the Rv. gelatinosus LH2
genes (Fig. 3e); this is similar to that seen in Fig. 3b for the WT Rb. sphaeroides LH2 in
strain DD13/G1(pRKCBC1). To some extent this similarity is not
unexpected since neurosporene is the major carotenoid synthesized by Rv. gelatinosus (Jirsakova and Reiss-Husson, 1993; Young,
1993). However, the level of the Rv. gelatinosus LH2 complex
was lower, and the B800 peak in this complex was also attenuated, in
comparison with the WT Rb. sphaeroides complex. Preliminary
results indicate that the same Rv. gelatinosus LH2 genes do
not give rise to an LH2 complex in the spheroidenone-containing strain
DD13, unlike the other LH genes described in this study. It is
interesting to note that the lack of a histidine residue in the Rv.
gelatinosus LH2 genes at a position where one is seen in the Rb. sphaeroides LH2 genes (
His
; Fig. 1) has not prevented the expression of a B800 pigment.
Previous studies have shown that mutagenesis of
His
Ser in the Rb. sphaeroides LH2 complex weakens
B800 binding but does not abolish it completely (Crielaard et
al., 1994; Visschers et al., 1994), The recent structural
data show that indeed
His
has a close
interaction with the B800 bacteriochlorophyll (McDermott et
al., 1995), but that it does not play a decisive role in binding
this pigment.
Bacteriochlorophyll and carotenoid pigments were
extracted from the transconjugant strains in order to investigate the
stoichiometry of these pigments with respect to one another. Fig. 4shows the absorption spectra of acetone:methanol extracts
of membranes from strains DD13(pRKCBC1), DD13/G1(pRKCBC1),
DD13(pRKACID), DD13/G1(pRKACID), and DD13/G1(pRKGEL). All spectra are
normalized to the Q absorbance maximum of
bacteriochlorophyll at 768 nm. It is clear that in the strains
containing the heterologously synthesized Rps. acidophila LH2
complexes (Fig. 4, a and b) the ratio of the
carotenoid to bacteriochlorophyll pigments is much higher than in those
containing the WT Rb. sphaeroides LH2 complexes (Fig. 4, c and d). In the former case this
reflects either a greater production of carotenoids by the cells, or,
more likely, a relatively low level of the assembled Rps.
acidophila light-harvesting complex, possibly arising from some
instability of the Rps. acidophila light-harvesting complex in Rb. sphaeroides. Thus there appears to be excess carotenoid
present, which is probably not associated with the heterologously
synthesized complexes. Whereas there is a stringent control of the
level of bacteriochlorophyll to match the level of LH2 apoprotein
(since there are no bacteriochlorophyll biosynthetic intermediates
present), this does not extend to control of the levels of carotenoids.
A similar situation is seen when Erwinia carotenoid
biosynthetic genes are expressed in Rb. sphaeroides, resulting
in new LH complexes with
-carotene and zeaxanthin as the
carotenoids. Again, carotenoid biosynthesis appears to be uncoupled
from the formation of both bacteriochlorophyll and LH apoproteins
(Hunter et al., 1994).
Figure 4: Absorbance spectra of acetone:methanol (7:2 v:v) extracted membranes from strains DD13(pRKACID) (a), DD13/G1(pRKACID) (b), DD13(pRKCBC1) (c), and DD13/G1(pRKCBC1) (d). All spectra are normalized to the bacteriochlorophyll peak at 768 nm.
Figure 5: The near infra-red region of the fluorescence excitation spectra recorded on membranes from the following strains: DD13(pRKCBC1) (dashed line), DD13/G1(pRKACID) (solid line), DD13(pRKACID) (bold line), and DD13/G1(pRKGEL) (dotted line). As the wavelength of excitation was varied from 750 to 850 nm, the emission of fluorescence was recorded at 900 nm.
In view of the
substitution for the ``native'' carotenoid, rhodopin
glucoside (see, for example, Gardiner et al.(1993)) by
spheroidene and neurosporene in the Rps. acidophila LH2
complexes, it was important to see if these carotenoids were capable of
transferring energy to the bacteriochlorophylls. Fig. 6compares
the excitation spectrum in the 350-750 nm region for
LH2-containing membranes from strains DD13/G1(pRKCBC1),
DD13/G1(pRKACID), and DD13/G1(pRKGEL), and measures the ability of the
carotenoids present to elicit fluorescence from the
bacteriochlorophylls, detected at 860 nm. The spectra are normalized at
the 590 nm excitation peak, which can be taken as representing fully
efficient transfer within the bacteriochlorophyll molecule, from the
Q band at 590 nm to the Q
band, which emits at
860 nm. From the carotenoid excitation peaks (455, 486, and 524 nm)
shown in Fig. 6, it can be seen that, in the membrane sample
where Rps. acidophila LH2 genes have been expressed in strain
DD13/G1(pRKACID), the carotenoid excitation peaks are of a much lower
intensity than those seen for the WT Rb. sphaeroides LH2
complex in DD13/G1(pRKCBC1), so it is probable that there is some
impairment in the energy transfer process. However, the excitation
peaks for the Rv. gelatinosus LH2 complex in DD13/G1(pRKGEL)
are of an intensity nearly equivalent to those seen for the WT LH2.
This indicates that if the heterologously expressed LH2 genes are
supplied with their native carotenoids (neurosporene is the major
carotenoid found in WT Rv. gelatinosus), then a significant
level of energy transfer can be observed.
Figure 6:
Visible region of the fluorescence
excitation spectra ( = 860 nm) recorded
on membranes from the following strains: DD13/G1(pRKCBC1) (a, plain line), DD13/G1(pRKGEL) (b, dotted
line), and DD13/G1(pRKACID) (c, dashed line),
showing the excitation bands arising from carotenoid pigments between
400 and 500 nm.
Figure 7:
Fluorescence excitation spectrum
( = 920 nm) recorded on membranes
prepared from strains DBC
/G5(pRKACID) containing Rb.
sphaeroides LH1 and reaction center complexes in addition to the Rps. acidophila LH2 complex (dashed line). (1 - T) spectra are shown for the same strain (solid line)
and for DBC
/G5 as a negative control (dotted
line).
Figure 8:
Electron micrograph of thin sections of
cells from Rb. sphaeroides strain DBC/G5 (LH1+RC) (a), cells from Rb. sphaeroides strain DBC
/G5
in which Rps. acidophila LH2 genes have been expressed
(DBC
/G5(pRKACID)) (b), and DBC
/G5(pRKCBC1) (c). Scale bars represent 200 nm.
In the future, it will be interesting to express more pucBA gene pairs from Rps. acidophila in Rb. sphaeroides since they may provide new information about the relationship between amino acid sequence and the position of the near infra-red bacteriochlorophyll absorption bands. The ability to express Rps. acidophila and Rv. gelatinosus LH2 genes in Rb. sphaeroides circumvents the problem of establishing a genetic system for these bacteria and will allow us to construct site-specific mutations in these foreign LH2 complexes. Such experiments are especially timely, given the recent x-ray crystallographic structure for the Rps. acidophila complex (McDermott et al., 1995). In the case of the Rv. gelatinosus LH2 genes, not only are the expression levels of the LH2 protein comparable to those found with WT Rb. sphaeroides LH2, the energy transfer function of the complex is maintained, illustrating the effectiveness of the expression system.