©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heterologous Expression of Genes Encoding Bacterial Light-harvesting Complexes in Rhodobacter sphaeroides(*)

(Received for publication, May 10, 1995; and in revised form, June 28, 1995)

Gregory J. S. Fowler (1)(§) Alastair T. Gardiner (2)(¶) R. Christopher Mackenzie (2) Stuart J. Barratt (2) Adrian E. Simmons (2)(¶) Willem H. J. Westerhuis (1) Richard J. Cogdell (2) C. Neil Hunter (1)

From the  (1)Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN and the (2)Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Most of the light-absorbing pigments in photosynthesis are found in the light-harvesting (LH) (^1)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 (alpha and beta) 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 alpha subunits and an outer ring of nine membrane-spanning beta 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 alphaTyr and alphaTyr residues as modulators of the bacteriochlorophyll Q(y) 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. (^2)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 puc^2BA 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 alpha-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 (LH2LH1RC), 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.




EXPERIMENTAL PROCEDURES

Media, Antibiotics, and Growth Conditions

E. coli strains were grown in Luria broth. Rb. sphaeroides strains were grown under semiaerobic/dark conditions in M22+ medium (Hunter and Turner, 1988) supplemented with 0.1% casamino acids for growth in liquid culture. For E. coli, tetracycline was used at a concentration of 10 µg/ml. For Rb. sphaeroides, antibiotic concentrations were: tetracycline, 1 µg ml; neomycin, 20 µg ml; and streptomycin, 5 µg ml.

Bacterial Strains and Plasmids

The bacterial strains used in this work include E. coli strain S17-1 (thi pro hsdRhsdMrecA RP4-2 (Tc::mu km::Tn7) (Simon et al., 1983), Rb. sphaeroides strains DD13 and DD13/G1 (genomic deletion of pucBA and pufBALMX; insertion of Sm^R and Km^R genes) and DBC/G5 (genomic deletion of pucBA genes; insertion of Sm^R genes leaving only the native LH1-RC core) (Jones et al., 1992). The DD13/G1 and DBC/G5 mutants each carry a mutation in the crtD gene that alters the normal complement of carotenoids, spheroidene and spheroidenone (yellow and red), to neurosporene and derivatives, which are green. The mobilizable plasmids used were based on pRKCBC1 (Tc^R; derivative of pRK415; 4.4kb fragment encompassing pucBAC); briefly this expression vector, described by Jones et al.(1992), contains the pucBA genes as a 420-base pair KpnI-BamHI insert. For the construction of the vectors for heterologous expression of the foreign LH2 genes (see, for example, DD13(pRKACID) and DD13/G1(pRKGEL); Table 1), the appropriate pucBA gene pair (containing similarly engineered KpnI-BamHI ends) were cloned into plasmid pRKCBC1 in place of the wild-type Rb. sphaeroides pucBA genes so that transcription through these Rps. acidophila and Rv. gelatinosus genes is driven by the Rb. sphaeroides puc promoter.

Conjugative Crosses

The LH2 pucBA gene pairs from Rps. acidophila and Rv. gelatinosus were amplified and tailed by polymerase chain reaction, and cloned as KpnI-BamHI fragments into the LH2 expression vector pRKCBC1; expression is driven by the Rb. sphaeroides puc promoter (Gibson et al., 1992). The constructs were introduced into Rb. sphaeroides by conjugative transfer. The mobilizable plasmids to be introduced into Rb. sphaeroides were first transformed into E. coli strain S17-1. Matings were then performed as described by Hunter and Turner (1988). Transconjugants were grown aerobically in the dark on plates of M22+ medium supplemented with appropriate antibiotics. In the case of matings performed to complement deletion/insertion mutants in trans using plasmids based on pRKCBC1, tetracycline was included in the growth medium. The nomenclature for the various Rb. sphaeroides transconjugant strains is given in Table 1.

Western Blotting

Blotting of sodium dodecyl sulfate-polyacrylamide gels (Western blots) were performed according to De Marcucci et al.(1985) and Sambrook et al. (1989), using antibodies to LH2 pigment-protein complexes prepared from Rps. acidophila LH2 (strain 7750) and Rb. sphaeroides.

Preparation of Intracytoplasmic Membranes

Membranes were prepared from cells grown semi-aerobically in the dark by disruption in a French pressure cell and were purified by harvesting from the interface of sucrose step gradients (15%/40%, w/w) after centrifugation.

Spectroscopy

Following conjugative transfer, antibiotic-resistant colonies were screened for the presence or absence of LH2 light-harvesting complexes using a Guided Wave model 260 fiber optic spectrophotometer (Guided Wave Inc., El Dorado Hills, CA). The fluorescence spectra were recorded on a Spex FluoroMAX spectrofluorimeter (Spex Industries, Inc., Edison, NJ). The membrane protein assay was performed using the DC protein assay kit (Bio-Rad).


RESULTS

The LH2 Genes Used and Verification of the Presence of Their Gene Products by Western Blotting

Fig. 1shows the sequence alignments for the LH2 alpha and beta polypeptides coded by the genes used in this study: the polypeptides are aligned around the conserved histidine residues (His^0) which co-ordinate the B850 pigments (McDermott et al., 1995). The Rps. acidophila and Rv. gelatinosus alpha sequences are 50% and 40% identical to the Rb. sphaeroides alpha sequence, respectively, whereas the Rps. acidophila beta and Rv. gelatinosus beta are, respectively, 50% and 70% identical to the Rb. sphaeroides beta sequence. The identities of the polypeptides produced by heterologously expressed genes were verified by Western blot analysis, which was performed on membranes from transconjugant strains into which the various LH2 genes had been introduced, using antibodies to either the native LH2 complex from Rb. sphaeroides or from Rps. acidophila strain 7750 (Fig. 2). With the Rps. acidophila LH2 antibody, the membranes containing the putative Rps. acidophila LH2 complex gave positive signals, whereas membranes from the Rb. sphaeroides WT and Rv. gelatinosus did not. In contrast, when the Rb. sphaeroides LH2 antibody was used, only the Rb. sphaeroides membranes gave a positive signal, whereas those containing heterologously synthesized Rps. acidophila and Rv. gelatinosus LH2 complexes did not.


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 alphabeta 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, (^3)for example, and that site-directed point mutations in the native genes can abolish assembly of the complex (Bylina et al., 1988), (^4)successful production of foreign LH2 complexes was not assured at the outset.

Absorbance Spectra of Heterologously Synthesized Complexes

The absorbance spectra of membranes prepared from LH2-only transconjugant strains are shown in Fig. 3, and they clearly demonstrate the presence of appreciable amounts of the Rps. acidophila LH2 complexes in strains DD13/G1(pRKACID) and DD13(pRKACID); the relevant strains are described in Table 1. The spectra are scaled to reflect the level of LH2 complex per amount of cellular membrane as quantified by total membrane protein, so it is clear that none of the heterologously expressed genes give rise to as much complex as do the WT genes, although there is ample material for biochemical and spectroscopic studies. In the red spheroidenone-containing strain DD13(pRKACID), Fig. 3d, the Rps. acidophila LH2 had absorbance properties ((max) = 853 nm; Fig. 3d) that differed considerably from WT Rb. sphaeroides LH2 in strain DD13(pRKCBC1) ((max) = 800, 850 nm; Fig. 3a). Similarly, in strain DD13/G1(pRKACID) containing the green carotenoid neurosporene, the Rps. acidophila LH2 showed altered absorbance properties ((max) = 800, 838 nm; Fig. 3c), not only with respect to the to the WT Rb. sphaeroides LH2 in strain DD13/G1(pRKCBC1) ((max) = 800, 850 nm; Fig. 3b), but also in comparison with the red version of this complex in Fig. 3d. Some synthesis of an Rps. acidophila LH2 complex was also observed in the phytoene-containing strain DD13/W1 (Jones et al., 1992), but this work will not be pursued further here. The change from a neurosporene (green) background to a spheroidene/spheroidenone (orange/red) background leads to marked reduction in the amount of the Rps. acidophila LH2 complex; this is accompanied by a marked attenuation of the ``B800'' band, together with a red shift of the 838 nm band of 15 nm, to 853 nm.


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 betaArg (betaArg) 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. (^5)It is interesting to note that in these last two cases, and in the present work, either the carotenoid or the residue betaArg 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 (alphaTyr Phe, alphaTyr Phe; Fowler et al.(1992)). The results of Raman spectroscopy carried out on the Rb. sphaeroides alphaTyr and alphaTyr 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 alphaTyr-Tyr Phe-Tyr, and the Rps. acidophila genes studied in the present work (which have the sequence alphaPhe-Met; see Fig. 1), the aromatic residue at position alpha 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 ((max) = 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 (betaHis; Fig. 1) has not prevented the expression of a B800 pigment. Previous studies have shown that mutagenesis of betaHis 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 betaHis 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(y) 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 beta-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.



Functional Tests for Energy Transfer within the LH2 Complexes

The value of a system for the heterologous expression of bacterial LH complexes depends on a demonstration that these complexes are still functional, that is that they can harvest and transfer light energy. Fig. 5shows the near infra-red region of the fluorescence excitation spectra for membrane-bound complexes from the LH2-only strains DD13(pRKCBC1), DD13(pRKACID), DD13/G1(pRKACID), and DD13/G1(pRKGEL). This technique measures the ability of the 800 nm-absorbing pigments to transfer energy to the 850 nm pigments, from which fluorescence is detected at 900 nm. In the positive control in Fig. 5a (dashedline), it can be seen that the 800 nm-absorbing pigments within the Rb. sphaeroides LH2 are able to elicit emission at 900 nm from the B850 band, as expected, showing that energy is efficiently transferred from the B800 to the B850 pigments. In Fig. 5b (solidline) for the Rps. acidophila LH2 genes, strain DD13/G1(pRKACID), the presence of an excitation shoulder at 800 nm demonstrates that this absorption band (see Fig. 3c) plays a role in the production of an emission associated with a B850-like bacteriochlorophyll; thus, it appears that in this heterologously synthesized complex, some energy is transferred from the B800 to the pseudo-B850 pigments. As expected, given the almost complete absence of 800 nm-absorbing pigments in the DD13(pRKACID) sample (see Fig. 3d), there is no evidence for such energy transfer for the heterologous Rps. acidophila LH2 complex in the spheroidenone background (Fig. 5c, boldline). In Fig. 5d (dottedline), for the Rv. gelatinosus LH2 complex, the relatively larger excitation peak at 800 nm demonstrates that some B800 B850 transfer occurs. Thus, in this last case in particular, there are indications of some energy transfer from the B800 to the B850 pigments, although no attempt has been made to quantify the efficiency of this process. This would suggest that the presence of the ``correct'' carotenoid for the Rv. gelatinosus LH2 complex, the relative abundance of B800, and the presence of B800 B850 transfer are all interdependent.


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(x) band at 590 nm to the Q(y) 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.



A Functional Test for Energy Transfer between the Rps. acidophila LH2 Complex and the Native LH1-RC Core Complex

The normal role of the peripheral LH2 antenna is to transfer energy to the LH1-RC core complex. This process can be monitored by exciting the LH2 complex at various wavelengths, from 750 to 900 nm, while monitoring any fluorescence emitted from the LH1 complex at 920 nm. The membranes used in this experiment were prepared from strain DBC/G5(pRKACID), which contains the Rps. acidophila LH2 complex together with the native LH1-RC core complex. Fig. 7shows the results, with the excitation spectrum as a dashedline; this profile is closely matched by the (1 - T) spectrum (solidline), which reflects the absorbing species present, and the extent of the alignment between the two spectra suggests that the Rps. acidophila LH2 complex is able to transfer energy to the LH1 complex. A lack of energy transfer from the foreign LH2 species to the native LH1 complex would have resulted in the absence of a shoulder in the excitation spectrum between 850-860 nm, despite the presence of such a shoulder in the equivalent (1 - T) spectrum. As a control, the (1 - T) spectrum for DBC/G5 membranes (no Rps. acidophila LH2 complex present) is shown (Fig. 7, dottedline), in which the absence of any contribution from an LH2 complex between approximately 850 and 860 nm is clearly seen.


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).



Ultrastructural Analysis of Cell and Intracytoplasmic Membrane Morphology in Transconjugant Strains

It has been known for nearly twenty years that the absence of the LH2 complex can produce alterations in the shape of the Rb. sphaeroides cell, as well as distortions in the intracytoplasmic membrane (Lommen and Takemoto, 1978; Drews and Oelze, 1981; Golecki et al., 1991; Hunter et al., 1988; Kiley et al., 1988; Gibson et al., 1992). It was therefore important to see if the restoration of either the native or foreign LH2 genes could restore normal morphology, especially since the type and amount of membrane invagination could influence the level of complex in the cell. In previous work on the ultrastructure of strain DBC (Gibson et al., 1992), the normal spherical membrane invaginations associated with WT Rb. sphaeroides appeared to be tubular, and in this and other mutants this alteration seemed to be associated with the absence of the LH2 complex. Since this effect could be reversed by the addition of native pucBAC genes expressed in trans, it was therefore of interest to see if the presence of the Rps. acidophila LH2 complex could produce a similar effect. Electron micrographs of thin sections of cells from strains DBC/G5 and DBC/G5(pRKACID) were taken (Fig. 8). Strain DBC/G5, which contains LH1 and reaction center complexes but lacks LH2 (Table 1), shows gross alterations in cell shape (relative to the WT) in that the cells are highly elongated (Fig. 8a). Upon introduction of the Rps. acidophila LH2 genes into strain DBC/G5 to give strain DBC/G5(pRKACID), the cells retained their highly elongated shape, but the tubular intracytoplasmic membranes usually associated with this LH2-minus strain were replaced by membranes containing the spherical vesicles more usually associated with WT Rb. sphaeroides (Fig. 8b). In the control (Fig. 8c), the introduction of Rb. sphaeroides LH2 genes into the LH2 deletion strain restored both the normal membrane morphology and cell shape. It has been noted that membrane morphology is probably affected by the presence of both the PucC protein and the LH2 complex (Gibson et al., 1992). In the present context, the combination of the native genes encoding PucC and the foreign genes encoding Rps. acidophila LH2, as well as the low expression level of the Rps. acidophila LH2 complex (relative to WT) has led to an abnormal cell shape, but normal vesicles. It is possible that higher levels of LH2 in the cell would have conferred a normal shape on the cell; alternatively, the low level of foreign Rps. acidophila LH2 might have had little or no effect, and the normal vesicles seen in the DBC/G5(pRKACID) strain could have arisen from the expression of the pucC genes contained in the pRKCBC1 expression plasmid. This point warrants further investigation.


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.



Concluding Remarks

We have demonstrated that it is possible to heterologously express foreign LH2 genes from Rps. acidophila and Rv. gelatinosus in Rb. sphaeroides and that the complexes are largely intact and functional, as judged by the spectroscopy reported here. This is the first report of the expression of foreign light-harvesting genes in a photosynthetic bacterium; it raises some interesting questions about the specificity of interaction between carotenoid and bacteriochlorophyll pigments in light-harvesting complexes, about the protein binding sites of carotenoid molecules, and about the interaction of LH2 and LH1 complexes. The fact that the Rps. acidophila genes are expressed at all is remarkable, given that this is a silent gene pair in Rps. acidophila (Gardiner, 1992). (^6)There could be several reasons why the Rps. acidophila gene pair is silent in the native bacterium, including the absence of the appropriate promoter sequence, or the absence of other important genes, an example being pucC, which normally lies downstream of pucBA in Rb. sphaeroides and Rb. capsulatus and which is essential for the formation of LH2 complexes (Tichy et al., 1989, 1991; Lee et al., 1989; Gibson et al., 1992).

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.


FOOTNOTES

*
This work was supported by grants from the Biotechnology and Biological Sciences Research Council, the Wellcome Trust, and the EC. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Author to whom correspondence should be addressed. Tel.: 44-114-2824191; Fax: 44-114-2728697; g.fowler{at}sheffield.ac.uk

Recipient of a postgraduate studentship from the Science and Engineering Research Council.

(^1)
The abbreviations used are: LH, light-harvesting; WT, wild-type; RC, reaction center.

(^2)
A. T. Gardiner, R. C. Mackenzie, S. J. Barratt, K. Kaizer, and R. J. Cogdell, manuscript in preparation.

(^3)
G. J. S. Fowler, L. McMaster, and C. N. Hunter, unpublished results.

(^4)
G. J. S. Fowler, J. D. Olsen, and C. N. Hunter, unpublished results.

(^5)
G. J. S. Fowler and C. N. Hunter, unpublished results.

(^6)
A. T. Gardiner, R. C. Mackenzie, K. Kaizer, S. J. Barratt, A. J. Simmons, and R. J. Cogdell, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Prof. Gordon Lindsay for help with the Western blotting.


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