Mutants of Rhodobacter sphaeroides lacking ... - Wiley Online Library

Molecular Microbiology (1992) 6(9), 1173-1184

Mutants of Rhodobacter sphaeroides lacking one or more pigment-protein complexes and complementation with reaction-centre, LH1, and LH2 genes M. R. Jones, G. J. S. Fowler, L. C. D. Gibson, G. G. Grief, J. D. Olsen, W. Crielaard^ and C. N. Hunter* Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2UH, UK. Summary The photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigmentprotein complex: the photochemical reaction centre and its attendant LH1 and LH2 light-harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reactioncentre genes. The resulting system allows each pigment-protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either lightharvesting complex in R sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids in the assembly of the photosynthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophytls.

Introduction The facuttative photosynthetic bacterium Rhodobacter sphaeroides has a retativety simple photosystem consisting of three types of membrane-bound pigment-protein complex. The core photosynthetic reaction centre (RC) is surrounded by the LH1 antenna comptex, which in turn is surrounded by a peripheral LH2 antenna complex (Vos ef Received 11 November, 1991; revised and accepted 20 January, 1992. tPreseni address: Department of Microbiology, University of Amsterdam, Nieuwe Achtergracht 128, 1018 WS Amsterdam, The Netherlands. 'For con-espondence, Tel, (0742) 768555, ext. 4191; Fax (0742) 728697.

ai, 1986). The reaction centre in R. sphaeroides consists of three subunits — H, L and M — and is the site at which photochemistry takes place (Okamura etal., 1974; Feher et ai, 1989). The protein subunits bind four motecutes of bacteriochtorophytl (BchI), two of bactenopheophytin, and two of ubiquinone. The three-dimensionat arrangement of the pigment and potypeptide components of the reaction centre is now known to a high resotution (Atten ef at., 1987a,b). The antenna comptexes are responsibte for Increasing the surface area and range of wavetengths over which tight energy can be absorbed, and for channetting excitation energy to a 'speciat pair' of Bchls within the reaction centre. The LH1 antenna comptex is constructed from a basic unit consisting of two smatt transmembrane polypeptides (a and p) encoded by the pufA and pufB genes (Kitey etat., 1987). The LH1 antenna binds carotenoid and Bcht, the latter absorbing mainty at 875nm (Bchl875). The LH2 antenna comptex is made up from a basic unit which again consists of one a and one 3 potypeptide, this time encoded by the puoA and puoB genes (Ashby efa/., 1987; Kitey and Kaptan,1987). tt binds carotenoid and two types of Bcht, one of which absorbs at 800 nm (Bcht800) and the other at 850 nm (Bcht850). A number of studies (Sundstrom et ai, 1986; van Grondelte ef ai, 1987; van Dorssen ef ai, 1988; Bergstrom ef ai, 1989; Freiberg et ai, 1989) have ted to the formutation of a scheme for energy transfer through the R. sphaeroides antenna (see Fig. 1). Energy is transferred from LH2 to LH1 via a minor component. Bcht870, which can be resotved at tow temperature (4K) (van Dorssen ef ai, 1988). This minor component can be identified in strains which tack LH1 and reaction centres and sc is a genuine component of the LH2 antenna system rather than a product o^ the association of LH2 with LH1 (Vos etai, 1988; Hunter efa/., 1990). It is atso possibte that energy is transferred directty from Bcht850 in LH2 to Bchi875 in LH1 (Freiberg etai, 1988; 1989). Energy is transferred from LH1 to the reaction centre via the minor component Bcht896, which again can be resotved at tow temperature (Bott ef ai, 1981; Borisov ef ai, 1982; Kramer et ai, 1984; Sebban ef ai, 1985). Bchl896 is present in strains which possess only LH1 (Vos ef ai, 1988; Hunter ef a/., 1990). This sequence of energy-transfer events was confirmed using three chemical mutants of R. sphaeroides deficient in one or

1174

M. R. Jones etal.

BchlSOO

Fig. 1. Scheme for energy transfer through the R. sphaeroides antenna.

Bchl850

LH2

LHl-

more of the pigment-protein complexes, i,e. NF57 (LH2 only), M21 (LHl +RC only) and M2192 (derived from M21, LHl only) (Vos etai. 1988; Hunter etai. 1990). Although the sequence and rates of energy transfer through the R sphaeroides antenna have been investigated in detail, relatively little is known about the structural features of the antenna system which determine the energy levels of the various populations of BchI molecules; these are manifested as different absorbance and emission spectra. One way of investigating these structural features is to use the technique of site-directed mutagenesis to construct mutant complexes in which the energy of a particular BchI species has been perturbed by changes in the surrounding protein environment. Electron transfer within the reaction centre can also be studied using this technique (Coleman and Youvan, 1990). In order to express mutant complexes in a suitable genetic background it is necessary to construct deletion/ insertion mutants of R. sphaeroides in which the genes of either the put operon (coding for the LHl a and 3 and RC L and M subunit poiypeptides) and/or the pucBA genes (coding for the LH2 p and « subunits) have been deleted and replaced by an antibiotic-resistance cassette. Two such strains have already been described. In strain DBC1 the pucBA genes were deleted and replaced by a cassette conferring resistance to kanamycin (Burgess etal., 1989). This strain can be used to express wild-type or mutant LH2 genes in an LH1/RC background. In strain DPF2 the pufBALMX genes were deleted and also replaced by a cassette conferring resistance to kanamycin (Hunter etai, 1991). In this paper we report the construction of two additional deletion/insertion mutants and the further characterization of strain DPF2. In strain DD13, pufBALMX and pucBA have been replaced by kanamycin and streptomycin cassettes, respectively. DD13 is devoid of all three pigment-protein complexes and can be used to express either LH1, LH2 or reaction centres as the sole complex. In the latter case, this is the first observation of this reaction centre complex in situ in R. sphaeroides. In strain DBCil the pucBA genes have been replaced by a streptomycin cassette, yielding a mutant which is equivalent to strain DBC1 but which is genetically consistent with the pucBA deletion in strain DD13. A number of derivatives of these deletion/insertion mutants with deficiencies in carotenoid biosynthesis have also been isolated which have provided interesting data on the role of

carotenoids in the assembly and function of the antenna complexes. Results Construction and analysis of the deletion/insertion mutant DBCQ A mutant of R. sphaeroides devoid of the LH2 antenna complex was constructed in a manner similar to that already described for strain DBC1 (Burgess ef ai, 1989), with the important exception that a streptomycin cassette was used in place of a kanamycin cassette. Briefly, a 1.1 kb region of ft sphaeroides DNA extending from the BamHI site upstream of pucB to the second SamHI site downstream of pucA was excised from plasmid pSCBCWT, and replaced with the 2 kb streptomycin/ spectinomycin cassette, Q (Prentki and Krisch, 1984). The resulting construct (plasmid pSCBCBfl) was transferred to ft sphaeroides NCIB 8253, and transconjugants were selected on the basis of resistance to streptomycin. Suitable colonies were further tested for the absence of the characteristic absorbance peaks of the LH2 antenna complex at 800 and 850nm (Fig. 2), All colonies examined displayed a major peak at 875 nm because of the Bchl875 of LHl (which appears as a shoulder in the wild-type spectrum (Fig. 2)), and minor peaks at 800 and 760 nm attributable to the reaction centre. The strain selected by this procedure was named DBCii. Plasmid pSUP202 is unable to replicate in ft sphaeroides and hence transconjugants which acquired streptomycin resistance and lost the LH2 complex must have done so following double homologous recombination, with the streptomycin cassette integrating into the chromosome in place of pucBA. Southern hybridization confirmed the presence of the i i cassette in a genomic digest (Fig. 3D; lane 3), and the absence of pucBA genes (Fig. 3A; lane 3) and pSUP202 (not shown). A spontaneous green-coloured derivative of DBCIi was subsequently isolated (DBC11/G5),

Analysis of the deletion/insertion strain DPF2 Strain DPF2 is a mutant which lacks the LH1/RC core complex but still has the LH2 complex. Details of this construction have been given elsewhere (Hunter ef ai. 1991). In brief, an approximately 3.2kb region of DNA extending from an engineered SamHI site proximal to

Deletion/complementation system for bacterial photosynthetic genes puc operon

1175

puf oparon

DBCft

0PF2

KmR

DD13/G1

700

950

Fig. 2. Near-infrared absorption spectra of membranes of wild-type and deletion/insertion mutant strains. The arrangement ot deletion/insertion mutations in the genomic DNA is shown in diagrammatic form. Only genes relevant to the subunits of the pigment-protein complexes are shown.

pufB down to the Sal\ site distal to pufXwas replaced by a 1.25 kb kanamycin cassette. The resulting construct in the vector pSUP202 (plasmid pJOl) was transferred into R sphaeroides wild-type strain NCIB 8253 by conjugative transfer from Escherichia coii strain 17-1. Kanamycinresistant transconjugants were screened for the absence of the characteristic LH1 absorbance peak at 875 nm (Fig. 2) and the inability to grow under photoheterotrophJc conditions. Southern blot analysis of strain DPF2 demonstrated the presence of the kanamycin-resistance gene (Fig. 3C; lane 2) and the absence of the pufL gene (Fig. 3B; lane 2) and piasmid pSUP202 (not shown). Strain DPF2 retained genomic copies of the bchA and pufQ genes, both of which are required for bacteriochlorophyll synthesis. These genes are located upstream of the pufBALtviX cluster but lie wholly outside the deleted region (Hunter et ai, 1991).

hybridization, which confirmed the presence of the kanamycin and streptomycin cassettes (Figs 3C and 3D; lane 4) and the absence of the pufL and pucBA genes (Figs 3A and 3B; lane 4) and pSUP202 (not shown). A spontaneous green-coloured derivative of DD13 was subsequently

Km

Construction and analysis of the double-deletion/ insertion mutant, DD13

20—

(A) puc

(B) pufL

20— 1 6—

20 1-6

10— 05 —

10 0-5-

(0

1-6-

A derivative of strain DPF2 also devoid of the LH2 antenna complex was constructed by mating plasmid pSCBCBIl into strain DPF2. Transconjugants were selected initially on the basis of resistance to both kanamycin and streptomycin, and subsequently screened for the lack of LH1, LH2 and reaction-centre absorbance peaks and an inability to grow photosynthetically. The selected transconjugant (named DD13; Fig. 2) was analysed by Southern

10 — 0-5 —

1

2

(D)

1

2

3

Sm

2

3

20 16 10 0-5

Rg. 3. Southern hybridization of genomic DNA (2)ig per lane) from wild-type and deletion/insertion mutan! strains. Lanes; 1. wild type; 2, DBCil; 3, DPF2; and 4, DD13. All genomic DNA was digested with SamHI. Probes used were a 420Dp BamH\-Kpn\ pucBA fragment (panel A); a 850bp Xbat-Sall pufL fragment (panel B); a 2.0kb EcoRI Sm" cassette (panel C); and a 1,25kb Psri Km" cassette (panel D).

1176 M. ft Jones etal. Fig. 4. Engineered R. sphaeroides DNA sequences used to complement pt*f and puc operon deletions. R. sphaeroides DNA was cloned into the broad host-range vector pRK415. Asterisks denote restriction sites that have been introduced by site-directed mutagenesis. Brackets denote sites that have either been removed by digestion with the appropriate enzyme foUowed by blunting and religation, or have been destroyed during the cloning procedure. Only genes relevant to the subunits of the pigment-protein complexes are shown.

(at LH1 expression vector pRKEKI

in pRK415

(b) LH2 expression vector pRKCBCI

(Bs)

(Bs)

(B)

lnpRK415

500 bp

isolated (strain DD13/G1) and a spontaneous whitecoloured derivative of DD13 was also isolated (DD13/W1). Strain DD13/W1 lacked coloured carotenoids (see below).

Construction of vector systems for the expression of altered light-harvesting complexes in deletion backgrounds The vector system used for the expression of wild-type and altered LH1 complexes in trans in strains containing genomic deletions in the puf operon was based upon a 1.8kb 5coRI-Kpnl fragment of the puf operon and the mobilizable broad host-range vector, pRK415 (Fig, 4a). In order to be able to subclone the pufBA structural genes for the purposes of site-directed mutagenesis, a SamHI site was engineered 73 bp upstream of the start of the pufB gene, as described by Hunter etal. (1991), and an Xba I site was created by engineering changes (T-^C and C—-T) at positions 61 and 60 bp upstream of the first base of the start codon for pufL (Fig. 4a). In order to clone the 1.8kb EcoR\-Kpn I fragment bearing these changes into plasmid pRK415 in such a way that the engineered SamHI and Xba I sites were unique within the final construct, pRK415 was digested with EcoR! and H;ndlll and then ligated to the 1,8kb £coRI-Kpnl fragment in a two-stage process. In the first step, the matching EcoR\ ends of the two DNA fragments were ligated together. The remaining free H/ndlll and Kpnl ends were then made blunt by filling-in and joined together in a second ligation reaction. The pufBA genes can be transferred between the resulting plasmid (named pRKEKI) and plasmids used for mutagenesis (M13mp18 and pBluescript) as a 480bp SamHI-X5al fragment. The 1.8kb EcoRI-Kpnl fragment bearing the unique SamHI and Xba\ sites was also cloned into a 6.8kb EcoRI-H/ndlll fragment encompassing the whole of the

puf operon, replacing the corresponding wild-type £coRI-Kpnl fragment. The resulting plasmid (pRKEHI) was used to restore both LHl and reaction-centre complexes to puf operon deletion strains. The vector system constructed for LH2 was also based on pRK415, together with a 3.8kb SscI fragment of ft. sphaeroides DNA encompassing pucSA (Fig. 4b). A Kpnl site was created by engineering changes (T—•G and T—*G) at positions 20 and 21 bp upstream of the first base of the start codon for pucB. To make the BamHI site located approximately 70bp downstream of the 3' end of pufA unique within the 3.8kb SscI fragment, the BamHI site located upstream of the puc operon was removed by restriction enzyme digestion, filling-in of the recessed termini and religation of the blunted ends. The 3.8kb SscI fragment altered in this way was transferred to pRK415 by excising it as a SscI fragment, filling-in the recessed termini, and blunt-end ligating it to pRK415 which had been digested with EcoRI and H/ndlll and had had its recessed termini filled-in. In the resulting plasmid (named pRKCBCI), the pucBA genes can be manipulated as a 420bp Kpnl-BamHI fragment (Fig. 4b). Complementation of strains DPF2, DBCQ and DD13/G1 with light-harvesting genes Strains DBCn, DPF2 and DD13/G1 were complemented in trans with the pufBA or pucBA genes using the expression vectors pRKEKI and pRKCBCI (see the Experimental procedures). As reported previously for strain DBC1 (Burgess et aS.. 1989), complementation oi strain DBCH with the 3.8kb SscI fragment of ft. sphaeroides chromosomal DNA encompassing the pucBA genes plus their promoter region (plasmid pRKCBCI) gave rise to transconjugant colonies exhibiting a near-infrared absorbance spectrum (Fig. 5a) similar to that of the wild type (Fig. 2). However it was noticeable that the 875 nm

Deletion/complementation system for baeterial photosynthetie genes

1177

puf operon

plasmid borne engineered puc operon

genomic DNA

[ R ] A)

plasmid borne engineered puf opeion

.//•

T

] B |A

genomic DNA plasmid tiorne engineered puc operon

genomic DNA plasmid borne engineered put operon

700

950 nm

Fig. 5. Near-infrared absorption spectra ot membranes from deletion/inserlion mutants complemented with expresston vectors bearing LH1 or LH2 genes, a, DBCK complemented with pRKCBCl; b, DPF2 complemented withpRKEKI; c, DD13/G1 complemented with pRKCBCI; and d, DDt3/G1 complemented with pRKEKI,

shoulder attributed to the LH1 complex was reduced in these transconjugants, suggesting overproduction of the LH2 connplex. This was possibly due to a copy-number effect, as multiple copies of the pueBA genes are present in these transconjugants. The actual copy number of these pRK415-based plasmids was not determined, but it is likely to be in the region of four to six, in accord with previous observations on pu^ operon expression vectors constructed using the closely related plasmid pRK404 (Davis ef ai, 1988). The overproduction of LH2 was not dependent on the orientation of the 3.8kb Bse\ fragment in pRK415, and hence the overproduction was not due to constitutive expression of the pueBA genes driven by the tet and/or tac promoter of pRK415. The ability of these transconjugant strains to grow under photoheterotrophic conditions was not impaired by the overproduction of the LH2 complex. Complementation of strain DPF2 with a 1 .Bkb chromosomal fragment encompassing pufBA plus their promoter region (plasmid pRKEKI) but not pufLMX yielded transconjugant strains which exhibited an apparently wild-type spectrum (Fig. 5b), The relative absorbance values at 850 and 875 nm provided no indication of any overproduction of the LH1 complex despite the presence of multiple

copies of the pufBA genes. As expected, these transconjugants would not grow under photoheterotrophic conditions, demonstrating the absence of reaction-centre complexes. Complementation of strain DD13/G1 with the 3.8 kb puc fragment or the 1.8 kb pu^fragment gave rise to transconjugants that exhibited a spectrum that was characteristic of either the LH2 complex (Fig, 5c) or the LH1 complex (Fig. 5d).

Complementation of strains DPF2 and DD13/G1 with reaction-centre and LH1 genes Strains DPF2 and DD13/G1 were complemented in trans with a 6.8kb EcoRI-H/ndllt fragment encompassing pufBALMX plus promoter regions (plasmid pRKEHI). In the former case, transconjugants exhibited a wild-type spectrum (Fig. 6a) and were capable ot photoheterotrophic growth. In the latter case, transconjugants exhibited an LH1 plus reaction-centre spectrum (Fig, 6b), with the 800 nm absorbance peak attributed to the monomeric bacteriochlorophylls of the reaction centre clearly visible as a minor component. These transconjugants were also capable of photosynthetic growth.

1178

M. R. Jones eial puc operon

genomic DNA

puf opef on

KmB

plasmid borne engineeied put ope'on

—

genomic DNA

b — plasmid borne engineered pul ope'on

I

C —

genomic DNA

Km"

plasmid borne engineeied pul opefon

I

genomic DNA

KmR

d — plasmid borne engineered pul opefon

Fig. 6. Near-infrared absorption spectra of membranes from deletion/insertion mutants complemented with expression vectors bearing LH1/RC genes, a, DPF2 complemented with pRKEHl; b, DD13/G1 complemented with pRKEHI; c, DPF2 complemented with pRKEH2; and d, DD13/G1 complemented with pRKEH2.

In addition to providing a means of transferring LH1 genes between plasmids, the engineered SamHI and Xbai restriction sites which flank pufBA in plasmid pRKEHI (see above) provided the opportunity to construct a puf operon which lacked the pufBA genes. Complementation of strain DD13/G1 with such an operon would be expected to yield transconjugants in which the reaction centre was the sole pigment-protein complex. Accordingly, plasmid pRKEH2 was constructed from pRKEHI by deletion of the region encompassed by the unique SamHI and Xba\ sites (achieved by digestion with Sam HI and Xba I followed by filling of the recessed termini and blunt-end ligation). Thus pRKEH2 lacked 477 bp of DNA including the entire coding sequence for both pufB and pufA. When strain DPF2 was complemented with this plasmid the transconjugants exhibited an absorbance spectrum similar to that seen for *LH2-only' strains (Fig. 6c c.f. Fig. 5c), but on this occasion the cells were capable of photoheterotrophic growth, demonstrating the presence of reaction centres. When strain DD13/G1 was complemented with pRKEH2, the transconjugants exhibited the characteristic reaction-centre spectrum with peaks at approximately 760, 800 and 860 nm (Fig. 6d). These cells

were also capable of photoheterotrophic growth despite the complete lack of antenna complex. However growth was considerably slower than that of wild-type cells under the same conditions of relatively low light intensity, doubling times being 12 h for strain DPF2(pRKEH2) and 4 h for the wild type. The doubling time of strain DPF2(pRKEH2) was approximately 8h under these conditions. The accelerated rate of growth relative to strain DD13/G1(pRKEH2) can be accounted for by transfer of energy from the LH2 complex to the reaction centre, a process which would normally require the presence of LH1 (Vos ef ai, 1986). Thus it appears that LH2, which does have some similarity with LH1 (at least at the level of primary sequence), can interact productively with the reaction centre. The demonstration of a reaction-centre spectrum in Fig. 6d provides the first absorbance profile of the R. sphaeroides complex in a native environment. Thus we have shown that reaction centres, which are normally closely associated with LH1 complexes at the levels of both genetic and physical organization, can assemble as a functional complex in the complete absence of a light-harvesting system.

Deletion/complementation system for bacterial photosynthetic genes

1179

more sharply defined peaks at approximately 454 and 485nm in DD13/G1/LH1, whilst with the DD13/W1/LH1 only a single peak was seen at 411nm. Preliminary indications are that the major carotenoids in DD13/Wl are phytoene, ^-carotene and phytofluene and that those in DD13/G1 are neurosporene plus the methoxy- and hydroxy-derivatives (R. J. Cogdell, personal communication). The absorbance peaks of the LHl bacteriochlorophylls of strains DD13//LH1, DD13/G1/LH1 and DD13/ W1/LH1 were at 876, 874 and 868nm, respectively (see inset. Fig. 7). This point deserves further investigation, but it is clear from these values that the carotenoids within LHl exert some modifying effect on the absorbance properties of the major bacteriochlorophyll species of the LHl complex.

400

500

600

700

800

900

When the double-deletion/insertion strains were complemented with plasmid pRKCBCI the results were more complex. Appreciable levels of the LH2 complex were detected in both the DD13 and DD13/G1 transconjugants, indicating that LH2 could assemble in the presence of both green and brown carotenoids (data not shown). However, in the DD13/W1 transconjugants only very low levels of LH2 were detected in cells grown aerobically in the dark on either solid or liquid medium. This result emphasizes an important difference between the LH1 and LH2 complexes; correct assembly of the LH2 complex is more strongly dependent on the carotenoid species available. The factors governing expression of the pucBA genes and assembly of the LH2 complex in these different carotenoid backgrounds are currently under more detailed investigation.

Wavelength (nm) Fig. 7. Absorption spectra of membranes from double-deletion mutants complemented with LHl genes.

Complementation with light-han/esting genes in different carotenoid backgrounds The isolation of spontaneous carotenoid mutants DD13/ GI and DD13/W1 from the brown-coloured strain DD13 gave rise to the opportunity to study the properties of light-harvesting complexes containing different types of carotenoid molecules. When the three strains were complemented with LHl genes using plasmid pRKEKI, LHl complexes exhibiting the characteristic single absorbance peak in the near-infrared were seen to assemble in all three carotenoid backgrounds (representative strains are referred to as DD13//LH1, DD13/G1/LH1 and DD13/W1/ LHl). LHl was present at comparable levels in all three strains. The differences in absorption due to the carotenoids were most clearly seen in spectra of membranes prepared from the transconjugant strains (Fig. 7). The broad absorbance peak in the 430-580 nm region of the spectrum in membranes from DD13//LH1 was replaced by

Discussion A system for the analysis of protein function by site-directed mutagenesis requires three major components: a sequenced target gene cloned into vectors suitable for the introduction of site-directed changes, a deletion/insertion strain to provide a null genetic background, and mobilizable vectors which facilitate complementation of the deletion strain with altered genes. Within the context of the photosynthetic bacteria the most notable application of this technique has been in the study ofthe photosynthetic reaction centre in ft sphaeroides and Rhodobacter capsulatus. These investigations have revealed a number of features associated with fast electron transfer from the special pair to the primary acceptor quinone (QA) (Coleman and Youvan, 1990; Kirmaierefa/., 1991; Mattoliefa/., 1991), and reduction and protonation of the secondary acceptor quinone (QB) (Paddock ef al., 1990; Takahashi and Wraight, 1990). They have been greatly assisted by the availability of an X-ray crystal structure for the Rhodopseudomonas viridis (Diesenhofer and Michel, 1989) and ft. sphaeroides (Allen ef al., 1987a,b) reaction centres.

1180

M. R. Jones etal.

These crystal structures allow potentially important residues to be identified with precision in a protein of several hundred residues and aid the interpretation of results from site-directed studies. A number of groups have described the construction of deletion/insertion mutants in thepu/operon (Youvanefa/., 1985; Farchaus and Oesterhelt, 1989; Chen etai, 1988; Davis ef af., 1988; Takahashi et ai, 1989; Paddock e( ai, 1989; Nagarajanefa/.. 1990; Hunter ef a/., 1991) and puc operon (Youvan et ai, 1985; Burgess et ai, 1989) of R. capsulatus and R. sphaeroides. In most cases the mutant will allow an altered reaction-centre or light-harvesting complex to be studied in cells and membranes which also possess the remaining pigment-protein complexes. The exception is the R. capsulatus U43 strain of Youvan et al. (1985) which has a kanamycin-resisfance cassette in place of the puf operon and a point mutation in the puc genes. This strain can be used to study altered reactioncentre or LH1 complexes in the absence of the LH2 spectral complex, although recent pulse-chase experiments have clearly demonstrated that the LH2 « polypeptide is present in intracytoplasmic membranes from this strain and in transconjugants arising from it (Stiehle et ai, 1990), The deletion/insertion mutants and plasmids described in this paper constitute the most comprehensive cassette mutagenesis system for the study of the bacterial photosynthetic apparatus to be constructed to date. Strain DD13 and its green and white derivatives are the first deletion/insertion mutants to be described with cassettes in both the puf and puc operons. Plasmids pRKEKI, pRKEHI and pRKCBCI together with the DD13 deletion strain allow either the LHI or LH2 complex to be studied in intact cells or membrane preparations as the sole pigment-protein complex or. in the case of LHI. in conjunction with the reaction centre. We can also study the effect of different types of carotenoid on the assembly and function of these complexes through the differently coloured deletion mutants. The absence of the genomic copies of both the puc operon and pu^operon ensures that the properties of the plasmid-borne light-harvesting complex cannot be influenced by the presence of polypeptides of the second light-harvesting complex; in strains of R. capsulatus devoid of both the LH1 and LH2 complex by virtue of multiple point mutations in the LHI and LH2 structural genes, pulse-chase experiments have shown the presence of both LH1 and LH2 potypeptides in the bacterial membrane, despite the absence of the LHI absorbance peak at 875 nm and the LH2 absorbance peaks at 800 and 850nm (Stiele etai, 1990). This olearly demonstrates that the absence of a characteristic absorbance peak does not provide evidence for the absence of the components of an antenna complex. In the light of this, a genetic background in v\/hich the structural genes coding

for the polypeptides of unwanted complexes have been deleted from the genome is preferable. Strain DPF2 can be used to construct transconjugants with either an LH1-HH2 or RC+LH1-i-LH2 phenotype. allowing alterations in the LH1 complex to be examined as part of a pseudo-wild-type antenna or an antenna devoid of reaction centres. Strain DBCU is similar to strain DBC1 described previously (Burgess et al., 1989), but carries a streptomycin rather than a kanamycin cassette in place of the LH2 genes. It has been constructed in order to be consistent with strain DD13 vi/hich also has a streptomycin cassette in the puc operon. Complementation of strain DBCn or DBC1 with plasmid pRKCBCI yields transconjugants with an RC-I-LH1+LH2 phenotype, allowing altered LH2 complexes to be studied as part of an intact photosystem. In R. capsulatus. the puc operon consists of the pucBA genes together with the pucCDE genes which are located downstream of the 3' end of pucA (Tichy ef ai, 1989), Unequivocal evidence for the existence oi pucCDE in R. sphaeroides has not been published to date, but it is known that interruption of the region immediately downstream of pucA leads to loss of the LH2 spectral complex (Lee etai, 1989). Furthermore, homologies in the DNA sequence immediately downstream of pucA indicate that the open reading frame designated pucC is also present in the latter species (L. C. D. Gibson and C. N. Hunter, unpublished). The probable start position of the pucC gene in R. sphaeroides lies downstream of the BamHI site which lies distal to pucA and which marks the 3' end of the puc deletion in strains DBCi i, DBC1 or DD13. Hence the puc deletion does not extend into the probable coding region of the pucC gene in any of these strains, although transcription of this gene could be interrupted. The engineered SamHt and Xba\ restriction sites which flank the pufBA genes provided an opportunity to construct a mutant that possessed reaction centres as the sole pigment-protein complex. The ability of the resulting transconjugants to grow under photoheterotrophic conditions indicates that the reaction centres in these cells were correctly assembled and functional despite the complete absence of LHI and LH2 antenna complexes. This is in general agreement with findings with R capsulatus {ByWna et ai, 1988; Dorgeetai, 1990) using mutants that cannot assemble both the LHI and LH2 antenna complex because of point mutations in the structural genes for the LH1 complex and in the pucC gene which is required for synthesis of the LH2 complex (Tichy et ai, 1991). The strain described here differs from these R. capsulatus strains in that membranes of the latter contain component polypeptides of both the LH1 and LH2 complexes, although they lack the assembled antenna complexes. Recent results further indicate that reaction centres can assemble in both the brown (DD13) and colourless (DD13AW1) carotenoid backgrounds in the

Deletion/compiementation system for bacterial photosynthetic genes 1181 Table 1. Bacterial strains and plasmids. Strain/Plasmid

Relevant characteristics

Source/Reference

supE44. MacU169. (