INTRODUCTION

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Purple nonsulfur bacteria such as Rhodobacter capsulatus are able to grow chemotrophically or phototrophically. Photosynthesis is anaerobic, and the components of the photosynthetic apparatus are formed gratuitously in the dark in response to oxygen limitation (5). An intracytoplasmic membrane system (ICM), contiguous with and derived from the cytoplasmic membrane, contains the apparatus necessary to sustain photosynthetic growth. In R. capsulatus the photosynthetic machinery includes the light-harvesting I (LHI) and II (LHII) complexes B875 and B800-B850, respectively, and the reaction center (RC) complex, each of which contains bacteriochlorophyll a (Bchl) and carotenoid pigments (15).

The LHI antenna complex contains two polypeptide subunits,  and , each spanning the ICM once due to a hydrophobic central region, which are encoded by the pufA and pufB genes. Conserved His residues bind Bchl within the transmembrane segments of and . The currently understood mechanisms of in vivo and in vitro assembly of the LHI complex were reviewed recently (14, 23). Strains of R. capsulatus defective in Bchl synthesis exhibit enhanced turnover of LH peptides, and binding of Bchl is believed to be essential for the proper membrane insertion of photosynthetic pigment-binding proteins (15). In one report on site-directed LHI peptide mutants, several (especially N-terminal) residues were implicated in appropriate pigment binding or complex assembly, and some mutations seemed to affect the structure of the RC (3). In other studies, site-directed mutations resulted in increased degradation rates of LHI peptides, and a model has been proposed in which electrostatic interaction between the negatively charged N terminus of the  polypeptide and the positively charged N terminus of the  polypeptide is a prerequisite for assembly of the LHI complex (14).

Following or concurrent with in vivo assembly in the R. capsulatus ICM, the LHI complex closely associates with the RC complex and efficiently transfers light energy to the RC. Chemical cross-linking experiments indicated a close association between these two complexes in the ICM of R. capsulatus (26), and it is likely that the LHI complex forms a ring around the RC (12, 33).

Little is known about proteins that might interact with LH complex components to facilitate membrane insertion of protein subunits, delivery of Bchl, complex assembly, or stabilization in the ICM. It is known that mutations of two homologous genes, pucC and ORF1696, result in analogous reductions in the LHII and LHI complexes, respectively and specifically. Although pucC mutants completely lack the LHII complex (15), secondary mutations in pucC deletion strains result in partial restoration of the LHII spectrum (21, 22). It was found that disruption of the open reading frame ORF1696 (which is located immediately 5' of and cotranscribed with the puhA gene; see Fig. 1) reduced the LHI complex steady-state level in the ICM of R. capsulatus (6, 41). However, it was not clear to what extent the consequences of ORF1696 disruption were due to cis or trans effects. Furthermore, if the ORF1696 gene product really is required to obtain maximal levels of the LHI complex, an important question is whether the ORF1696 protein acts to inhibit LHI complex turnover or to enhance LHI assembly.

In this paper we report the results of disruption-complementation analyses of several ORF1696 mutants generated by insertion of antibiotic resistance cartridges at different sites within ORF1696. We address the question of the function (assembly versus stabilization) provided by the ORF1696 protein in kinetic analyses of LHI assembly and decay rates in an ORF1696 mutant compared to the parental ORF1696⁺ strain.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The wild-type R. capsulatus strain SB1003 (39), the ORF1696 mutant ZY1 (6), and the expression plasmid pJAJ9 (19) were described previously. The Escherichia coli strain C600 r m⁺ (9) was used for the routine cloning of plasmids. Strains SM10 (30) and HB101(pRK2013) (11) were used for the diparental and triparental conjugational transfer of plasmids, respectively, into R. capsulatus. Other bacterial strains and plasmids used in this study are described below.

Growth conditions and media. E. coli cultures were grown in Luria-Bertani medium (28) at 37°C. Antibiotics were added as required at the following concentrations (in micrograms per milliliter): ampicillin, 200; kanamycin sulfate, 50; tetracycline-HCl, 10; and trimethoprim, 40. R. capsulatus cultures were grown in RCV minimal medium as described previously (22) with antibiotics added as required in the following concentrations (in micrograms per milliliter): kanamycin sulfate, 10; tetracycline-HCl, 0.5; and spectinomycin-2HCl, 10.

In vitro DNA techniques and plasmid constructions. Restriction endonuclease digestion, DNA ligation, agarose gel electrophoresis, transformation of E. coli, and other recombinant DNA procedures were carried out essentially as described previously (28).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   (A) Restriction map of ORF1696 and flanking genes found in an 8-kb region of the photosynthesis gene cluster of the R. capsulatus genome (2). Abbreviations: B, BamHI; Bc, BclI; E, EcoRI; H, HindIII; M, MunI; N, NaeI; P, PstI; St, StuI; X, XhoI. (B) Schematic illustration of ORF1696 and flanking genes with sites of antibiotic resistance cartridge insertion. Solid lines below the genes represent antibiotic resistance cartridge DNA molecules. Stem-loop symbols represent transcriptional terminators. Dashed lines indicate the site of cartridge insertion in ORF1696. The direction of transcription is from left to right, and the puhA promoter (puhAp) is indicated by the bent arrow.

Strain construction. Plasmids were mobilized by conjugation from E. coli into the R. capsulatus gene transfer agent (GTA) overproducer strain DE442 (38) by use of the mobilizing vector pDPT51, present in E. coli Tec5, as described previously (32). The ORF1696::Km^r insertions in plasmids pCYNAE and pCYMUN were transduced into the R. capsulatus puc operon deletion strain, LHII (22), by GTA-mediated interposon mutagenesis as described previously (37). GTA filtrate containing the ORF1696::Km^r insertion between the StuI sites was a gift from C. Bauer's lab. The ORF1696:: insertion in plasmid pRR3 was transduced into the R. capsulatus LHII mutant strain, MW442 (29). The ORF1696 mutants of LHII were named according to the site(s) at which the Km^r (or ) cassette was inserted in the ORF1696 gene, i.e., Nae, Stu, Stu::, and Mun. Thus, the Km^r cartridge was inserted 1,392, 411, and 87 nucleotides downstream of the putative ATG start codon for ORF1696 in strains Nae, Stu, and Mun, respectively. The Mun and Stu insertions are upstream of the reported site of the puhA promoter, puhA_p (6), and the Ble^r gene segment is translationally out of frame with the 3' segments of ORF1696. The Nae insertion is downstream of puhA_p and also creates a translationally in-frame fusion between the remaining 60 nucleotides of ORF1696 and a 5' segment of the Ble^r gene on the SmaI fragment of the KIXX Km^r cartridge (4).

LHI decay kinetics experiments. Strains LHII and Stu were grown to early stationary phase (absorbance at 650 nm [A₆₅₀] = 1.5 to 2.0) in RCV medium under anaerobic, photosynthetic conditions in 900-ml Roux bottles. Zero hour samples were removed from these cultures, and the cells were pelleted by centrifugation and stored at 80°C for later analysis. Portions (500 ml) of the photosynthetic cultures were used to inoculate 9.5 liters of RCV medium in a 20-liter fermenter with aeration maintained at or near 20% partial O₂ pressure by sparging with air at a constant rate of 3 liters/min as well as by automatic adjustment of the impeller revolutions-per-minute value. Triplicate samples were removed every 2 h, and the cells were pelleted by centrifugation and stored at 80°C prior to analysis.

LHI assembly kinetics. Strains LHII and Stu were grown under highly aerated conditions in 100 ml of RCV medium in a 1-liter Erlenmeyer flask incubated in a gyratory shaker at 300 rpm to an optical density of 2.0 to 2.5 A₆₅₀ units. A zero hour sample (5.0 ml) was removed from these highly aerated cultures, the remaining portion (95 ml) of the cultures was used to inoculate 700 ml of fresh RCV medium in separate 1-liter flasks, and the flasks were incubated at 150 rpm in a gyratory shaker. Samples were removed from these semiaerobic cultures every 30 min, and the cells were pelleted and stored at 80°C.

Analytical methods. All measurements were done on a minimum of two independent cultures and were highly reproducible (10 to 15% variation). For spectroscopy, cell samples were resuspended in 24% bovine serum albumin in RCV medium and analyzed for pigment-protein complex levels as previously described (22). LHI complex levels were determined as the integrated area under the absorbance peak at 875 nm, normalized to cell numbers by multiplying spectra by a factor to give an A₆₅₀ (due to light scattering) of 0.2, with SpectraCalc and GRAMS 386 software packages (Galactic Industries Corp.). The contribution of the RC to the 875-nm peaks was assumed to be negligible. In the kinetics experiments, the values of the A₈₇₅/A₆₅₀ ratios obtained were plotted graphically as a function of time, and the slopes of the resultant lines were calculated. Total cellular Bchl content was determined by acetone extraction as described previously (31). The -galactosidase activities were determined as described previously (22).

	RESULTS

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ORF1696 gene disruption-complementation experiments. Disruption of ORF1696 with a Km^r cartridge resulted in a decrease in LHI antenna complex levels in strains ZY1 and ZY3, but it was not clear if this phenotype was due solely to the loss of an ORF1696 gene product since trans-complementation experiments were not done (6). Therefore, the plasmid pCY42 (which contains the ORF1696 gene transcribed from the puf promoter) was introduced into ZY1, and the absorption spectroscopy profile of intact cells of this strain, grown under semiaerobic conditions, was compared with those generated from strain ZY1(pJAJ9) and the wild-type strain SB1003(pJAJ9) grown under the same conditions. The LHI complex level in ZY1(pCY42), manifested as a shoulder on the long-wavelength slope of the 850-nm LHII absorbance peak, was restored nearly to that of the wild-type strain (40). This trans-complementation experiment indicates that the ORF1696 gene product is required to obtain the wild-type level of the LHI complex in an LHII⁺ background.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Absorption spectra obtained with intact cells grown under semiaerobic conditions. (A) Parental strain LHII(pJAJ9) and the Kmr disruption mutants Nae(pJAJ9), Stu(pJAJ9), and Mun(pJAJ9). (B) Parental strain LHII(pJAJ9) and pCY42-complemented Nae, Stu, and Mun strains. (C) Strains Mun and Stu compared with Stu::.

Effect of a Km^r cartridge disruption of ORF1696 on the transcription and translation of a pufB::lacZ fusion. Northern blot analysis demonstrated that a ORF1696 Km^r disruption mutation had no effect on the levels of pufBA mRNA, and so the ORF1696 protein should not modulate transcription of the puf operon or mRNA decay (6). We tested the possibility that the ORF1696 protein acts to regulate pufB gene expression at the level of transcription and/or translation by introducing plasmid pXCA::935 (1), which encodes a pufB'::lac'Z translational fusion driven by the puf promoter, into strains LHII and Stu. After growth under semiaerobic conditions, the -galactosidase activities obtained with LHII(pXCA::935) were 754 (±44) U and with Stu(pXCA::935) were 677 (±2) U. This experiment confirms that the ORF1696 protein does not significantly affect transcription of puf genes and indicates that it does not modulate translation of pufB mRNA. Thus, the ORF1696 protein regulates LHI complex levels at a posttranslational level.

Kinetic analyses of LHI formation and decay. In principle, the ORF1696 protein could function to stabilize (e.g., protect from proteolysis) the otherwise-assembled LHI complex in the ICM or it could be a catalyst to assemble the LHI complex from the - and -polypeptide subunits and pigment molecules. We differentiated between these two possibilities by kinetic analyses of the LHI complex formation and the decay in the LHII and Stu strains.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Representative graphs of growth (A) and of LHI assembly (B) over time for Stu (dashed line) and LHII (solid line) strains. Inocula were grown under highly aerated conditions and then transferred to growth conditions with reduced aeration to induce LHI synthesis.

View larger version (13K): [in this window] [in a new window]   FIG. 4.   Representative graphs of culture growth (A) and of LHI decay (B) over time for Stu (dashed line) and LHII (solid line) strains. Inocula were grown under anaerobic-photosynthetic growth conditions and then transferred to highly aerated growth conditions to repress LHI synthesis.

	DISCUSSION

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ORF1696 is an open reading frame encoding 477 amino acids that is located 3' of the bchFNBHLM genes and immediately 5' of the puhA gene (Fig. 1) within the photosynthesis gene cluster of R. capsulatus (2). Transcription of these genes initiates at two promoters, one located upstream of the bchF gene and another located within ORF1696 (6). Hydropathy analyses of the primary amino acid sequence of ORF1696 show it to be very hydrophobic, and gene fusion experiments indicate that it is an integral membrane protein with 12 transmembrane segments (40). Therefore, the ORF1696 protein is likely to be located in the ICM, which contains the LHI antenna complex.

Our data demonstrate that Km^r interposon mutations of the ORF1696 gene in R. capsulatus Nae and Stu reduced the amount of the LHI complex and were complemented in trans with a plasmid-borne copy of ORF1696 (Fig. 2). The -galactosidase activities of Stu and LHII mutants harboring plasmid pXCA::935, in which the lac'Z gene is fused translationally in frame with pufB and transcribed from the puf promoter, were similar, indicating that pufB mRNA transcription and translation are not significantly affected by the mutation in ORF1696. Therefore, restoration of the LHI complex in the Stu(pCY42) and Nae(pCY42) strains to near-normal levels shows that a ORF1696 gene product enhances the steady-state level of the LHI complex in the ICM.

Several reasons might account for the incomplete restoration of the LHI complex observed in the complemented Nae and Stu strains. For example, the ORF1696 gene may not be expressed as strongly from the pCY42 plasmid as it is expressed from its natural chromosomal location. Although the Km^r cassette could have a marginally polar effect in Stu and Nae (see below), it is clear that the absence of the ORF1696 gene product is the primary reason for the reduced LHI content of these strains.

The 50% reduction of the LHI complex level observed in the Nae mutant suggests that the loss of as few as the 13 C-terminal amino acid residues of ORF1696 impairs its function. Since the 5' remnant of the Ble^r gene on the Km^r cartridge was fused translationally in frame to the 3' codons of ORF1696 in Nae, no ORF1696 amino acid residues were deleted per se. That is, 97 heterologous amino acids were added to Ala-464 of the N-terminal segment of ORF1696 at the disruption site, and the C-terminal 13-amino-acid segment of ORF1696 (starting with Gly-465) was fused to the truncated Ble^r protein. Nevertheless, this disruption greatly interfered with the function of the transected ORF1696 protein (Fig. 2).

Since all of the ORF1696 mutants contained small amounts of LHI, the N-terminal segments remaining in the three mutants described here, as short as 31 amino acid residues in the Mun mutant, conceivably could contribute to LHI assembly, or else LHI is assembled inefficiently in the complete absence of ORF1696 activity. We suggest that ORF1696 is a major factor in LHI complex assembly but that either there are additional assembly factors or LHI forms spontaneously to a limited degree in vivo, as has been observed in vitro (23).

The low level of the LHI complex in Stu::, compared to the levels in Stu and Mun (Fig. 2C), is attributed to a combination of the direct effect of the disruption of ORF1696 and the indirect polar effect of the  disruption. The RC and LHI complexes are closely associated in the ICM, which could provide mutual stabilization as a result of protein-protein interactions (26, 33, 37). In fact, the amounts of the RC complex 800-nm peak in these three mutants correspond to the following order: Stu > Mun > Stu:: (Fig. 2C). Thus, the  cartridge insertion in Stu:: and the Mun Km^r cartridge insertion seem to have polar effects to different degrees on transcription of puhA and genes located 3' of puhA, such as ORF214, which have been observed to reduce RC and LHI levels when mutated (37). This conclusion is supported by the results of the trans-complementation experiments on the Mun strain (Fig. 2B). We suggest that a rho-dependent transcription termination site exists between the MunI site and the nearest of the two StuI restriction sites within the ORF1696 sequence and gives rise to the polar effect observed in Mun but not in Stu or Nae. Our results indicate that transcription initiated at the bchF promoter is required for normal expression of puhA, ORF214, and perhaps other genes located 3' of ORF214. This interpretation supports the proposal that the bch-puhA superoperon, previously thought to end immediately after puhA (6), extends beyond the puhA gene (7, 37).

It is difficult to account for the appearance of the broad, heterogeneous region of absorbance extending from approximately 750 to 790 nm in the Mun and the Stu:: spectra that accompanied the reduction in the 800-nm RC peak (Fig. 2). This absorbance could in principle arise from Bchl degradation products due to pigment-protein complex turnover or to biosynthetic intermediates, but absorption spectra of pigments in acetone extracts of Mun and LHII cells were superimposable (40). Thus, this spectrum seems to be due to Bchl molecules abnormally associated with proteins.

Our comparisons of the kinetics of LHI assembly and decay in strains LHII and Stu show that ORF1696 functions to enhance LHI assembly (Fig. 3 and 4; Table 1). It is conceivable that ORF1696 can act reversibly in an equilibrium-driven process to promote loss of Bchl from or disassembly of the LHI complex in the absence of Bchl synthesis, as indicated by the LHI decay kinetics (Fig. 4B; Table 1).

Although the differences between the LHII and the Stu slopes for LHI accumulation are significant (Fig. 3B and Table 1), the chromosomal disruptions of ORF1696 did not result in the complete loss of LHI under steady-state conditions (Fig. 2). This partial phenotype resulting from the mutation of ORF1696 raises questions as to how many factors are involved in maintaining the LHI complex at normal steady-state levels. Although the exact step at which ORF1696 exerts its role in the assembly of the LHI complex, whether by interacting with LHI polypeptides or with pigments, is unknown, we propose below two nonexclusive models.

Firstly, in vivo translocation of the LHI  and  polypeptides into the ICM has been hypothesized to involve an integral membrane protein, such as ORF1696, as well as DnaK and GroEL homologs (14, 24). These cytoplasmic chaperonins may bind to and escort the LHI polypeptides to the ICM, where a membrane-bound translocation apparatus (which may consist of ORF1696 alone or include additional factors) enhances the stable insertion of LHI  and  polypeptides into the membrane. This process would be followed by, or coincide with, ORF1696-independent binding of Bchl and carotenoid molecules to the membrane-spanning portions of the LHI  and  polypeptides to yield the mature LHI holocomplex.

Secondly, ORF1696 might act as an intermediary, transferring Bchl molecules from the terminus of the Bchl biosynthetic pathway to the LHI apoproteins, to enhance formation of the mature LHI holocomplex. Amino acid sequence comparisons between the R. capsulatus ORF1696 protein, homologs, and PucC sequences found in other purple nonsulfur bacteria reveal the presence of several histidines that are conserved to different degrees, including an invariant histidine residue at position 152 of the R. capsulatus ORF1696, which could participate in transient binding of Bchl (40). However, site-directed mutagenesis of the ORF1696 His-152 residue to Asn or Phe did not significantly affect LHI complex levels in trans-complementation experiments (40).

Homologs of ORF1696 have been found in the purple nonsulfur bacteria Rhodopseudomonas viridis (35), Rhodospirillum rubrum (8), and Rhodobacter sphaeroides (6, 13) and are similarly located 5' to the puhA gene of these species. Furthermore, the homologous pucC gene is required for LHII formation and is present in bacteria that contain the LHII complex (15-17), and ORF1696 homologs have been discovered in the cyanobacterium Synechocystis sp. strain PCC6803 (20) and in Prochlorococcus marinus (18). It will be interesting to see if proteins encoded by these open reading frames play a role in LH complex assembly in these species as does the ORF1696 protein in R. capsulatus.

It is clear that ORF1696 is a gene that encodes a protein involved in LHI complex assembly in R. capsulatus, and so we propose that the name be changed to the genetic designation lhaA, for light-harvesting complex assembly.