INTRODUCTION

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Rhodobacter sphaeroides 2.4.1 is a purple nonsulfur photosynthetic bacterium which displays a versatile metabolic lifestyle. It is able to grow by aerobic and anaerobic respiration and photosynthetically in the light under anaerobic conditions. The major determinant for the synthesis of the photosynthetic apparatus is oxygen. When oxygen tensions fall below a certain threshold value (~2.5%), the photosynthetic apparatus is synthesized. In addition, the levels and the ratios of the light-harvesting complexes are determined by the incident light intensity.

Transcriptional regulation of the photosynthesis (PS) genes in R. sphaeroides involves the coordinate action of at least four major regulatory systems (the PrrBA two-component activation system, the AppA-PpsR antirepressor-repressor system, FnrL, and TspO) which are responsible for redox sensing (25). Previous studies in our laboratory have shown that mutations in the ccoNOQP operon encoding the cbb₃ oxidase result in the induction of PS gene expression under aerobic growth conditions (18, 19, 28). This observation was interpreted to suggest that a pathway involving the cbb₃ oxidase in response to O₂ serves as an oxygen sensor which generates an inhibitory signal for PS gene expression. This signal is mediated by the PrrBA two-component regulatory system, which is a major switch controlling PS gene expression (17).

However, we could not rule out the possibility that FnrL, at least in part, is somehow involved in transmission of the inhibitory signal emanating from the cbb₃ oxidase, since expression of the puc operon and hemA gene, which are in part regulated by FnrL, is partially derepressed in the Cco mutants under aerobic conditions (18, 27-29).

The construction of a CcoP and FnrL double mutant enabled us to answer this question. In this study, we demonstrate that FnrL does not mediate the derepression signal derived from the inactivation of the cbb₃ oxidase under aerobic conditions. Furthermore, we do present evidence that the bchE, hemN, and hemZ genes, which have previously been identified as potential targets on the basis of sequence analyses of their upstream regions for regulation by FnrL, are in fact regulated by both FnrL and the PrrBA two-component system. These observations, as well as earlier results, will be discussed in conjunction with the role of FnrL and its relation to other regulatory circuits in controlling PS gene expression.

	MATERIALS AND METHODS

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Strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. R. sphaeroides 2.4.1 strains were grown at 30°C on Sistrom's medium A (SIS) (3) containing succinate as the carbon source and supplemented, as required, with the following antibiotics: tetracycline, 1 µg/ml; kanamycin, 25 µg/ml; trimethoprim, 50 µg/ml; and streptomycin and spectinomycin, 50 µg/ml each. Chemoheterotrophic cultures were grown either aerobically, sparged with 30% O₂-69% N₂-1% CO₂, or semiaerobically, sparged with 2% O₂-97% N₂-1% CO₂. Photosynthetic cultures were grown at a low incident light intensity of 3 W/m² in completely filled screw-cap glass tubes. The tubes containing photosynthetic cultures were rotated by using a rotary drum to keep cells suspended and mixed. Photosynthetic growth of R. sphaeroides was monitored with a Klett-Summerson colorimeter with a no. 66 filter. Anaerobic growth with dimethyl sulfoxide (DMSO) as a terminal electron acceptor was performed in SIS medium supplemented with 0.1% (wt/vol) yeast extract in the presence of DMSO (0.5% vol/vol) in screw-cap tubes in the dark. Escherichia coli strains were grown at 37°C on Luria-Bertani medium supplemented, when required, with the following antibiotics: ampicillin, 50 µg/ml; kanamycin, 50 µg/ml; tetracycline, 20 µg/ml; and streptomycin and spectinomycin, 50 µg/ml each.

DNA manipulations and conjugation techniques. Genomic DNA from R. sphaeroides was isolated by the method of Ausubel et al. (1). Standard protocols or the manufacturer's instructions were followed for recombinant DNA manipulations (20).

Construction of mutants and lacZ transcriptional fusions. For the construction of the CCOP1FNRL double mutant, the ccoP gene was mutated in the FNRL background using plasmid pUI2801, which had been used for the construction of the original CCOP1 mutant (18).

Quantitative analysis of spectral complexes. The harvested cells were resuspended in ICM buffer (10 mM KH₂PO₄/K₂HPO₄, 1 mM EDTA [pH 7.2]) and disrupted by passage through a French pressure cell (ca. 0.9-cm-diameter piston) at 90 MPa. Cell-free crude extracts were obtained following centrifugation in a benchtop microcentrifuge at 13,000 rpm for 15 min at 4°C to remove unbroken cells and cell debris. Spectra were recorded with a UV 1601PC spectrophotometer (Shimadzu Corp., Columbia, Md.). The B800-850 and B875 complex levels were determined by the method of Meinhardt et al. (12) from the spectral data collected.

Crt and Bchl analyses. For extraction of photopigments, 0.5 ml of crude cell extract was mixed with 1.5 ml of acetone-methanol (7:2, vol/vol), followed by vigorous vortexing for 1 min. After centrifugation in a benchtop microcentrifuge at 13,000 rpm for 15 min, the supernatant was used for quantitation of carotenoid (Crt) and bacteriochlorophyll (Bchl) as described previously (3).

-Galactosidase assays and protein determinations. -Galactosidase assays were performed on crude cell extracts as described elsewhere (19). Protein concentration was determined by the bicinchoninic acid protein assay (Pierce, Rockford, Ill.) using bovine serum albumin as the standard protein.

	RESULTS AND DISCUSSION

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Formation of spectral complexes in the CCOP1FNRL double mutant under aerobic conditions. In order to address the question of whether the signal emanating from the cbb₃ oxidase involves the participation of the FnrL protein, a double mutant carrying mutations in both fnrL and ccoP was constructed (strain CCOP1FNRL). When grown on SIS agar plates aerobically, the double mutant formed colonies with red pigmentation, the extent of which was intermediate between the appearance of the wild type and the high levels of pigmentation of the CCOP1 mutant (18). To confirm that the increased pigmentation of the CCOP1FNRL mutant under aerobic conditions was due to the presence of photosynthetic spectral complexes, crude extracts from cultures of this mutant strain grown aerobically were prepared, and the levels of the light-harvesting complexes (B800-850 and B875) as well as the photopigments Bchl and Crt were determined spectrophotometrically (Table 2). For comparison, crude extracts were also prepared from the wild-type, CCOP1, and FNRL strains grown under the same conditions. As expected, the wild-type and FNRL mutant strains produced virtually no light-harvesting complexes under aerobic conditions. In contrast, the CCOP1FNRL mutant strain exhibited 50 and 64% of the levels of the B800-850 and B875 complexes, respectively, relative to the levels which were detected in the CCOP1 mutant. In agreement with these results, the cellular levels of the photopigments Bchl and Crt also increased in both CCOP1 and CCOP1FNRL strains compared with those levels in the wild-type and FNRL strains. These data demonstrated that the mutation in ccoP led to the oxygen-insensitive formation of the photosynthetic complexes even in the FnrL-minus background. It should be recalled that an FnrL mutant strain of R. sphaeroides cannot produce spectral complexes even under inducing conditions such as 2% oxygen (26).

FnrL is not involved in the cbb₃-mediated signal transduction pathway under aerobic conditions. Previous studies revealed that mutations of the cco operon in R. sphaeroides lead to the aerobic derepression of not only those PS genes regulated by the PrrBA two-component system, such as puc and puf, but also hemA, which encodes the 5-aminolevulinic acid (ALA) synthase and which is, at least in part, under the control of FnrL (6, 18, 28). An interpretation of these results raised the possibility that FnrL is an additional regulator which communicates with the cbb₃ oxidase. To address this question, we compared the promoter activities of genes which are both regulated by FnrL and derepressed by mutations in the cco operon under aerobic conditions. These experiments were performed with the wild-type, CCOP1, and CCOP1FNRL mutant strains grown under aerobic conditions. We speculated that if FnrL mediates the derepression signal resulting from inactivation of the cbb₃ oxidase under aerobic conditions, then the derepression of PS gene expression observed in the CCOP1 mutant strain should be abolished in the CCOP1FNRL mutant. As shown in Table 3, the puc operon and hemA gene were strongly derepressed under aerobic conditions in the CCOP1 mutant by factors of 2.8- and 7.2-fold, respectively, compared with the wild type. The disruption of fnrL in the background of the CCOP1 mutant resulted in a further 1.4- and 2.3-fold increase in puc and hemA promoter activities, respectively, compared with the CCOP1 mutant. This result suggested that FnrL does not mediate the derepression signal resulting from inactivation of the cbb₃ oxidase under aerobic conditions, which is in good agreement with the oxygen-insensitive formation of the spectral complexes in the CCOP1FNRL mutant under aerobic conditions. The higher promoter activity of the hemA gene in the CCOP1FNRL mutant grown under aerobic conditions than in the CCOP1 mutant can be explained by the fact that the transcription start point of hemA under aerobic conditions lies within the predicted FnrL binding motif (16), and therefore suggests that FnrL might be a repressor of hemA under aerobic conditions. In the case of increased puc operon expression, the FnrL binding sequence overlaps the integration host factor binding sequence, and removal of FnrL could permit enhanced activation of puc operon expression by integration host factor (11). These results raise an additional question. If hemA and puc operon expression is increased in the CCOP1FNRL mutant background, why do we not see at least the level of spectral complexes in the double mutant as observed for the CCOP1 mutant? One possible answer is that there might be some PS genes which are regulated by FnrL and which are not derepressed by the inactivation of the cbb₃ oxidase under aerobic conditions. Thus, these gene products might be limiting factors for spectral complex formation.

Regulation of the bchE gene. A putative FnrL-binding motif is located 53 nucleotides upstream of the translational start codon of the bchE gene, whose product catalyzes the conversion of Mg-protoporphyrin monomethyl ester to divinyl-protochlorophyllide in the Bchl biosynthetic pathway. The presence of an FnrL-binding motif upstream of the bchE gene prompted us to examine whether this gene is regulated by FnrL. To address this question, we assayed the promoter activity of bchE in the FnrL-minus background. Since the FNRL mutant is unable to grow under anaerobic conditions, we performed an oxygen shift experiment in which cultures grown with 30% oxygen were shifted to 2% oxygen (Fig. 1). At the time intervals indicated, samples were harvested, and their -galactosidase activities were determined. In the wild-type strain, the levels of bchE::lacZ expression increased steadily and appreciably after the cultures were shifted to 2% oxygen. In contrast, the promoter activity of bchE in the FNRL mutant showed a marginal increase up to 4 h after the shift to 2% oxygen. This indicated a clear requirement for FnrL for the induction of bchE expression in response to lowering of the oxygen tension.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Induction kinetics of bchE expression from the bchE::lacZ transcriptional fusion plasmid pBCHE in the wild-type strain () and FNRL mutant strain () following a shift from 30 to 2% oxygen (indicated by the vertical arrow). Strains were grown under 30% O2 to an optical density at 600 nm (OD600) of 0.15 to 0.2, and the partial pressure of O2 was reduced to 2%. Cultures were sampled at the times indicated, and crude extracts were assayed for -galactosidase activity.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   -Galactosidase activities in R. sphaeroides strains containing the bchE::lacZ transcriptional fusion plasmid pBCHE. The wild-type (2.4.1) and prrCBA-minus (PRR) strains were grown aerobically (30% oxygen, black bars) to an OD600 of 0.3 to 0.4 or anaerobically in the dark with 0.5% DMSO (gray bars). All values are the averages of two independent determinations. Vertical bars represent the standard deviations from the mean.

Regulation of the hemZ and hemN genes. Sequence analyses have also revealed the presence of FnrL binding sequences 35 and 34 bp upstream of the presumed translational start codons of the hemN and hemZ genes, respectively, encoding isoenzyme forms of the coproporphyrinogen III oxidase involved in the tetrapyrrole biosynthetic pathway. Previous work by Yeliseev and Kaplan (24) revealed that both genes are derepressed under semiaerobic and anaerobic conditions and that the hemN gene appears to encode a form of the enzyme that may be active aerobically. To examine the role of FnrL in the regulation of hemZ and hemN, the corresponding transcriptional lacZ fusions were introduced into the wild-type strain 2.4.1 as well as the FNRL mutant strain. The strains were grown either under 30% oxygen conditions or under 2% oxygen conditions, in which many of the genes known to be under the control of FnrL appear to be fully induced.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   -Galactosidase activities in R. sphaeroides strains containing either the hemZ::lacZ or hemN::lacZ transcriptional fusion plasmid. The wild-type (2.4.1) and fnrL-minus (FNRL) strains were grown aerobically (30% oxygen, black bars) to an OD600 of 0.3 to 0.4 or semiaerobically (2% oxygen, gray bars) to an OD600 of 0.4 to 0.5. All values provided are the averages of two independent determinations. Vertical bars represent the standard deviations from the mean.

Regulation of the tetrapyrrole biosynthetic pathway. As outlined in Fig. 4, both the Bchl and heme biosynthetic pathways have the same intermediates from ALA through protoporphyrin IX, where they branch (22). Hemes are absolutely required for both aerobic and anaerobic growth of R. sphaeroides. In contrast, the synthesis of Bchl should be inhibited in cells grown under high-oxygen conditions, since free Bchl in the presence of light and O₂ produces toxic free radicals and the expression of the puf and puc operons is minimal under these conditions. In fact, the levels of detectable Bchl found in R. sphaeroides grown under high-oxygen conditions is minimal. Therefore, the "opening" of the pathway from protoporphyrin IX to Bchl a must be stringently controlled and exquisitely responsive to the levels of O₂ as well as light present in the environment. The results presented here and those published earlier (25) indicate that R. sphaeroides has developed overlapping regulatory circuits to enable such controls (Fig. 4). R. sphaeroides has two isoenzymes for the synthesis of ALA (HemA and HemT) (15, 16) and two coproporphyrinogen III oxidases (HemN and HemZ) (4, 27), which have been identified as points of FnrL regulation in the tetrapyrrole biosynthetic pathway prior to protoporphyrin IX. The hemT product was shown to be fully functional under both aerobic and anaerobic conditions in the absence of HemA (in the HemA-minus mutant), and hemT seems not to be regulated by FnrL due to the lack of an observed FnrL-binding motif in the control region (15, 16).

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Bchl and heme biosynthetic pathways. The relevant genes, the regulation of which is either established or predicted on the basis of sequence analyses, are depicted. The question mark to the right of a regulator symbol indicates that regulation by the corresponding regulator is inferred from sequence analyses and is therefore not proven experimentally. The Bchl biosynthetic branch is shown in a yellow box. To the left of the yellow box, the regulation of four inferred operons which are involved in Bchl biosynthesis is represented. The red and blue arrows represent induction under oxygen-limiting conditions (semiaerobic or anaerobic conditions) and repression under high-oxygen conditions, respectively. The outer membrane TspO protein affects the expression of PpsR target genes by acting on HemN posttranscriptionally (24). At the bottom, the regulation of the puc and puf operons as well as the puhA gene, which encode the structural polypeptides of the reaction center and light-harvesting complexes, is presented. Abbreviations: MV, monovinyl; DV, divinyl; CoA, coenzyme A.

Inactivation of the cbb₃ oxidase bypasses the requirement for FnrL in the formation of spectral complexes. Spectral complex formation under inducing conditions, such as 2% oxygen, is severely affected in the FNRL mutant of R. sphaeroides, indicating that FnrL is an essential regulator in the formation of spectral complexes (26). However, the oxygen-insensitive formation of spectral complexes in the CCOP1FNRL double mutant also suggested that the requirement for FnrL in spectral complex formation can be at least partially alleviated by the inactivation of the cbb₃ oxidase. This finding raised the question of whether the CCOP1FNRL mutant is able to grow photosynthetically. Zeilstra-Ryalls and Kaplan (27) have reported that the FNRL mutant can grow neither photosynthetically nor anaerobically with DMSO as a terminal electron acceptor for anaerobic respiration. Surprisingly, we found that the CCOP1FNRL double mutant regains the ability to grow, albeit slowly, under photosynthetic conditions. In liquid medium in completely filled screw-cap glass tubes, a continuous increase in cell density was observed for the CCOP1FNRL mutant after approximately the same lag phase as for the wild type when the medium was inoculated with aerobically grown cells, although its growth rate was slower than that of the wild-type and CCOP1 mutant strains (Fig. 5). As expected, the FNRL mutant cannot grow photosynthetically.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Growth characteristics of the wild-type and mutant strains under photosynthetic conditions. Photosynthetic cultivations were performed at low light intensity (3 W/m2) in completely filled screw-cap glass tubes to minimize the repression effect of PpsR. Cultures were inoculated with aerobically grown cells. The levels of spectral complexes in the strains grown under the same conditions to an OD600 of 0.3 to 0.4 are given above the growth curves for B800-850 and (B875). OD600 = (Klett unit + 3.06)/102.04.