Aerobic and anaerobic Mg-protoporphyrin monomethyl ester cyclases in purple bacteria: a strategy adopted to bypass the repressive oxygen control system.

Two different mechanisms for Mg-protoporphyrin monomethyl ester (MgPMe) cyclization are shown to coexist in Rubrivivax gelatinosus and are proposed to be conserved in all facultative aerobic phototrophs: an anaerobic mechanism active under photosynthesis or low oxygenation, and an aerobic mechanism active only under high oxygenation conditions. This was confirmed by analyzing the bacteriochlorophyll accumulation in the wild type and in three mutant strains grown under low or high aeration. A mutant lacking the acsF gene is photosynthetic, exhibits normal bacteriochlorophyll accumulation under low oxygenation and anaerobiosis, and accumulates MgPMe under high oxygenation. The photosynthesis-deficient bchE mutant produces bacteriochlorophyll only under high oxygenation and accumulates MgPMe under low oxygenation and anaerobiosis. The double knockout mutant is devoid of photosystem and accumulates MgPMe under both conditions indicating the involvement of the two enzymes at the same step of the biosynthesis pathway. Oxygen-mediated expression of bchE was studied in the wild type and in a regulatory mutant. The reverse transcriptase-PCR and the bchE promoter activity results demonstrate that the expression of the bchE gene is oxygen-independent and suggest that it is rather the enzyme activity that should be oxygen-sensitive. No obvious sequence similarities were found between oxygen-dependent AcsF and the oxygen-independent anaerobic Mg-protoporphyrin monomethylester cyclase (BchE) enzymes. However, common to all BchE proteins is the conserved CXXX-CXXC sequence. This motif is essential for 4Fe-4S cluster formation in many anaerobic enzymes. Expression and purification of BchE were achieved, and the UV-visible spectral analyses confirmed the presence of an active 4Fe-4S cluster in this protein. The use of different classes of enzymes catalyzing the same reaction under different oxygen growth conditions appears to be a common feature of different biosynthetic pathways, and the benefit of possessing both aerobic and anaerobic systems is discussed.

Two different mechanisms for Mg-protoporphyrin monomethyl ester (MgPMe) cyclization are shown to coexist in Rubrivivax gelatinosus and are proposed to be conserved in all facultative aerobic phototrophs: an anaerobic mechanism active under photosynthesis or low oxygenation, and an aerobic mechanism active only under high oxygenation conditions. This was confirmed by analyzing the bacteriochlorophyll accumulation in the wild type and in three mutant strains grown under low or high aeration. A mutant lacking the acsF gene is photosynthetic, exhibits normal bacteriochlorophyll accumulation under low oxygenation and anaerobiosis, and accumulates MgPMe under high oxygenation. The photosynthesis-deficient bchE mutant produces bacteriochlorophyll only under high oxygenation and accumulates MgPMe under low oxygenation and anaerobiosis. The double knockout mutant is devoid of photosystem and accumulates MgPMe under both conditions indicating the involvement of the two enzymes at the same step of the biosynthesis pathway. Oxygen-mediated expression of bchE was studied in the wild type and in a regulatory mutant. The reverse transcriptase-PCR and the bchE promoter activity results demonstrate that the expression of the bchE gene is oxygen-independent and suggest that it is rather the enzyme activity that should be oxygen-sensitive. No obvious sequence similarities were found between oxygen-dependent AcsF and the oxygen-independent anaerobic Mg-protoporphyrin monomethylester cyclase (BchE) enzymes. However, common to all BchE proteins is the conserved CXXX-CXXC sequence. This motif is essential for 4Fe-4S cluster formation in many anaerobic enzymes. Expression and purification of BchE were achieved, and the UV-visible spectral analyses confirmed the presence of an active 4Fe-4S cluster in this protein. The use of different classes of enzymes catalyzing the same reaction under different oxygen growth conditions appears to be a common feature of different biosynthetic pathways, and the benefit of possessing both aerobic and anaerobic systems is discussed.
Purple bacteria perform anoxygenic photosynthesis on the basis of a bacteriochlorophyll-mediated process. It takes place within the membrane photosynthetic apparatus, composed of three pigment-protein complexes as follows: the two light-harvesting antennae and the reaction center, associated with carotenoids and bacteriochlorophylls. Bacteriochlorophyll a is the most widely distributed bacteriochlorin pigment and is found in most photosynthetic bacteria. The early steps in bacteriochlorophyll a biosynthesis up to chlorophyllide a are common to the biosynthesis pathway of chlorophyll a, a pigment present in all organisms capable of oxygenic photosynthesis. The chelation of Mg 2ϩ into protoporphyrin IX to form Mg-protoporphyrin IX (MgP) 1 is catalyzed by the products of the bchD, bchI, and bchH genes. Methylation of MgP to form MgP monomethyl ester (MgPMe) is then catalyzed by the product of bchM (for review see Refs. 1 and 2). MgPMe, a key intermediate in the biosynthesis of chlorophylls and bacteriochlorophylls, is the substrate for the oxidative cyclase(s) responsible for the formation of the isocyclic ring V of protochlorophyllide (Pchlide) a (1,2). In phototrophic bacteria, this cyclase is encoded by the bchE gene. Recently, mutational analyses of selected loci from the photosynthesis gene cluster of the purple nonsulfur bacterium Rubrivivax gelatinosus allowed us to identify the acsF gene as responsible for the cyclization of MgPMe (3). Pinta et al. (3) generated a photosynthetic competent mutant accumulating a pigment identified as MgPMe only under high oxygenation. The data showed that under high oxygenation, bacteriochlorophyll a biosynthesis is controlled by AcsF at the level of MgPMe oxidative cyclization and raised the question as to how bacteriochlorophyll a is synthesized under low oxygenation or anaerobic photosynthetic conditions. We have suggested the coexistence of two pathways leading to the bacteriochlorophyll a biosynthesis in this bacterium. This assumption is in agreement with biochemical studies in Rhodovulum sulfidophilum claiming that this bacterium incorporates atomic oxygen from either H 2 O or O 2 to form the 13(1)-oxo group of the bacteriochlorophyll a isocyclic ring, and suggesting that two different cyclization mechanisms coexist in R. sulfidophilum involving both an oxygenase and a hydratase to form the 13(1)-oxo group of the bacteriochlorophyll a isocyclic ring (4). However, in Rhodobacter sphaeroides cells shifted from respiratory to photosynthetic growth conditions, H 2 O was shown to be the source of oxygen for the cyclization reaction (5). The derivation of the 13(1)-oxo group from water showed that the formation of the isocyclic ring from the 13-propionic acid methyl ester side chain of MgPMe is an anaerobic process involving a hydratase. This bacterium produces bacteriochlorophyll a also under aerobic * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AY309082.
The ability to produce bacteriochlorophyll a both aerobically and anaerobically represents a significant advantage for these bacteria as they become competent to produce the photosystem even under aerobiosis and to shift rapidly to photosynthetic growth following reduction of oxygen tension and exposure to light.
To elucidate the mechanisms of the bacteriochlorophyll isocyclic ring formation in the photosynthetic bacterium R. gelatinosus, we have identified the bchE gene and analyzed bchE and acsF knockout mutants. The observed phenotypes and the characteristics of the bacteriochlorophyll precursors in the mutants strongly support the involvement of the two genes at the same level of the biosynthetic pathway and explain the presence of bacteriochlorophyll a either under aerobiosis or anaerobiosis in R. gelatinosus and in other facultative aerobic photosynthetic bacteria.

EXPERIMENTAL PROCEDURES
Bacterial Strains, Plasmids, and Growth Media-Escherichia coli was grown at 37°C on LB medium. R. gelatinosus strain 1 (6) and the mutants were grown in malate (ML) medium (7) at 30°C, photosynthetically (anaerobiosis and light) or in the dark in 50-ml flasks filled with 50 ml of medium (low oxygenation conditions), or 250-ml flasks containing 20 ml of medium (high oxygenation conditions). Shaking was at 100 rpm. Antibiotics were used at the following concentrations: chloramphenicol 3 g/ml, ampicillin 50 g/ml, kanamycin (Km) 50 g/ml, spectinomycin 50 g/ml, streptomycin 50 g/ml, tetracycline 2 g/ml. Bacterial strains and plasmids used in this work are listed in Table I.
Molecular Biology Techniques-Standard methods were used according to Sambrook et al. (8), unless otherwise indicated. Plasmid DNAs were purified using QIAprep spin miniprep kit (Qiagen) or Quantum prep plasmid midiprep kit (Bio-Rad). DNA was treated with restriction enzymes and other nucleic acid-modifying enzymes (Klenow fragment, T4 DNA polymerase, and T4 DNA ligase) according to the manufacturer's specifications. DNA fragments were analyzed on agarose gels, and different restriction fragments were purified using the Geneclean kit (Bio 101, Inc., Vista, CA).
Library Screening for bchE-The PCR was carried out using genomic or plasmid templates in a 50-l reaction mixture containing PCR buffer, 1.5 M MgCl 2 , 200 M of each deoxynucleoside triphosphate, 1 M of each primer, 5% Me 2 SO, and 2.5 units of Taq DNA polymerase. 20 cycles were performed in a Hybaid thermal cycler, each cycle comprising 30 s at 92°C, 40 s at 60°C, and 60 s at 72°C. Primers are listed in Table II.
Gene Transfer and Strain Selection-Transformation of R. gelatinosus cells was carried out by electroporation as described previously (9). Transformants were selected on malate plates supplemented with the appropriate antibiotic.
Construction of the bchE-lacZ Reporter Fusion Plasmid-To construct a bchE-lacZ fusion, the promoter region of bchE containing the putative PpsR-binding site (TGT N12 ACA) was amplified by PCR (380-bp DNA fragment) from plasmid pSO2770. The following primers were designed to create XbaI and BamHI sites at 5Ј and 3Ј ends of the amplified fragment, respectively: Ol-bchE-FBamHI and Ol-bchE-RX-baI. The sequence and the orientation of the amplified DNA fragment were confirmed by sequencing after cloning into the BamHI and XbaI site of the lacZ transcriptional fusion vector plasmid pSO3001 to generate the fusion plasmid pSO3027 (bchE::lacZ).
RT-PCR Analysis of bchE Gene Expression-Total RNA from WT cells grown photosynthetically or aerobically under high or low oxygenation was extracted according to Ref. 10, treated with RNase-free DNase I, and then purified by phenol chloroform extraction and ethanol precipitation. A reverse transcription reaction was performed to synthesize the cDNA from 3 g of total RNA using Superscript II RNase enzyme (Invitrogen) at 60°C for 1 h with oligonucleotide Ol-bchE-R2 (extending from position ϩ1110 to ϩ1091 of the bchE gene). The prod-

Construction of Plasmids for High Level Expression of BchE in E.
coli-A His 6 -tagged construct was prepared using the pET-15b plasmid (Novagen). The bchE gene was amplified by PCR from pSO2770 plasmid using the Ol-HbchE-FNdeI and Ol-HbchE-RBamHI primers. NdeI and BamHI sites were introduced at the ATG translation initiation codon and after the TGA stop codon, respectively. The NdeI-BamHI PCR fragment was inserted into the NdeI-BamHI site of pET-15b. Two plasmids were isolated and sequenced. Plasmid pSO2775 contains the wild type sequence of bchE and should encode a full-length protein. In plasmid pSO2776, a spontaneous mutation (G to A) was randomly introduced during PCR, introducing a premature stop codon at position 1098 (TGA/TGG) of the bchE coding sequence.
Overexpression, Purification, and Analysis of a His-tagged BchE Protein-E. coli BL21(DE3) cells harboring either pSO2775 or pSO2776 were grown in LB medium at 30°C until they had reached an A 600 of 0.6. Then isopropyl-␤-D-thiogalactopyranoside was added to a final concentration of 1 mM, and growth was continued for 4 h. Isopropyl-␤-Dthiogalactopyranoside-induced cells from 200-ml cultures were suspended in 50 ml of binding buffer containing 20 mM Tris/HCl (pH 7.9), 5 mM imidazole, and 0.5 M NaCl and broken by a French pressure cell. Unbroken cells and debris were removed by centrifugation at 600 ϫ g for 10 min. Inclusion bodies were obtained by centrifugation at 5000 ϫ g for 10 min. The expressed proteins were extracted from the inclusion bodies using 6 M urea in the binding buffer. To purify the proteins, the supernatants were applied to nickel nitrilotriacetic acid-agarose (Qiagen) affinity columns equilibrated with binding buffer. The columns were then washed with this buffer and the wash buffer (20 mM Tris/HCl (pH 7.9), 0.5 M NaCl, and 15 mM imidazole). The bound proteins were eluted from the columns with elution buffer containing 500 mM imidazole. The overexpressed His-tagged BchE proteins were resolved by 12% SDS-PAGE and stained with Coomassie Blue.
Membrane Protein Preparation and Spectrophotometric Measurements-The membranes were prepared by cell disruption with a French press in 0.1 M sodium phosphate buffer (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride, followed by differential ultracentrifugation. The membranes were then resuspended in the same buffer. Absorption spectroscopy was performed with a CARY 500 spectrophotometer.
Preparation of Pigment Extracts-Cells were harvested in their exponential growth phase, when the culture optical density at 680 nm reached 0.7. Pigments were extracted from cell pellets by resuspending cells in 20 volumes of ice-cold acetone/methanol 7:2 (v/v). The cell debris was eliminated by centrifugation. Further extractions were made until the cell debris pellet was completely depigmented. For the HPLC analyses, the solvent and residual water were then totally evaporated. The dried pigments were resolubilized in a small volume of acetone/methanol 7:2 (v/v) and concentrated by evaporation.
Secreted pigments were extracted from 10 ml of growth medium using Sep-Pak C 18 silica cartridges (Waters) according to the manufacturer's specifications. The pigments were eluted by adding 1 ml of ice-cold acetone/methanol 7:2 (v/v).
HPLC Analysis-20 l of pigment preparation were injected on a 5-m Kromasil C18 column (4.6 mm inner diameter, length 250 mm) and eluted at a 1 ml/min solvent flow rate. HPLC solvent mixture was acetonitrile/methanol/dichloromethane 75:15:15 (v/v/v). Detection was carried out at 436 nm, and collected fractions were further analyzed by spectrophotometric absorption measurements.

RESULTS
Sequencing of the entire photosynthesis gene cluster of R. gelatinosus followed by mutational analysis allowed the identification of the acsF gene as responsible for the aerobic oxidative cyclization of MgPMe (3). In Rhodobacter species, this reaction is catalyzed under anaerobiosis by the BchE Mg-protoporphyrin IX monomethyl ester oxidative cyclase (11). To elucidate the mechanism of the bacteriochlorophyll isocyclic ring formation in R. gelatinosus under anaerobiosis conditions, we have undertaken the study of bchE involvement in this process.
Cloning and Sequence Analysis of the bchE Genomic Region in R. gelatinosus-The purple photosynthetic bacterium R. gelatinosus has within its genome a cluster of ϳ59 kb in length containing most of the photosynthesis related genes (12). Two genes involved in bacteriochlorophyll biosynthesis are missing from this cluster. In an attempt to identify these genes, namely bchE and bchJ involved in the early stage of bacteriochlorophyll biosynthesis, a R. gelatinosus genomic DNA library was screened using a couple of primers (Ol-bchE-F and Ol-bchE-R) based on the published Rhodobacter bchE gene sequences (AJ010302 and Z11165). A plasmid (pSO2770) containing bchE and bchJ genes was isolated and sequenced. Sequence similarity searches were performed using the BLAST network service (13). The bchE gene is 1746 bp long and encodes a 582-amino acid polypeptide with a predicted molecular mass of about 63 kDa. A putative PpsR-binding motif was identified upstream from the bchE gene suggesting an oxygen-dependent expression of bchE. BlastP and Clustal analyses indicated that the BchE polypeptide from R. gelatinosus shows similarities to BchE from R. rubrum (68% identity and 80% similarity) and from R. sphaeroides (61% identity and 74% similarity). Further sequence analyses indicate that BchE possesses a vitamin B 12binding motif in its N-terminal domain suggesting that this protein uses vitamin B 12 as cofactor. A potential S-adenosyl-Lmethionine (SAM)-binding motif characteristic of methyltransferases is present in the central region. Moreover, cysteine residues Cys-205, Cys-209, and Cys-212, which are part of a conserved CXXXCXXC motif, were found in many BchE proteins. This motif was also found and shown to be essential for a 4Fe-4S cluster formation in many oxygen-independent oxidases.
bchE Mutant Construction and Characterization-To gain insight into the role of bchE gene in the bacteriochlorophyll isocyclic ring formation, a bchE mutant was generated. For that purpose the bchE gene fragment cloned in plasmid pSO2771 was disrupted with the kanamycin resistance cartridge inserted at the BssHII site of bchE to construct plasmid pSO2772. Wild type cells electroporated with pSO2772 were plated on selective medium under respiratory conditions to select transformants resulting from double crossover events. The BCHE mutant colonies showed a brown pigmentation on plates. The BCHE mutant was photosynthesis-deficient, and interestingly, when grown aerobically under high oxygenation, the mutant resembles wild type, i.e. it synthesizes bacteriochlorophyll and assembles the photosystem like a photosynthetic competent strain. Furthermore, the culture medium was pigmented (greenish) only when cells were grown under low oxygenation conditions, suggesting the presence of a pigment secreted by the cells and indicating that they have an altered pigment production. The involvement of bchE in the MgPMe-cyclase reaction was first investigated by absorption spectroscopy. Because the BCHE mutant is photosynthetic deficient, the growth properties of this mutant and its ability to synthesize pigments and photosystem were investigated, in comparison to the WT grown under respiratory conditions, under high or low oxygenation. Under high oxygenation conditions, both strains had the same generation time and assembled photosynthetic complexes as indicated by the absorbance peaks at 800 and 860 nm (Fig. 1A). Nevertheless, under low oxygenation conditions, and unlike the WT, the BCHE mutant assembled no or a very low amount of photosynthetic complexes (Fig. 1B). Moreover, this strain also accumulated an additional compound absorbing at 416 nm characteristic of protochlorophyllide in aqueous solution.
Analysis of acetone/methanol pigment extracts from WT and BCHE strains grown under high oxygenation conditions showed identical spectra, with absorption at 770 nm for bacteriochlorophyll a, 420 -442-472 nm for carotenoids, and 362 nm for the bacteriochlorophyll a Soret absorption band ( Fig.  2A). Under low oxygenation, the WT spectrum still presented the same characteristics. In contrast, only traces of bacteriochlorophyll a could be detected in the low oxygenated BCHE mutant, whereas a majority peak appeared at 416 nm (Fig.  2B). These spectral characteristics of the BCHE mutant grown under low oxygenation recall the spectral characteristics of the SIX9Km (acsF::Km) mutant grown under high oxygenation (3).
Functional Complementation of BCHE Phenotype-pSO2773, a replicative plasmid bearing the bchE gene, was introduced into BCHE mutant cells. All the resulting colonies were photosynthetic, produced bacteriochlorophyll a, and assembled spectral complexes under photosynthetic as well as under low oxygenation conditions. These experiments confirmed that the loss of bacteriochlorophyll a and photosystem in the BCHE mutant grown under low oxygenation was strictly due to the disruption of the bchE gene.
HPLC Analysis of Pigments from High and Low Oxygenated bchE Mutant Cells-Even though the above data suggest that

FIG. 2. Absorption spectra of pigment extracts from R. gelatinosus WT (black) and BCHE mutant (gray) cells grown under high oxygenation (A) and low oxygenation (B) conditions.
the BCHE mutant accumulates the MgPMe intermediate of bacteriochlorophyll biosynthesis, like the (acsF::Km) mutant, further analysis of the bacteriochlorophyll intermediates pool accumulated by the bchE disrupted mutant was subsequently undertaken to determine the nature of these pigments. For this analysis, the pigment content of the BCHE mutant grown under either low or high oxygenation was analyzed by HPLC. Isolated pigment fractions were identified on the basis of their UV-visible absorption spectra and of the retention time. In the isolated pigment extract from cells grown under high oxygenation, two fractions were collected (peak 1 and 4) in addition to the carotenoid OH spheroidenone (peak 3) (Fig. 3A). According to their retention time and UV-visible absorption spectra, fraction 1 was identified as Mg-protoporphyrin (416 -552-590 nm), whereas fraction 4 was identified as bacteriochlorophyll a (770 nm).
In the extracted pigment from cells grown under low oxygenation, three fractions were collected and analyzed by UV-visible absorbance (Fig. 3B). Fraction 1 elutes after 2.6 min and was identified as Mg-protoporphyrin (MgP) (416 -552-590 nm) or its monomethyl ester (MgPMe). Based on the data published previously (3) concerning the retention time of MgPMe and MgP, this compound is proposed to be the MgPMe. Fraction 2 elutes after 4 min and absorbs at 402-504-538 -574 and 680 nm, it was identified as protoporphyrin IX. Fraction 4 corresponds to the smallest peak; it elutes after 11 min and exhibits a maximal peak at 770 nm; it was concluded that it corresponds to bacteriochlorophyll a. These results showed that the main accumulated metalloporphyrins in the BCHE mutant grown under low oxygenation are similar to those found in the R. gelatinosus acsF mutant grown under high oxygenation. This is the first direct evidence that both enzymes are involved in MgPMe cyclization and catalyze the same step of bacteriochlorophyll biosynthesis. Nevertheless, AcsF acts strictly under high oxygenation conditions, whereas BchE is involved when the oxygen tension drops. If this assumption is correct, then a double bchE-acsF mutant should be devoid of bacteriochlorophyll a and photosystem irrespective of growth conditions. bchE-acsF Double Mutant Construction and Characterization-To inactivate the acsF gene in the WT and in the BCHE backgrounds, a plasmid (pSO2774) bearing the acsF gene with the ⍀ cartridge inserted at the StuI site was used to electroporate the WT and the BCHE cells. Electroporated cells were plated on selective medium to select transformants resulting from double crossover events. The resulting disrupted strains were named ACSF⍀ and BCHE-ACSF, respectively. ACSF⍀ mutant has almost the same phenotype as SIX9Km (3). It was able to grow under both photosynthetic and respiratory conditions; it accumulated MgPMe only under high oxygenation, and the spectroscopic characterization of the metalloporphyrins was similar to that of MgPMe isolated from the SIX9Km mutant (not shown). ACSF⍀ mutant however was blue green and devoid of carotenoids because of the polar effect of the ⍀ cartridge on the downstream-localized crtB gene.
As expected the BCHE-ACSF double mutant was photosynthesis deficient, and the culture medium was greenish when the cells were grown either under low or high oxygenation. The secreted pigments were extracted from the growing medium using Sep-Pak cartridges, and the collected fraction was analyzed by absorption spectroscopy. The spectrum shows a major peak at 416 nm (data not shown). Spectral analyses of the membranes prepared from cells grown either under low or high oxygenation show the absence of the characteristic absorbance peaks of the photosynthetic complexes, as well as the presence of a pronounced peak at 416 nm.

FIG. 3. Comparison of HPLCs performed on pigment extracts from R. gelatinosus BCHE mutant grown under high oxygenation (A) or low oxygenation (B) conditions. Compound 1 (MgPMe)
is the major bacteriochlorophyll pigment accumulated under low oxygenation. HPLC solvent mixture is acetonitrile/methanol/dichloromethane 75:15:15 (v/v/v). I, injection; AU, arbitrary unit. Acetone/methanol pigment extracts from these membranes showed identical spectra for both growth conditions. A main peak is observed at 416 nm, and neither the peak at 770 nm nor the peak at 362 nm characteristic of the bacteriochlorophyll a absorption and its Soret band, respectively, were detected (Fig.  4). These results confirm that the bacteriochlorophyll biosynthesis pathway is fully blocked in the BCHE-ACSF double mutant irrespective of the growth conditions. In other respects, these results show that the bchE and acsF genes are involved at the same step of the bacteriochlorophyll a biosynthesis pathway. This implies that, despite their sequence and probably their structural differences, both enzymes recognize and transform the same substrate (MgPMe) to generate Pchlide a. We concluded that R. gelatinosus possesses two pathways leading to the MgPMe cyclization, an aerobic pathway controlled by the aerobic MgPMe oxidative cyclase AcsF and the anaerobic pathway controlled by the anaerobic MgPMe oxidative cyclase BchE (Fig. 5). The benefit of possessing the two genes as well as the mechanistic differences between the two enzymes will be covered under "Discussion." Concerning the oxygen-dependent/ independent cyclization of MgPMe by AcsF and BchE, respectively, many levels of control might be considered, particularly (i) gene expression control, (ii) enzyme cofactor availability, and (iii) redox potential.
Effect of Oxygen on bchE and acsF Genes Expression, Role of ppsR-It has been demonstrated that oxygen regulates bacteriochlorophyll biosynthesis in purple photosynthetic bacteria at the transcriptional level. Rhodobacter species control the synthesis of their bacteriochlorophyll in the presence of oxygen by the use of transcriptional factors. PpsR (CrtJ) was shown to repress the expression of many bacteriochlorophyll genes (14,15). The absence of bacteriochlorophyll a in the acsF mutant grown under high oxygen tension despite the presence of the bchE gene suggests that the expression of bchE gene is repressed. The presence of a putative PpsR-binding motif upstream of the bchE gene prompted us to examine whether this gene is repressed by the transcriptional regulator PpsR. The gene coding for the transcriptional factor PpsR was isolated from R. gelatinosus, 2 and the corresponding disrupted mutant was used to clarify the mechanism by which oxygen regulates the bacteriochlorophyll a biosynthesis pathway at the level of MgPMe cyclization. The ACSF⍀ mutant and the double mutant ACSF-PPSR were grown under low or high oxygenation, and their pigment contents were compared with the pigments produced in the carotenoid-less control strain SIB1.
Under low oxygenation conditions, acetone/methanol extracts from all strains showed identical spectra with absorbance at 770 nm for bacteriochlorophyll a and 362 nm for the bacteriochlorophyll a Soret absorption band. However, under high oxygenation, the SIB1 spectrum still presented the same characteristics, whereas the ACSF-PPSR and ACSF⍀ mutant showed identical spectra, with absorption maxima at 416 nm. These results suggest that under high oxygenation disruption of ppsR does not derepress the bchE expression and that this factor is not involved in the control of the expression of the bchE gene. These results should be confirmed at the level of gene expression using a reporter fusion plasmid and RT-PCR to assess the bchE promoter activity under different conditions.

Oxygen Regulation of bchE Gene Expression, bchE Promoter
Activity-To sustain the obtained genetic results, a bchE-lacZ reporter fusion plasmid pSO3027 was introduced into the wild type and the PPSR mutant. Wild type and PPSR mutants containing the bchE-lacZ fusion grown under high or low oxygenation were assayed for ␤-galactosidase activity. As shown in Fig. 6, under both conditions the level of expression of the bchE-lacZ fusion was not different between the wild type and the PPSR mutant, confirming that the expression of the bchE gene is not regulated by PpsR. It is of note that the promoter activity of bchE is only slightly increased when the oxygen tension drops. In both the WT and the PPSR mutant, the bchE promoter activity was ϳ1.5 times greater when cells were grown under low oxygenation conditions. We conclude that bchE gene is expressed under both high and low oxygenation and independently of the PpsR transcription factor.
Oxygen Regulation of bchE Gene Expression, RT-PCR Analysis-To confirm further the expression of the bchE gene under either high or low oxygenation conditions, Northern hybridization was performed to examine the production of transcripts under these conditions; however, we were unable to detect any transcript. Expression of bchE was then determined by RT-PCR. Total mRNA was extracted from WT cells grown photosynthetically or aerobically under high or low oxygenation and subjected to RT-PCR. The results are shown in Fig. 7. In the positive genomic control a PCR product of the expected size of 384 bp was obtained, and no PCR product was detected in any of the negative control reactions. DNA bands corresponding to the expected size from all RT samples were obtained, demonstrating that in all investigated oxygen conditions, the bchE gene was expressed (Fig. 7). Taken together, the RT-PCR and the bchE promoter activity results revealed that R. gelatinosus bchE gene expression is oxygen-independent and that its transcription is not repressed under aerobic conditions. These findings raised the questions of why the BchE enzyme fails to catalyze the formation of the isocyclic ring V of protochloro-phyllide under high oxygenation conditions in the ACSF mutant. Many hypotheses should be considered; the enzyme activity may be controlled either at the translational level, i.e. stability of the protein, or at the post-translational level, i.e. post-translational modifications. BchE sequence analyses revealed the presence of an iron-sulfur cluster-binding motif (CXXXCXXC), suggesting that BchE is an iron-sulfur protein.
The assembly of the iron sulfur cluster within the protein may be oxygen-dependent, and the loss of the Fe-S cluster by exposure to oxygen may result in an inactive form of the enzyme. Finally, the redox state of the putative iron-sulfur cluster in BchE and hence the catalytic activity may be oxygen-sensitive, accounting for an enzyme active only under low oxygenation and anaerobic conditions.
Overexpression, Purification, and Analysis of a His-tagged BchE Protein-As a first step toward understanding the BchE functioning, we overexpressed the gene encoding bchE and initiated its biochemical studies to check the presence of the iron-sulfur cluster. A full-length recombinant His 6 -BchE (575 residues) was produced in inclusion bodies in E. coli. The preparation was yellow-brown in color; however, only a small fraction could be solubilized with urea (6 M), and most of the iron-sulfur cluster was degraded upon solubilization as judged by the loss of the color and the loss of the absorption peak around 410 nm, characteristic of oxidized 4Fe-4S (not shown). A truncated form of His 6 -BchE* (384 residues) was also produced in inclusion bodies (Fig. 8A). Both the inclusion body preparation and the 6 M urea solubilized fraction were yellowbrown in color, indicating that solubilization did not degrade the iron-sulfur cluster. The presence of intact iron-sulfur centers in the solubilized fraction was verified by UV-visible spectroscopy. Fig. 8B shows that the truncated His 6 -BchE* preparation displays a spectrum between 300 and 600 nm characteristic of iron-sulfur proteins, with a major absorption peak around 410 nm and a weak additional broad absorption around 580 nm. Addition of sodium dithionite resulted in a shift (414 -424 nm) of the absorbance in the visible region, demonstrating that the iron-sulfur cluster was redox-active. These features are typical of many proteins containing 4Fe-4S clusters (17)(18)(19) but not of a 2Fe-2S cluster. The 2Fe-2S cluster usually shows distinct absorption peaks around 330, 420, 460, and 560 nm (20). The truncated His 6 -BchE* was purified from the solubilized fraction by column affinity (Fig. 8A, lane 1). The purified protein migrated as a band of ϳ43 kDa corresponding to the predicted molecular size of the tagged construct, and a second band that could correspond to the dimeric (86 kDa) form was also revealed. Similar UV-visible spectra were observed for the purified protein, with a peak around 410 nm for the oxidized form and a peak around 418 nm for the reduced form (Fig. 8B, inset) indicating the presence of a redox-active ironsulfur cluster.

DISCUSSION
By using H 2 18 O and 18 O labeling in vitro assays and mass spectrometry, Porra et al. (4) examined bacteriochlorophyll a formation in R. sulfidophilum, a strain that produces bacteriochlorophyll a both aerobically and anaerobically. Labeling experiments showed that under anaerobic conditions, the oxygen atom incorporated into the keto group on ring V of the MgPMe molecule is derived from H 2 O, whereas under aerobic conditions, the oxygen is derived from molecular oxygen (4). These data suggested two different mechanisms for MgPMe cyclization in this bacterium. Pinta et al. (3) have identified in R. gelatinosus AcsF as the aerobic Mg-protoporphyrin monomethyl ester cyclase and showed its involvement in the cyclization of MgPMe. In this report we have identified the anaerobic Mg-protoporphyrin monomethyl ester cyclase encoded by the bchE gene, and we showed that BchE is active only under low oxygenation or anaerobiosis. On the other hand, analysis of the mutants allowed the confirmation of the proposed two mechanism model for the cyclization and the identification of the involved acsF and bchE genes. Despite considerable sequence and structural dissimilarities between AcsF and BchE, both enzymes are very likely able to recognize the same substrate, MgPMe, and catalyze its conversion to Pchlide a. We propose that bacteriochlorophyll a biosynthesis is a branched pathway in R. gelatinosus and presumably in other facultative aerobic phototrophs, at the level of MgPMe (Fig. 5).
AcsF homologues were found in many chlorophyll-containing organisms as follows: in purple bacteria (R. sphaeroides and R. palustris), in green non-sulfur bacteria (Chloroflexus aurantiacus), in cyanobacteria (Prochlorococcus marinus, Anabaena sp. PCC 7120 and Synechocystis sp. PCC 6803), in red algae (Porphyra purpurea), in green algae (Chlamydomonas reinhardtii), and in plants (Oryza sativa and Arabidopsis thaliana). In all these organisms AcsF and homologues would be required for chlorophyll biosynthesis as shown in C. reinhardtii (21), and very likely for MgPMe cyclization to produce protochlorophyllide a under aerobic conditions as recently demonstrated for the CHL27 gene from Arabidopsis. 3 The acsF homologue was not found in the strict anaerobe Chlorobium tepidum (AE006470). Analysis of the deduced amino acid sequence of AcsF and its homologues showed the presence of two metallocenter iron ligand motifs (D/E)EXXH. This motif is characteristic of monooxygenases, a class of metalloproteins including the E. coli aerobic coproporphyrinogen oxidase (HemF) and the ribonucleotide reductase (NrdB), where two copies of the motif (D/E)X 28 -37) DEXXH provide ligands to bind a binuclear iron cluster (23). It is noteworthy that in the absence of oxygen, E. coli activates the anaerobic forms of coproporphyrinogen oxidase (HemN) and the ribonucleotide reductase (NrdG).
Among photosynthetic chlorophyll containing organisms, homologues of the bchE gene were found only in photosynthetic bacteria (purple, green, and cyanobacteria). Up to now, bchE gene homologues were not found in plants or in the green alga C. reinhardtii. By analogy with other anaerobic oxidoreductases (HemN and NrdG), BchE would catalyze the cyclization reaction via radical chemistry requiring the contribution of an iron-sulfur cluster. The iron-sulfur-binding motif including three cysteine residues (CXXXCXXC) was found in the BchE sequences, and the presence of the iron-sulfur center in the purified BchE protein was verified by UV-visible spectroscopy. This motif was also found in the amino acid sequences of all HemN proteins as well as in other proteins ( Fig. 9) including the anaerobic ribonucleotide reductase-activating enzyme (NrdG), biotin synthase (BioB), and lipoate synthase (LipA) (18,24). In all the enzymes these three cysteine residues are part of an unusual Fe-S cluster, and all these proteins have in common that they use SAM as a cofactor to form a radical involved in catalysis. A fourth conserved cysteine is found in all anerobic Mg-protoporphyrin monomethyl ester cyclases (Fig.  9); its role in the iron-sulfur binding should be verified by mutagenesis. It was shown by site-directed mutagenesis that the three cysteines of the conserved CXXXCXXC sequence in the anaerobic ribonucleotide reductase NrdG are sufficient for iron-sulfur binding (17).
Detailed analysis of heme metabolism, deoxyribonucleotide synthesis, and now the bacteriochlorophyll biosynthesis pathways revealed that the oxidative reaction step proceeds via different mechanisms depending on the oxygen growth conditions (Fig. 10). Oxygen-dependent reactions requiring molecular oxygen are catalyzed with di-iron-containing enzymes, whereas anaerobic enzymes requiring water contain SAM and iron-sulfur. The protein sequences derived from the genes of the aerobic enzymes are completely different from the se- quences derived from genes specifying anaerobic enzymes. The use of different classes of enzymes catalyzing the same reaction appears therefore to be a common feature of different biosynthesis pathways (Fig. 10). For instance, there have been reports of two coproporphyrinogen oxidase systems, one oxygen-requiring type (HemF) found in aerobic organisms using a binuclear iron center and another type found in anaerobic systems (HemN) requiring H 2 O as electron acceptor and functioning with an iron-sulfur cluster (19). In E. coli, three classes of ribonucleotide reductases have been classified according to the radical generator and oxygen growth conditions. Class I enzyme (NrdB) produces a tyrosyl radical through the action of a binuclear iron center, and class III enzyme (NrdG) produces a glycyl radical through the action of an iron-sulfur cluster and SAM (25,26).
In the case of bacteriochlorophyll biosynthesis, it was proposed that under anaerobiosis BchE catalyzes the cyclization of MgPMe via radical chemistry (27). The reaction should be SAM and vitamin B 12 -dependent, consistent with the reported phenotype for the R. capsulatus vitamin B 12 biosynthesis-deficient mutant accumulating MgPMe (27). Under aerobiosis, the aerobic enzyme AcsF is a di-iron-containing enzyme requiring molecular oxygen for the catalysis. A three-step model in which the Pchlide a is produced after hydroxylation and oxidations of MgPMe was proposed by Porra et al. (28).
Oxygen regulation and expression of genes encoding the aerobic or the anaerobic enzymes were studied in many bacteria. In Bradyrhizobium japonicum, no significant expression of the hemN gene was detected in aerobically grown cells; however, the gene expression was strongly induced under microaerobic or anaerobic conditions (29). In Pseudomonas aeruginosa expression of both hemF and hemN was induced during anaerobic growth (30). In E. coli, expression of the nrdB gene was also shown to decrease under anaerobiosis to a lower basal level, but the gene is still expressed under both conditions (31). Expression of the bchE gene was studied by assessing the promoter activity and the mRNA production under both aerobiosis and anaerobiosis, and the results clearly showed that the bchE gene is expressed irrespective of the oxygen growth conditions. However, the BchE Mg-protoporphyrin monomethyl ester cyclase is inactive under high oxygenation. This is probably due the sensitivity of the iron-sulfur cluster to oxygen. Further biochemical studies should be performed to identify the post-transcriptional steps that control the enzyme activity under aerobic conditions. To bypass this undesirable oxygen FIG. 9. Comparison of R. gelatinosus (R. gel) BchE sequence to different homologues and to proteins involved in anaerobic processes in E. coli. R. sph: R. sphaeroides; C. tep: C. tepidum; Synech: Synechocystis sp. strain PCC 6803. The conserved cysteines constituting the putative 4Fe-4S clusterbinding motif are indicated as boldface letters. The alignment was generated using ClustalW. N-and C-terminal parts of the proteins are not shown.
FIG. 10. Schematic representation of the two classes of enzymes catalyzing the same reaction under different oxygen conditions (aerobiosis versus anaerobiosis). Enzymes of the aerobic class (HemF, NrdB, and AcsF) require oxygen as a substrate and act through a binuclear iron center. Enzymes of the anaerobic class (HemN, NrdG, and BchE) use water as substrate, require SAM, and act through a 4Fe-4S cluster. BchE requires also vitamin B 12 (27). Sequences required for the formation of the di-iron or the 4Fe-4S centers are shown. effect, the aerobic organisms have acquired or maintained the aerobic form of the enzyme.
Coexistence of both aerobic and anaerobic cyclases in the facultative aerobic phototrophic bacteria raises an interesting question regarding the mechanism of chlorophyll biosynthesis in strict aerobes and strict anaerobes. The wide distribution of acsF homologues among the strict aerobic phototrophs concomitant to the lack of bchE homologues in the genomes analyzed so far leads us to suggest a simple scheme whereby strict aerobes possess and use only the aerobic Mg-protoporphyrin monomethyl ester cyclase, whereas the strict anaerobes devoid of acsF homologues possess and use only the anaerobic Mgprotoporphyrin monomethyl ester cyclase to produce chlorophyll (Fig. 5). That way, aerobic restricted plants do not need the anaerobic form of the Mg-protoporphyrin monomethyl ester cyclase and will make use of their aerobic cyclase; the strict anaerobic bacterium C. tepidum has no need of the aerobic AcsF enzyme and will exclusively retain the anaerobic cyclase form and make use of it. As for facultative aerobes, they enjoy the selective benefit of having both aerobic and anaerobic cyclases providing the competitive advantage of producing chlorophylls under both aerobic and anaerobic conditions. Surprisingly, no homologue of bchE gene was found in the C. reinhardtii, although this organism can grow and produce photosystem under oxygen-limited conditions. This alga possesses two AcsF homologue isoforms (crd1 and cth1); the crd1 was shown to be induced under low oxygen tension, and a crd1 mutant is chlorotic under such conditions (21,32) suggesting that chlorophyll biosynthesis is supplied by Crd1 under low oxygen tension.
In purple bacteria, the puf and puc operons, encoding proteins of the photosynthetic apparatus, are oxygen-regulated (14,15). A drop in the oxygen tension in the environment leads to an increase in the levels of puf and puc expression. Availability of bacteriochlorophylls, even in a low amount under aerobic conditions, will then be a significant advantage to rapidly build up the photosystem, and a sudden shift from aerobic to anaerobic conditions will result in cells qualified to immediately start photosynthetic growth following exposure to light and further reduction of oxygen tension.