©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Altered Monovinyl and Divinyl Protochlorophyllide Pools in bchJ Mutants of Rhodobacter capsulatus
POSSIBLE MONOVINYL SUBSTRATE DISCRIMINATION OF LIGHT-INDEPENDENT PROTOCHLOROPHYLLIDE REDUCTASE (*)

(Received for publication, August 22, 1994; and in revised form, November 23, 1994)

Jon Y. Suzuki (§) Carl E. Bauer (¶)

From the Molecular, Cellular, and Developmental Biology Program, Department of Biology, Indiana University, Bloomington, Indiana 47405

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In land plants in particular, it has been well established that chlorophyll intermediates, Mg-protoporphyrin, Mg-protoporphyrin monomethylester, protochlorophyllide, and chlorophyllide occur as monovinyl and divinyl forms. The pool of monovinyl and divinyl intermediates differ according to species, age of tissue, and light regime. In this study, we investigated the monovinyl and divinyl characteristics of protochlorophyllide and chlorophyllide in the purple non-sulfur photosynthetic eubacterium Rhodobactercapsulatus. Our results indicate that mutations in genes known to completely block the reduction of protochlorophyllide to chlorophyllide (such as bchN, bchB, and bchL mutants), accumulate a pool of monovinyl and divinyl forms of protochlorophyllide just as observed in plants. However, we also observed that directed insertion and deletion mutations in bchJ, a gene located in the photosynthesis gene cluster, affected the ratio of monovinyl and divinyl protochlorophyllide. Specifically, bchJ-disrupted strains accumulate reduced levels of bacteriochlorophyll concomitant with the accumulation of divinyl protochlorophyllide. Mutants of bchJ in combination with a second mutation in bchL still produce a mixed pool of monovinyl and divinyl protochlorophyllide; however, the ratio of monovinyl to divinyl protochlorophyllide is skewed in favor of divinyl protochlorophyllide. These results thus identify bchJ as the first sequenced gene that affects the divinyl to monovinyl ratio of photopigment intermediates in any photosynthetic organism. In addition, the results of our study also suggest that light-independent protochlorophyllide reductase is discriminatory for a monovinyl substrate.


INTRODUCTION

In plants and algae, it has been demonstrated that chlorophyll intermediates exist in monovinyl and divinyl forms. Divinyl intermediates contain vinyl side groups at rings A and B, whereas monovinyl forms contain a vinyl group at ring A and an ethyl group at ring B. (The structure of mono- and divinyl forms of the intermediate protochlorophyllide are shown (Fig. 1)). The ratio of accumulated monovinyl to divinyl intermediates have been shown to be altered by physiological and environmental factors such as age and light exposure for several intermediates, especially protochlorophyllide(1, 2, 3, 4, 5, 6, 7, 8, 9) . It has also been demonstrated that the character of these alterations are unique for different photosynthetic organisms(1, 2, 3) . However, it is as yet unclear as to the significance of having pools of monovinyl and divinyl intermediates as monovinyl chlorophyll a is the final product in the biosynthetic pathway for most oxygenic phototrophes. The only exception so far is abundant marine prochlorophytes for which divinyl chlorophyll is the final major product of the biosynthetic pathway(11, 12, 13) .


Figure 1: Pathway of bacteriochlorophyll biosynthesis in R. capsulatus from protochlorophyllide as discussed in text. BchJ is shown to denote possible involvement in determining the divinyl (DV) to monovinyl (MV) ratio of the protochlorophyllide pool in R. capsulatus. Blackboxes denote relevant vinyl groups. Bacteriochlorophyll biosynthesis genes involved various steps of the pathway are indicated adjacent to the arrows.



Even though the functional significance of divinyl intermediates is not known, the nature of divinyl reductase(s) is important to investigate from the standpoint of understanding the complexity of chlorophyll biosynthesis. For example, a number of studies propose that the monovinyl and divinyl pools of different intermediates represent separate routes for chlorophyll biosynthesis and that there exists multiple unique divinyl reductases that are responsible for reducing different divinyl intermediates (for review, see (8) and (14) ). Alternative models propose that there is a single divinyl reductase enzyme with unique intermediate-specific components or one enzyme exhibiting broad substrate specificity(1) . It has also been proposed that reduction of the ring B vinyl group may also occur by initial reduction of a pyrrole subunit double bond followed by double bond migration that gives rise to 4-vinyl group reduction(15) . The existence of divinyl pools and the implication of multiple divinyl reductases or intermediate-specific components thus leads to another layer of regulation requiring maintenance and perhaps coordinated developmental expression of the respective genes in the genome.

In a previous study, our laboratory reported that mutants of Rhodobacter capsulatus, which completely block bacteriochlorophyll a biosynthesis at the step of protochlorophyllide reduction, accumulated both monovinyl and divinyl forms of protochlorophyllide(16) . The existence of both monovinyl and divinyl intermediates indicated that purple photosynthetic bacteria, like that reported for plants, may also contain a 4-vinyl reductase enzyme. Genetic investigations on bacteriochlorophyll biosynthesis in R. capsulatus has also indicated that all of the known genes involved in the magnesium tetrapyrrole branch of the bacteriochlorophyll biosynthetic pathway are clustered to a 45-kilobase pair region of the genome termed the photosynthesis gene cluster(17) . The photosynthesis gene cluster has been sequenced, and identified open reading frames have been disrupted by directed mutagenesis(10, 16, 18, 19, 20) . During the course of these studies, we observed that a mutation in open reading frame orf213 (bchJ) resulted in a mutant that accumulated protochlorophyllide as well as functional bacteriochlorophyll(10) . The phenotype of the bchJ mutant, which involved only a partial blockage of protochlorophyllide reduction, is thus distinct from that of mutations that disrupt the protochlorophyllide reductase enzyme complex that effectively block bacteriochlorophyll biosynthesis at this step of the pathway(10, 16, 19) . From this initial observation, bchJ was postulated to be required for maximal activity of the light-independent protochlorophyllide reductase enzyme complex. In this study, we reinvestigated the effect of bchJ on bacteriochlorophyll biosynthesis and present evidence that disruptions of this open reading frame leads to alterations in the monovinyl/divinyl pools of protochlorophyllide. bchJ therefore appears to code for a polypeptide that is involved in reduction of the 4-vinyl group of protochlorophyllide rather than in protochlorophyllide reduction perse. We also discuss evidence that light-independent protochlorophyllide reductase may be discriminatory for monovinyl protochlorophyllide versus that of divinyl protochlorophyllide.


MATERIALS AND METHODS

Plamids, Strains, Media, and Growth Conditions

Plasmid and strains utilized in this study are listed in Table 1. Strains of R. capsulatus were routinely grown in RCV 2/3 PY medium (21) under semiaerobic conditions (50 ml of liquid medium in a 125-ml Erlenmeyer flask shaken at 200 rpm) or under photosynthetic (anaerobic) conditions in a screw cap tube. A light source of approximately 5,000 lux was utilized for cultures grown photosynthetically, except where noted in the text. Growth rate of photosynthetic cultures was monitored by light scattering at 660 nm in a Klett-Summerson photometer. Relevant R. capsulatus strains were grown in 2.5 µg/ml streptomycin and in some cases 5.0 µg/ml kanamycin for plasmid maintenance. Escherichia coli strain NM522 (22) was used routinely for cloning procedures and plasmid DNA amplification.



Polymerase Chain Reaction Amplification

A total of four polymerase chain reaction (PCR) (^1)amplifications were performed in this work. Two PCR reactions were performed to amplify the structural gene of bchJ (oRF213), the coding sequence of which is positioned at coordinates 18511-19152 of the R. capsulatus photosynthetic gene cluster (EMBL accession number Z11165). The first PCR product of 690 base pairs was designed to include a region upstream of the bchJ structural gene including the putative endogenous Shine-Dalgarno sequence as well as to engineer KpnI and BglII restriction sites flanking the gene. For this amplification the 27-mer primer 5`BchJKpn (5` CGC GGT ACC TCG ACG CCG CGG AAT AAG 3`) was used as the ``upstream'' primer, and the 28-mer 3`BchJBglII (5` GCG AGA TCT TCA GCC GGA GTG GAC AGA G 3`) was used as the ``downstream'' primer. The trinucleotide sequence TCG of the 5`BchJKpn primer represents the region starting at 18,480 on the photosynthesis gene cluster (PGC) whereas the sequence TCA of primer 3`BchJBglII is complementary to the bchJ stop codon TGA, which ends at 19,152 of the PGC. The plasmid RPS404 (17) was digested with XhoI and BglII and used as the template for amplification at a concentration of 0.14 or 0.014 µg/PCR reaction. Vent polymerase (New England Biolabs) was used as per manufacturers instructions without MgS0(4) and without added bovine serum albumin. Temperature regime used was as follows: 98 °C for 5 min, 72 °C for 4 min during which Vent polymerase was added along with oil, 98 °C for 2.5 min followed by 30 cycles of 97 °C for 20 s, 60 °C for 30 s, and 72 °C for 1 min. The reaction was completed with an incubation at 72 °C for 7 min followed by a holding period at 4 °C. The PCR product was ligated into the SmaI site of pUC19(23) . The resulting plasmid, pUC19BchJ, was found to contain bchJ with the 5` end adjacent to the EcoRI site of pUC19. A second PCR reaction of bchJ was performed to amplify a product that excludes all noncoding regions upstream and downstream of the structural gene. For this reaction, the 26-mer 5`NdeIBchJ (5` GCG CAT ATG AGC GGT GCC GCG CCT GC 3`) and the 29-mer 3`BchJ (5` GCG AGA TCT TCA GCC GGA GTG GAC AGA GC 3`) were used as amplification primers. The plasmid pTZ19uBchJ, a derivative of pUC19BchJ, was digested with PstI and EcoRI-purified with Elu-Quik (Schleicher and Schuell) and utilized as the PCR template. The amplification regime described above was utilized for amplification using Vent DNA polymerase (2 units/100 µl reaction). The PCR product was subsequently cloned into the HincII site of vector pTZ19r (U. S. Biochemical Corp.) resulting in the clone named pTZ19rBchJ.

Two sets of primers were utilized to PCR amplify the 5` and 3` regions exclusive to the bchJ gene. For the 5` region amplification, the 27-mer JS5`RI (5` CGA ATT CGC CGA CAT CTT CAC CGA AGC 3`) and the 28-mer JS5`BAM (5` CGG ATC CGC CGT CAC TCC TTC TTA TTC C 3`) were used along with the plasmid pDB30 ((14) , BamHI fragment 15468-18791 of the PGC) as the template. PCR product size was estimated to be about 733 base pairs. For the 3` region amplification, the 26-mer JS3`BAM (5` CGG ATC CAG GCC CCT TCG GGC CCT TG 3`) and 27-mer JS3`BglII (5` GAG ATC TAC ATC ACC ACGCCG GTG CC 3`) were used along with the plasmid pDB26 ((10) , BamHI, PstI fragment of coordinates 18,791 and 20,827, respectively, of the photosynthesis gene cluster) as the template. PCR product size was expected to be about 907 base pairs. The 100-µl PCR amplification reactions were performed using Tricine buffer (24) and Taq polymerase. The following regime was utilized for amplification: 98 °C for 5 min, 72 °C for 4 min during which Taq polymerase and oil was added, followed by 30 cycles of 98 °C 30 s, 60 °C for 30 s, 72 °C for 1 min. The amplification reaction was completed with an incubation at 72 °C for 7 min and a final incubation at 4 °C. The PCR products from the above two reactions were ligated into the TA cloning vector pT7Blue T-Vector (Novagen) and named p5`Delta213 for the clone containing the region 5` exclusive to bchJ and p3`Delta213 for the clone containing the region 3` exclusive to bchJ.

Plasmid Construction and Mobilization

All enzymes were purchased from New England Biolabs, except where noted. Molecular biological techniques were performed as described by Ausubel et al., (25) except where noted. To create the plasmid construct with a complete deletion of bchJ for interposon (Kan^R) selection (pPUFDelta213Kan), a EcoRI, BglII fragment insert from p3`Delta213 was first ligated into the corresponding sites of the pPUFHC vector to create pPUF3`Delta213. The 733-base pair EcoRI, BamHI fragment from p5`Delta213 was then ligated to the corresponding sites in pPUF3`Delta213 to create the plasmid pPUFDelta213, which contains the 5` and 3` regions of the bchJ gene exclusive to the bchJ gene. Finally, the BamHI fragment containing the Km^R gene from pUC4KanKixx (Pharmacia Biotech Inc.) was ligated into the BamHI site of pPUFDelta213 resulting in plasmid pPUFDelta213Kan. The bchJ expression plasmid pPUF:BchJ was created by ligating the BglII, EcoRI insert fragment from pTZ19uBchJ (derivative of pUC19BchJ) into the BamHI, EcoRI sites of vector pPUFP1. Relevant plasmids were mobilized into R. capsulatus from E. coli strain XL1BlueMR (Stratagene) containing the mobilizing plasmid pDPT51 (26) or from strain S17-1(27) .

Directed Mutations in the R. capsulatus Genome at the bchJ (orf213) Locus

Directed insertion or deletion mutations of bchJ (DB213 or Delta213, respectively) in various strain backgrounds were performed by gene transfer agent-mediated interposon mutagenesis as described previously(10, 28) . For the novel Delta213 mutation created in this work, gene transfer agent was prepared from the gene transfer agent-overproducing strain CB1127 (21) containing the plasmid pPUFDelta213Kan.

Southern Blot Analysis

DNA from parent and selected mutant strains of R. capsulatus were isolated by the cetyltrimethylammonium bromide method, and CsCl(2) gradient purified(24) . Two sets of digestions, BglII and SalI, were performed as per manufacturers instructions (New England Biolabs). DNA equivalent to about 1 µg/lane was loaded on 0.7% agarose gel and Southern blotted in duplicate. Two probes were used in separate hybridization experiments; the NdeI, BglII fragment containing the bchJ structural gene from pTZ19rBchJ5 and the BamHI fragment containing the Km^R gene from pUC4KanKixx. DNA fragments were isolated by Elu-Quik (Pharmacia) and random primer-labeled using 25 µCi of 3000 Ci/mmol of [P]dATP (Amersham Corp.) as per manufacturer's instructions (Random Primed DNA labeling kit, U. S. Biochemical Corp.). Hybridization was performed in 0.2% Blotto, 1.0% Nonidet P-40, 10 µl of antifoam/100 ml of hybridization solution, at 65 °C overnight. The blots were washed twice in 2 times SSC, 0.1% SDS. for 20 min at room temperature followed by two washes in 0.2 times SSC, 0.1% SDS, for 20 min at 65 °C.

Whole Cell Absorption Spectrum Profiles

1.0-1.5 ml of cells were pelleted in an Eppendorf centrifuge tube. The cell pellet was resuspended in 50 µl of RCV 2/3 PY media and mixed with 0.5-1.5 ml of 30% bovine serum albumin and scanned as described previously (29) using a Beckman DU-50 recording spectrophotometer.

Pigment Extraction and Low Temperature Spectral Analysis

10-ml samples from 1-day-old saturated 50-ml cultures grown semiaerobically in RCV 2/3 PY were pelleted in Falcon 2059 tubes for 10 min at 10,000 rpm in an SS-34 (DuPont) rotor. Cell pellets were frozen and held at -80 °C until extraction. Thawed samples were resuspended in 7.0 ml of ammoniacal acetone (acetone/0.1 N NH(4)OH) and centrifuged in a JA20 fixed angle rotor 18,000 rpm for 12 min to pellet debris. An equal volume of hexane was added to the supernatant and mixed by perfusion. The phases were separated in a clinical centrifuge, and the hexane phase was removed. This was repeated a second time with 2.0 ml of hexane. The hexane extracted supernatant was treated as described previously to partition pigments into ether as preparation for spectroscopic analysis(2) . Low temperature (77 K) spectroscopy was performed, and results were calculated and recorded in the lab of Dr. Constantin Rebeiz (University of Illinois, Champaign, Illinois) as described previously(30, 31) .


RESULTS

Spectral and Growth Characteristics of bchJ Mutants

A directed insertion mutation in bchJ (strain DB213) was previously constructed by our laboratory while performing systematic insertion mutational analyses of the photosynthesis gene cluster(10) . The insertion mutation in DB213 disrupts bchJ at codon 144 (out of a total of 213 codons). Unlike cultures of wild type R. capsulatus, which exhibit red coloration, cultures of strain DB213 have a green hue indicating that they have an alteration in pigment production. Whole cell spectral analysis (Fig. 2) indicates that DB213 is capable of bacteriochlorophyll synthesis as indicated by the absorbance peaks at 800 and 850 nm; however this strain also accumulates an additional compound at 633 nm, which is characteristic of protochlorophyllide in aqueous solution. One of the obvious effects of this mutation is that DB213 also accumulates reduced levels of bacteriochlorophyll as evidenced by a reduction in the 800 and 850 nm absorbing pigment-protein complexes. Identical insertion mutations in bchJ were also made in various genetic backgrounds that only allowed synthesis of light harvesting-I (875 nm absorbing), or light harvesting-II (800, 850 nm absorbing) complexes, and in both cases the cells also synthesized lower amounts of bacteriochlorophyll (data not shown). We concluded therefore that the reduction in these pigment-protein complexes is most likely due to the limited available bacteriochlorophyll caused at the expense of accumulated protochlorophyllide. Strain DB213 is capable of photosynthetic growth, thereby indicating that this strain synthesizes a functional photosystem. However, as shown by growth curves in Fig. 3, the photosynthetic growth rate of DB213 is reduced over that observed with the parent strain SB1003. The observed reduction in the photosynthetic growth rate is more pronounced as light intensity is decreased (Fig. 3). Since the cells grow normally under high light intensity it would indicate that the cells have the capability to synthesize a functional photosystem. We therefore presume that the observed reduction in growth rate reflects limitation in bacteriochlorophyll biosynthesis rather than toxicity of the accumulated protochlorophyllide.


Figure 2: Whole cell absorption spectra of wild-type, SB1003, and bchJ mutant DB213 of R. capsulatus grown under semi-aerobic conditions. The dottedline represents the spectrum of SB1003. The solidline represents the spectrum from DB213. Absorption maximum at 590, 800, and 850 nm represent bacteriochlorophyll absorbance, whereas the indicated absorption peak at 633 nm represents accumulated protochlorophyllide by strain DB213.




Figure 3: Photosynthetic growth rate profile of R. capsulatus strain SB1003 (open boxes) or the bchJ mutant strain DB213 (closed boxes). A, moderate light (5380 lux); B, high light (10,760 lux).



Since DB213 contains an insertion mutation in the latter half of the gene, there is the formal possibility that the observed phenotype is the result of only a partial disruption of a polypeptide that is essential for protochlorophyllide reduction. It was therefore important to construct a null mutation in bchJ so that we could properly assess the role of bchJ in bacteriochlorophyll biosynthesis. A complete deletion of bchJ from the genome of R. capsulatus (strain Delta213) was therefore constructed by replacing the entire bchJ coding region with a gene cassette containing the Tn5 km^r structural gene (Table 1; see ``Materials and Methods'' for details of this strain construction). Disruption of bchJ was confirmed by performing Southern blot analysis of digested genomic DNA that was obtained from the parent strain SB1003 and the bchJ mutant strains DB213 and Delta213 (Fig. 4). A similar analysis was also performed with strains BPY69-DB213 and BPY69-Delta213, which are strains containing the identical insertion and total deletion mutations of bchJ as described for DB213 and Delta213 with the exception that the genetic background of these additional strains (BPY69) also contains a mutation that inhibits carotenoid biosynthesis (Table 1). As observed in Fig. 4A, bchJ deletion strains Delta213 and BPY69-Delta213 exhibit a complete loss of bchJ as indicated by the absence of hybridization of the genomic DNA to the bchJ probe. This is in contrast to wild type and DB213 genomic DNA preparations that did exhibit hybridization to the bchJ probe. Fig. 4B shows the result of hybridizing the identical Southern blot with the Km^R gene that was utilized to disrupt the bchJ locus. As expected, only strains mutant for bchJ exhibit hybridization to this probe. Spectral analysis and growth characteristics of the bchJ deleted strains were observed to be indistinguishable to that described above for the bchJ insertion strain DB213, thereby indicating that loss of the bchJ gene product results in only a partial blockage of bacteriochlorophyll biosynthesis at the protochlorophyllide reduction step of the pathway (data not shown).


Figure 4: Southern blot analysis of genomic DNA from R. capsulatus strains which are wild-type (SB1003, BPY69), insertion mutants (DB213, BPY69-DB213) or deletion mutants (Delta213, BPY69-Delta213) of bchJ. A, Southern blot hybridized with a radiolabeled probe for bchJ. B, identical Southern blot hybridized with the radiolabeled probe of the Km^R gene isolated from pUC4KanKixx (see ``Materials and Methods.''



Even though the above results indicate that a disruption of bchJ leads to only a partial inhibition of bacteriochlorophyll biosynthesis there is also the formal possibility that the observed phenotype is a result of polarity of the mutations causing reduced expression of an essential downstream gene that is involved in this step of the pathway. To ensure that polarity was not causing the observed phenotype, we also constructed a plasmid that trans-expresses only the bchJ gene product (pPUF:BchJ). When plasmid pPUF:BchJ is mated into bchJ-disrupted strains DB213, Delta213, BPY69-DB213, and BPY-Delta213, we observed a complete restoration of bacteriochlorophyll biosynthesis to wild type levels and an absence of detectable amounts of protochlorophyllide (data not shown). From these results we can conclude that the phenotype described above for DB213 is the true phenotype of a null mutation in bchJ. This is contrasted to the phenotype observed with mutations in the light-independent protochlorophyllide reductase subunits BchL, BchB, or BchN, which result in the complete blockage of bacteriochlorophyll biosynthesis at the step of protochlorophyllide reduction concomitant with the loss of photosynthetic growth capability(10, 16, 19) .

Monovinyl and Divinyl Pools of Protochlorophyllide-accumulating Mutants

Further analysis of the protochlorophyllide intermediate accumulated by bchJ-disrupted strains was subsequently undertaken to determine the nature of the ring B side group (4-vinyl versus 4-ethyl). For this analysis, we performed low temperature (77 K) fluorescence excitation spectral analysis of protochlorophyllide that was extracted from R. capsulatus cell cultures. At low temperature, monovinyl protochlorophyllide exhibits a maximal fluorescence emission at 625 nm (F) when excited at 437 nm, as well as minor F emission when excited at 443 nm. On the other hand, divinyl protochlorophyllide exhibits maximum F emission when excited at 443 nm and a minor F emission when excited at 451 nm. (A formula for calculating the molar amounts of monovinyl and divinyl protochlorophyllide as based on the fluorescence excitation spectra has been described previously(30, 31) ). Shown in Fig. 5A is the results of low temperature spectral analysis of protochlorophyllide extracted from strain BPY4, which contains a mutation in the protochlorophyllide reductase subunit BchL. (The spectrum shown is of a BPY4 strain that contains plasmid pPUFP1 as a control for complementation analysis described below). The spectra shows two excitation peaks, one at 437 nm (monovinyl) and the other at 443 nm (monovinyl + divinyl) as well as a shoulder at 451 nm (divinyl). The calculated monovinyl to divinyl protochlorophyllide ratio from this spectrum is 1.4 (Table 2). A similar ratio of monovinyl to divinyl protochlorophyllide is observed with strains JDA, JDB, ZY5, and Y80 (Table 1), which are strains that also contain mutations in one of the three protochlorophyllide reductase subunits (bchB, bchL, or bchN).


Figure 5: Tracings of the low temperature 625 nm fluorescence excitation spectra (F625) of nonesterified pigments from various R. capsulatus strains in ether, with excitation at wavelengths between 380 nm-500 nm at 77K. A, extracts from strain BPY4, which also contains a control vector plasmid pPUFP1. B, extracts from strain Delta213. C, extracts from strain BPY4-Delta213 that also harbored a plasmid control vector pPUFP1. D, extracts from strain BPY4-Delta213, which was complemented in trans with plasmid pPUF:BchJ.





The excitation spectrum shown in Fig. 5B is of protochlorophyllide that was extracted from the bchJ-disrupted strain Delta213. This spectrum shows the absence of a 437-nm peak as well as a more pronounced 451-nm peak, characteristic features of divinyl protochlorophyllide(30, 31) . Indeed, as indicated in Table 2, protochlorophyllide accumulated by both Delta213 as well as strain DB213 is essentially 100% divinyl. This result indicates that bchJ mutants are not only distinct from light-independent protochlorophyllide reductase mutants in that a bchJ mutation results in only a partial inhibition of this step of the pathway, but, in addition, the protochlorophyllide accumulated by bchJ mutants is of differing chemical nature (all divinyl). It is unlikely therefore that the bchJ gene product is simply affecting the activity of light-independent protochlorophyllide reductase, since if that were occurring then one would expect that the pool of protochlorophyllide accumulated by bchJ mutants would be of both monovinyl and divinyl forms, which is not the observed case.

As noted above, the pool of protochlorophyllide that is accumulated by bchJ mutant strains is dissimilar from protochlorophyllide accumulated by light-independent protochlorophyllide reductase mutants in two respects: (i) bchJ mutants accumulate protochlorophyllide that is exclusively 4-vinyl and, (ii) unlike protochlorophyllide reductase mutants, which completely inhibit protochlorophyllide reduction, the bchJ disruption causes only a partial blockage at this step of the pathway and thus these cells accumulate both bacteriochlorophyll as well as the additional pool of divinyl protochlorophyllide. Since the final product of the pathway, bacteriochlorophyll a, is reduced at the 4-vinyl position(15) , it is possible that the bchJ mutants fail to accumulate monovinyl protochlorophyllide as a consequence of this intermediate being rapidly utilized as a substrate for bacteriochlorophyll biosynthesis. To test the possibility that monovinyl protochlorophyllide is being bled off into the synthesis of bacteriochlorophyll, we constructed a double mutant strain of R. capsulatus (BPY4-Delta213; Table 1) that contained a mutation in protochlorophyllide reduction (bchL) in addition to bchJ. As shown in Fig. 5C, fluorescence emission spectral analysis of BPY4-Delta213 shows a decrease in the monovinyl 437-nm peak and an increase in the divinyl 451-nm shoulder relative to that observed for BPY4 (Fig. 5A). Importantly, the emission spectrum is not all divinyl as is the case for strain Delta213 (Fig. 5B). This indicates that bchJ-disrupted strains still have the capability of making some monovinyl protochlorophyllide. Quantitation of the spectral results (Table 2) indicate that the ratio of monovinyl to divinyl protochlorophyllide decreases from 1.7 for strain BPY4 to a value of 0.31 for the double mutant strain BPY4-Delta213. Also indicated in Table 2and in the emission spectrum of Fig. 5D is the observation that the pool of monovinyl protochlorophyllide can be returned to near normal levels when bchJ is added in trans to strain BPY4-Delta213. These results demonstrate that although bchJ affects the ratio of monovinyl to divinyl protochlorophyllide, bchJ is not absolutely required for the accumulation of monovinyl protochlorophyllide. The significance of this observation in regards to possible substrate level discrimination of light-independent protochlorophyllide reductase will be covered in more detail under ``Discussion.''

Monovinyl and Divinyl Characterization of Chlorophyllide

We also investigated the monovinyl and divinyl pools of chlorophyllide that are formed as the product of the light-independent protochlorophyllide reductase reaction. We investigated the vinyl nature of this pool to ascertain whether light-independent protochlorophyllide reductase may favor monovinyl protochlorophyllide as a substrate over that of divinyl protochlorophyllide. If substrate level discrimination is indeed occurring then it would be expected that chlorophyllide would be predominately of monovinyl form. To study the monovinyl/divinyl characteristics of chlorophyllide, we performed room temperature spectral analysis (Fig. 6) and low temperature fluorescence excitation spectral analysis on the bchA-bchF-disrupted strain CB1200, which accumulates chlorophyllide. As indicated in Table 2, we observed that the chlorophyllide pool accumulated by CB1200 is indeed predominantly monovinyl (>98%). Interestingly, we also observed that when the bchJ mutation was introduced into the CB1200 strain (creating CB1200-DB213) this construct also accumulated a pool of protochlorophyllide in addition to chlorophyllide (Fig. 6). Furthermore, the pool of protochlorophyllide that is accumulated by CB1200-DB213 is predominantly divinyl despite the fact that the chlorophyllide pool is predominantly monovinyl (Table 2).


Figure 6: Whole cell absorption spectra of strain CB1200 (bchZ, bchF) and CB1200-DB213 (bchZ, bchF, bchJ) of R. capsulatus grown under semi-aerobic conditions. The dottedline represents the spectrum of CB1200. The solidline represents the spectrum from CB1200-DB213. The absorption maximum at 633 nm represents accumulated protochlorophyllide, whereas the absorption maximum at 670 nm represents chlorophyllide.




DISCUSSION

Despite numerous spectral and enzymatic studies, the sequence of events that leads to 4-vinyl reduction is still an area of uncertainty. What is clear from our study, as well as those of Rebeiz and co-workers (4, 8) , is that there exists a complex pattern of monovinyl and divinyl magnesium tetrapyrrole intermediates that appears to be present in most phototrophes. From enzymatic studies in plant systems, it is still unclear whether there are a set of distinct different enzymes responsible for reducing the 4-vinyl group of different magnesium tetrapyrrole intermediates or, alternatively, whether there is one enzyme that exhibits reduced substrate specificity or possibly a ``core enzyme'' that interacts with secondary subunit(s) that confers differing substrate specificities. Genetic investigations have also not shed much light on this reaction since there are only a few reports of mutants which affect the 4-vinyl step of magnesium tetrapyrrole biosynthesis(32) .

Although we cannot unequivocally rule out that bchJ codes for a polypeptide that directly affects the light-independent protochlorophyllide reductase enzyme complex, we favor a model that involves bchJ in 4-vinyl reduction. Our reasoning for this conclusion is based on several observations. Foremost is that the bchJ phenotype is quite distinct from that of light-independent protochlorophyllide reductase mutants, specifically, our analysis indicates that null mutations of bchJ result in a phenotype exhibiting only partial blockage of bacteriochlorophyll biosynthesis at the point of protochlorophyllide reduction. This phenotype differs from protochlorophyllide reductase mutations that exhibit a complete blockage of bacteriochlorophyll biosynthesis at this step of the pathway. Furthermore, protochlorophyllide reductase mutants accumulate a mixed pool of monovinyl and divinyl forms of protochlorophyllide, a phenotype that also differs from bchJ mutants that accumulate only the divinyl form of this intermediate. To a large extent, the bchJ phenotype is similar to that observed by Shioi et al.(33) in which they demonstrated that culturing R. sphaeroides cells in the presence of nicotinamide resulted in accumulation of divinyl protochlorophyllide. They reasoned that nicotinamide was perhaps a competitive inhibitor of a putative protochlorophyllide 4-vinyl reductase enzyme. In fact, similar experiments performed in our laboratory indicate that the whole cell absorption spectrum of wild type R. capsulatus grown in media supplemented with nicotinamide is similar to that of a bchJ mutant (data not shown). Assuming that light-independent protochlorophyllide reductase is indeed discriminatory for a monovinyl substrate, this would explain why a pool of divinyl protochlorophyllide accumulates in a bchJ mutant background, given that disruption of bchJ leads to elevated levels of divinyl protochlorophyllide.

If bchJ is indeed directly involved in the reduction of the 4-vinyl group of protochlorophyllide, then several adhoc assumptions will have to be made about the role of the polypeptide in this reaction. For example, it is clear that bchJ is not the only factor that is involved in reduction of this side group. This is evident from our observation that whereas strains, which are unable to reduce protochlorophyllide (such as bchL, B, or N mutants), accumulate a mixed pool of monovinyl and divinyl protochlorophyllide, the introduction of a second bchJ mutation in these strains does not result in the sole accumulation of divinyl protochlorophyllide. Instead, the introduction of a bchJ mutation to a strain blocked in protochlorophyllide reduction only skews the pool toward an elevated level of divinyl protochlorophyllide. Taken alone, these results suggest that there may exist a second 4-vinyl reductase enzyme that is able to convert earlier divinyl intermediates to monovinyl form. Alternatively, bchJ may code for a nonessential subunit of a 4-vinyl protochlorophyllide reductase enzyme complex that is able to partially function in its absence. We favor the former model since we have also observed that the magnesium protoporphyrin monomethyl ester pool in R. capsulatus also appears to contain both monovinyl and divinyl forms, the nature of which does not appear to be greatly altered by the presence or absence of bchJ (data not shown).

Our conclusion that light-independent protochlorophyllide reductase may be discriminatory for a monovinyl substrate also provides some insight to observations made in plants where pools of divinyl and monovinyl intermediates are known to vary according to species, growth conditions, age of tissue, and importantly light regime. For example, it has been recently been shown that cyanobacteria, algae, and nonflowering land plants exhibit two forms of protochlorophyllide reductase (for review, see (34) ). One form requires light for catalysis and is therefore termed light-dependent protochlorophyllide reductase, whereas the other form functions irrespective of light and is termed light-independent protochlorophyllide reductase (plant light-independent protochlorophyllide reductase is structurally and functionally related to the R. capsulatus protochlorophyllide reductase enzyme complex). In contrast to the presence of both forms of protochlorophyllide reductase in ``dark greening'' organisms, angiosperms (flowering land plants) appear to only contain the light-dependent version. As a consequence, these plants require light for greening (i.e. for production of chlorophyll). (Interestingly, it has been shown that the light-dependent version appears to favor divinyl protochlorophyllide as a substrate over that of monovinyl protochlorophyllide(1) , which is the inverse of what may be occurring for the light-independent enzyme.) Taking these observations into account, a metabolic grid pertaining to monovinyl and divinyl pools of protochlorophyllide and chlorophyllide can be drawn as shown in schematic in Fig. 7. For dark greening organisms, one would predict that a pool of divinyl protochlorophyllide would accumulate under dark growth conditions since the light-independent enzyme would preferentially utilize monovinyl protochlorophyllide as a substrate. Dark synthesis of chlorophyll that occurs in the in these organisms would, in essence, remove monovinyl protochlorophyllide from the pool in a manner analogous to that observed by the bchJ-disrupted strains of R. capsulatus. Indeed, one characteristic feature of dark-greening plant species is that they are known to accumulate protochlorophyllide in darkness that is predominately of the divinyl form(2) . On the other hand, when angiosperms are growing in the dark, they lack the capability to reduce protochlorophyllide and would therefore be expected to accumulate a mixed pool of monovinyl and divinyl protochlorophyllide. One would presume that the composition of the resulting protochlorophyllide pool in these plants would simply reflect the activity of 4-vinyl reductase enzyme(s). Not surprisingly, it has been observed that the ratio of monovinyl to divinyl protochlorophyllide that is accumulated by dark grown angiosperms varies widely among dicotyledonous and monocotyledenous species(1, 3) .


Figure 7: Proposed scheme for the latter steps in chlorophyll biosynthesis in phototrophes containing both light-independent (PCR) and light-dependent (POR) protochlorophyllide reductase activities. Thickness of arrowlines represent proposed relative enzyme activities.




FOOTNOTES

*
This work was supported in part by a Research Career Development Award from the National Institutes of Health (to C. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship administered by the Institute for Molecular Biology, Indiana University. Present address: Center for Gene Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-01, Japan.

To whom correspondence should be addressed: Dept. of Biology, Indiana University, Jordan Hall, Bloomington, IN 47405. Tel.: 812-855-6595. Fax: 812-855-6705.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; KmR, Kanamycin resistance; PGC, photosynthesis gene cluster.


ACKNOWLEDGEMENTS

We thank Dr. Constantin Rebeiz and Ramin Parham for generous use of their fluorescence spectrophotometric facilities and for helpful discussions.


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