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

Polymerase Chain Reaction Amplification

()

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`213 for the clone containing the region 5` exclusive to bchJ and p3`213 for the clone containing the region 3` exclusive to bchJ.

Plasmid Construction and Mobilization

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

Southern Blot Analysis

Whole Cell Absorption Spectrum Profiles

Pigment Extraction and Low Temperature Spectral Analysis

RESULTS

Spectral and Growth Characteristics of bchJ Mutants

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 213) was therefore constructed by replacing the entire bchJ coding region with a gene cassette containing the Tn5 km 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 213 (Fig. 4). A similar analysis was also performed with strains BPY69-DB213 and BPY69-213, which are strains containing the identical insertion and total deletion mutations of bchJ as described for DB213 and 213 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 213 and BPY69-213 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 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 (213, BPY69-213) of bchJ. A, Southern blot hybridized with a radiolabeled probe for bchJ. B, identical Southern blot hybridized with the radiolabeled probe of the Km 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, 213, BPY69-DB213, and BPY-213, 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

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 213. C, extracts from strain BPY4-213 that also harbored a plasmid control vector pPUFP1. D, extracts from strain BPY4-213, 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 213. 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 213 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 (BPY4-213; Table 1) that contained a mutation in protochlorophyllide reduction (bchL) in addition to bchJ. As shown in Fig. 5C, fluorescence emission spectral analysis of BPY4-213 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 213 (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 BPY4-213. 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 BPY4-213. 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

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.

()

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

ACKNOWLEDGEMENTS

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