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