Introduction

Chlorophyllous pigments are essential for photosynthetic organisms to harvest light energy and drive photochemical reaction centers (RCs). Phototrophic species in the phylum Proteobacteria (so-called purple bacteria) produce either bacteriochlorophyll (BChl) a or BChl b, depending on species and utilize these pigments for photochemistry1,2. The difference in chemical structures between BChls a and b occurs at the C8 position, an ethyl group on BChl a and an ethylidene group on BChl b (Fig. 1). The characteristic C8-ethylidene group on BChl b provides the extension of the π-conjugated system on the parental bacteriochlorin ring and gives rise to the red shift in the longest wavelength absorption band (Qy band) of BChl b with respect to that of BChl a. Consequently, BChl b is the sole natural pigment that can efficiently absorb light energy of near-infrared wavelength (≥ 800 nm) in the monomeric state, although BChl a has the Qy band slightly overlapping the near-infrared light region. The pigment is useful for developing artificial light-harvesting systems, such as dye-sensitized solar cells and photodynamic therapy, which are in demand to utilize uncaptured photons in longer wavelength.

Figure 1
figure 1

Late steps of BChl a biosynthesis in R. sphaeroides (top) and of BChl b biosynthesis in B. viridis (bottom).

The presented λmax peak positions are values obtained in diethyl ether. E = COOCH3.

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Purple bacteria capture sunlight energy by light-harvesting proteins, so-called LH1 and LH2 complexes (some species have only LH1 complexes) and transfer the light energy into the type-II RC, where conversion of light energy into chemical potential energy occurs3. The RC and LH1 complexes bind BChl a or BChl b, depending on species and form a supercomplex in the cytoplasmic membrane. When BChl a (λmax = 770 nm in monomer) is incorporated into LH1 proteins, an absorption band of LH1 holoproteins usually occurs at < 900 nm in Rhodobacter species4. On the other hand, LH1 complexes binding BChl b (λmax = 795 nm in monomer) show a significantly red-shifted absorption band at > 1000 nm in Blastochloris viridis5. Although the difference in λmax wavelength in the monomeric state is 25 nm between BChls a and b, the difference between Qy bands of LH1 complexes binding these two pigments is 140 nm.

Model organisms in the genus Rhodobacter (e.g., R. capsulatus and R. sphaeroides) are genetically amenable, have versatile ways of growing and produce BChl pigments even when grown under dark microoxic conditions. Biosynthetic pathways for BChl a are well established in the Rhodobacter species6,7. On the other hand, B. viridis can be grown only under light anoxic conditions and is not genetically amenable. Study of the BChl b-containing RC complex of B. viridis has not progressed very far since the crystal structure of the RC was solved at the atomic level by Deisenhofer et al.8, for which they were awarded the Nobel Prize in 1988. The biosynthetic step to form the characteristic C8-ethylidene group on BChl b had remained unknown until our in vitro enzymatic assays revealed the enzyme responsible for the ethylidene formation9. Canniffe and Hunter later investigated the enzymatic activity by in vivo complementation experiments10.

Biosynthetic pathways for BChl a and BChl b are branched at the step catalyzed by chlorophyllide a oxidoreductase (COR)9. COR, a nitrogenase-like enzyme, is composed of three subunits: BchX is an electron-donating component and BchY and BchZ form a heterotetramer (BchYZ) and work as a catalytic component. We have demonstrated that COR of the BChl a-producing bacterium R. capsulatus (a-COR) has dual functions: the 8-vinyl reduction of 8-vinyl-chlorophyllide (8V-Chlide) a and the C7 = C8 double bond reduction of resultant chlorophyllide (Chlide) a, forming 3-vinyl-bacteriochlorophyllide (3V-BChlide) a as a product (Fig. 1)9,11,12. We have also revealed that, in contrast, COR from BChl b-producing B. viridis (b-COR) recognizes only 8V-Chlide a, not Chlide a, as its substrate and catalyzes the direct formation of 3V-BChlide b ( = BChlide g) possessing the C-8 ethylidene group (Fig. 1)9. It is noteworthy that the two types of CORs are well conserved in their amino acid sequences, up to 89% similarities9. The plasticity of the nitrogenase-like enzyme not only causes the committed pathways for biosynthesis of BChls a and b, but also show a unique example of subtle amino acid substitutions in enzyme(s) that results in profound changes between the energetics of photosystems with BChl a and BChl b.

Reduction of the 8-vinyl group of chlorophyll intermediates is performed by 8-vinyl reductase, also called divinyl reductase (DVR)6,13,14,15. DVR is divided into two types: one is plant-type BciA using NADPH as electron donors and the other is cyanobacterial-type BciB using ferredoxin as electron donors13. In addition, as mentioned above, COR of R. capsulatus can work like DVR, i.e., it has the 8-vinyl-reduction ability9,12. Until the function of BciA was revealed in 200714, BchJ had been considered to be DVR16. Indeed, a bchJ-deletion mutant of R. capsulatus accumulated a large amount of an intermediate pigment, 8-vinyl-protochlorophyllide (8V-PChlide) a16. BchJ is known to be involved in BChl biosynthesis, although how is still unclear.

In this study, we introduced and overexpressed BchYZ of B. viridis in a series of R. sphaeroides mutant strains. The mutant of R. sphaeroides lacking functions of intrinsic BciA and COR and overexpressing extrinsic BchYZ of B. viridis produced BChl b under dark microoxic conditions. We also constructed the R. sphaeroides mutant lacking BciA and BchJ and overexpressing BchYZ of B. viridis, resulting in the strain producing both BChl a and BChl b. The proposed function of BchJ will be discussed.

Results and discussion

Construction and pigment analysis of the platform R. sphaeroides mutant strains

The wild-type strain of R. sphaeroides was used as a host strain to construct the single mutants, ΔbchZ and ΔbchJ (Fig. S1). The ΔbciA/bchZ and ΔbciA/bchJ mutants of R. sphaeroides were constructed in the same manner using the ΔbciA mutant12 as a host strain. Analytical PCR experiments confirmed that the bchZ or bchJ allele was completely segregated in each mutant strain (Figs. S1CD, details of the analytical PCR are described in Supplementary Information).

Figure 2A shows high performance liquid chromatography (HPLC) elution profile of the authentic BChl a extracted from the R. sphaeroides wild-type strain (trace i). R. sphaeroides is known to mainly produce BChl a esterified with phytol as a hydrocarbon tail (trace i, peak at 10.5 min), although it contains trace amounts of BChl a esterified with unreduced (geranylgeranyl, dihydrogeranylgeranyl and tetrahydrogeranylgeranyl) tails (trace i, asterisks)17. In-line absorption spectrum of the elution peak at 10.5 min represents a typical BChl a absorption spectrum with λmax at 770 nm (Fig. 2A, inset). Pigments of R. sphaeroides mutant strains grown under dark microoxic conditions were also extracted and analyzed by HPLC. The ΔbchZ and ΔbciA/bchZ mutants did not produce any hydrophobic BChl compound (Fig. 2A, traces ii and iii), but accumulated hydrophilic Chlide-like pigments (Fig. 2B, traces i and ii). The Chlide-like component in the ΔbchZ mutant (Fig. 2B, peak 1) eluted at the same time as the standard Chlide a did (peak 3). In-line absorption and mass spectra of the pigment (Figs. 2CD, traces 1) were identical to those of the standard Chlide a (Figs. 2CD, trace 3). This indicates that the ΔbchZ mutant lacks the function of COR and therefore accumulates Chlide a, an intermediate pigment in biosynthetic pathways for BChl a (see Fig. 1). The Chlide-like hydrophilic component from the ΔbciA/bchZ mutant (Fig. 2B, peak 2) eluted 3-min later than Chlide a (peak 3) but at the same time as the standard 8V-Chlide a (peak 4). The in-line absorption spectrum of peak 2 (Fig. 2C, trace 2) was almost identical to that of the standard 8V-Chlide a (Fig. 2C, trace 4). Also, the in-line mass spectrum of peak 2 (Fig. 2D, trace 2) was almost identical to that of the standard 8V-Chlide a (Fig. 2D, trace 4). These indicate that the ΔbciA/bchZ mutant accumulates 8V-Chlide a, a precursor for Chlide a (Fig. 1).

Figure 2
figure 2

Reverse-phase HPLC-MS analysis of pigments extracted from the wild-type, ΔbchZ and ΔbciA/bchZ strains.

(A, traces i-iii) HPLC elution profiles of hydrophobic pigments from the wild-type, ΔbchZ, and ΔbciA/bchZ strains, respectively, monitored at 770 nm. (A, inset) In-line absorption spectrum of the elution peak at 10.5 min shown in trace i. Minor elution peaks shown with asterisks in Fig. 2A are BChl a esterified with unreduced (geranylgeranyl, dihydrogeranylgeranyl and tetrahydrogeranylgeranyl) tails at the C17 position, according to the previous study17. (B, traces i and ii) HPLC elution profiles of hydrophilic pigments extracted from the ΔbchZ and ΔbciA/bchZ mutants, respectively, monitored at 435 nm. (B, trace iii) HPLC elution profile of pigment standard mixtures containing Chlide a (peak 3) and 8V-Chlide a (peak 4), monitored at 435 nm. (C) In-line absorption spectra of peaks 1-4 shown in Fig. 2B. (D) In-line mass spectra of peaks 1–4 shown in Fig. 2B.

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In vitro COR activity assays using heterologous BchX and BchYZ

COR is composed of three subunits, BchX, BchY and BchZ. BchX is an electron-donating component and BchYZ works as a catalytic component11. In the previous study, we constructed plasmids to overexpress BchX and BchYZ components in E. coli9. In this study, we separately purified BchX and BchYZ of R. capsulatus (a-X and a-YZ) and those of B. viridis (b-X and b-YZ) and assayed COR activities in vitro in the heterologous combination. COR activities were assayed by absorption changes in 80% acetone extracts, according to our previous studies9,18. The heterologous combination of b-X and a-YZ was mixed with Chlide a (Fig. 3A). After 60-min incubation, the substrate peak from Chlide a at 666 nm decreased, concomitantly with the appearance of a new peak from the assay product of 3V-BChlide a at 732 nm (Fig. 3A). When the b-X and a-YZ components were mixed with 8V-Chlide a, the same phenomenon was observed (Fig. 3B). These results are almost identical to the previous results of assays using all BchXYZ components from a BChl a-producing bacterium9. As another combination, a-X and b-YZ components were mixed with Chlide a, then no product peak was observed in the region of 700–750 nm (Fig. 3C). But the assay mixture of a-X and b-YZ with 8V-Chlide a showed a new peak of the assay product of 3V-BChlide b ( = BChlide g) at 762 nm (Fig. 3D). These also support the results in the previous study using all BchXYZ components from B. viridis for the assay9. These assay results clearly indicate that BchX is able to transfer electrons to heterologous BchYZ components to form active CORs and that the pattern of catalytic activities of a- and b-type CORs is BchYZ-dependent.

Figure 3
figure 3

Changes in electronic absorption spectra of COR assay mixtures.

Absorption spectra were recorded in 80% acetone (20% aqueous buffer). The b-X and a-YZ components were mixed with Chlide a (A) and 8V-Chlide a (B). The a-X and b-YZ components were mixed with Chlide a (C) and 8V-Chlide a (D). The Qy absorption peaks of the substrates and products are shown in parentheses.

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R. sphaeroides recombinant strains overexpressing BchYZ of B. viridis

Taking the results of the in vitro enzymatic assays using heterologous COR components into consideration, we introduced only the bchYZ genes of B. viridis into the wild-type and mutant strains of R. sphaeroides. The plasmid pJ7-BvYZ-Gm carrying the bchYZ genes of B. viridis was incorporated into the wild-type, ΔbchZ and ΔbciA/bchZ strains of R. sphaeroides, resulting in strains named WT+BvYZ, ΔbchZ+BvYZ and ΔbciA/bchZ+BvYZ, respectively. After conjugation, transconjugant colonies on gentamycinr selective plates were re-streaked on selective plates two times and then a single colony was picked up and grown in liquid medium. The cultures grown under dark microoxic conditions were harvested and pigments were extracted and analyzed by HPLC.

The pigment extracted from the WT+BvYZ strain showed the same elution time (Fig. 4A, trace i) as the authentic BChl a extracted from wild type did (Fig. 2A, trace i), indicating that the intrinsic a-COR still dominantly works in the mutant strain even when B. viridis BchYZ proteins are overexpressed. The ΔbchZ+BvYZ strain did not produce any hydrophobic BChl compound (Fig. 4A, trace ii), again even though B. viridis BchYZ components were overexpressed. This suggests that the intrinsic BciA of R. sphaeroides dominantly reacts with 8V-Chlide a and therefore 8V-Chlide a, a suitable substrate for the B. viridis BchYZ, is not available any longer. The suggestion can be proved by making the ΔbciA/bchZ+BvYZ mutant. Figure 4A, trace iii, shows that the BChl component extracted from the ΔbciA/bchZ+BvYZ strain had an elution time at about 10 min, 0.5-min earlier than that of BChl a. The authentic BChl b extracted from B. viridis showed the same retention time (Fig. 4A, trace iv). The BChl components eluting at 10 min from the ΔbciA/bchZ+BvYZ mutant and B. viridis were collected by preparative HPLC and electronic absorption spectra were measured. The absorption spectra of the collected pigments were identical and both showed λmax at 797 nm (Figs. 4A, insets), clearly indicating that the ΔbciA/bchZ+BvYZ strain produced BChl b. These results indicate that the loss of DVR and the replacement of intrinsic BchYZ catalytic components by BchYZ of BChl b-producing bacteria are required in order to make R. sphaeroides produce BChl b: i.e., exchanging all of the BchXYZ subunits is not necessary. The average amount of BChl b molecules produced in the ΔbciA/bchZ+BvYZ mutant was ~30 mg per 1-L culture. The R. sphaeroides mutant can grow and synthesize BChl b even under dark microoxic conditions and it could be a good platform for industrial production of BChl b.

Figure 4
figure 4

Reverse-phase HPLC-MS analysis of pigments extracted from the R. sphaeroides mutant strains expressing B. viridis BchYZ and from the wild type of B. viridis.

(A, traces i-iv) HPLC elution profiles of hydrophobic pigments from WT+BvYZ, ΔbchZ+BvYZ, ΔbciA/bchZ+BvYZ and the wild-type B. viridis, respectively, monitored at 770 nm. (A, top and bottom insets) Electronic absorption spectra of the pigments from the ΔbciA/bchZ+BvYZ mutant and B. viridis, respectively, in methanol. The pigment elution peaks shown in traces iii and iv of Fig. 4A were collected by preparative HPLC and the absorption spectra were measured with a Hitachi U-3500 spectrophotometer (Tokyo, Japan). (B) A HPLC elution profile of hydrophilic pigments accumulated in the ΔbchZ+BvYZ cultures monitored at 665 nm. (C) In-line absorption spectra of peaks 1-4 shown in Fig. 4B. (D) In-line mass spectra of peaks 1-4 shown in Fig. 4B.

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Although the ΔbchZ+BvYZ strain produced no hydrophobic BChl compound, it accumulated hydrophilic Chlide-like pigments (Fig. 4B). The HPLC elution profile of hydrophilic Chlide-like pigments extracted from the ΔbchZ+BvYZ mutant showed one major elution peak and three minor peaks (Fig. 4B). In-line absorption spectra of peaks 1 and 2 were identical and both showed the Qy absorption band at 659 nm and the Soret band at 429 nm (Fig. 4C, traces 1 and 2), 7-nm and 4-nm blue-shifted from those of the standard Chlide a, respectively (Fig. 2C, trace 3). Also, the pigments eluted as peaks 1 and 2 had a mass of 633.4 (Fig. 4D, trace 1 and 2), 18 mass units larger than the mass of Chlide a (615.3, Fig. 2D, trace 3). These results, together with the presence of a fragment 615.4 mass peak (Fig. 4D, traces 1 and 2) and with the fact that peaks 1 and 2 eluted earlier than the standard Chlide a (Fig. 2B, peak 3), indicate that these two elution peaks are ascribable to 3-(1-hydroxyethyl)-Chlide a with 31R- and 31S-configurations. In-line absorption spectrum of the minor peak 3 in Fig. 4B showed the Soret band at 408 nm (Fig. 4C, trace 3), about 20-nm blue-shifted from those of peaks 1 and 2 (traces 1 and 2). The clear appearance of Qx bands at 500–550 nm were observed in the absorption spectrum of peak 3 (Fig. 4C, trace 3). The pigment eluting as peak 3 gave m/z = 611.4 as its parent mass peak (Fig. 4D, trace 3), which is 22 mass units smaller than that of 3-(1-hydroxyethyl)-Chlide a (Fig. 4D, traces 1 and 2). These suggest that the pigment eluting as peak 3 is 3-(1-hydroxyethyl)-pheophorbide a lacking the central magnesium. In-line absorption and mass spectra as well as elution time of peak 4 (Figs. 4CD, traces 4 and Fig. 4B) were almost identical to those of the standard Chlide a (Figs. 2CD, traces 3 and Fig. 2B, peak 3), indicating that the pigment eluting as peak 4 was Chlide a.

These results indicate that the ΔbchZ+BvYZ mutant mainly accumulated 3-(1-hydroxyethyl)-Chlide a. On the other hand, Canniffe and Hunter10 recently constructed a similar mutant using a different method, homologous gene recombination and reported different results. The mutant of R. sphaeroides they made, in which all the intrinsic bchXYZ genes were deleted and then the exogenous bchXYZ genes of B. viridis were incorporated into the genome, produced BChl a. However, our previous study9 has clearly indicated that the suitable substrate for the B. viridis COR proteins is 8V-Chlide a, not Chlide a and the present study also supports the idea that BchYZ components of B. viridis do not react with Chlide a (Figs. 3 and 4). Amino acid sequences of a-COR and b-COR proteins have very high similarities9. There is a possibility that their mutant could have second mutations in the amino acid sequence that changes the hydrogenation mode of CORs, from 1,4-addition to 1,2-addition. This could happen by the change of even a few amino acid residues. Incorporated exogenous bchXYZ genes in the genome of their mutant were only confirmed by PCR amplification, but the sequence of the PCR product was not sequenced10. It would be very interesting if the incorporated bchXYZ were sequenced and amino acid substitutions were found.

The hydration of the C3 vinyl group to complete BChl biosynthesis is catalyzed by BchF hydratase (Fig. 1)19. In the ΔbchZ mutant, the pigment biosynthetic pathway was almost suspended at the Chlide-a/BchXYZ step, although the mutant probably produced a small amount of 3-(1-hydroxyethyl)-Chlide a; in-line absorption and mass spectra of a minor elution peak from the ΔbchZ mutant (Supplementary Fig. S2) are almost identical to those of 3-(1-hydroxyethyl)-Chlide a from the ΔbchZ+BvYZ strain (Fig. 4CD, traces 1). On the other hand, the ΔbchZ+BvYZ mutant mainly accumulated 3-(1-hydroxyethyl)-Chlide a (Fig. 4B, peak 1): it seemed that BchF carried out its catalytic reaction in the mutant rather than in the ΔbchZ mutant. The difference between the two mutants is only the presence of overexpressed BchYZ of B. viridis in the ΔbchZ+BvYZ mutant and this therefore would be the reason for the difference in accumulated pigment intermediates. The overexpressed B. viridis BchYZ proteins would probably sequester Chlide a in the ΔbchZ+BvYZ mutant, but no reaction happens because the suitable substrate for b-COR to achieve its reaction is 8V-Chlide a9. There might then be a substrate exchange from BchYZ to BchF, which forms a 1-hydroxyethyl group at the C3 position.

Electronic absorption spectrum of the BChl b-producing R. sphaeroides strain

Figure 5A shows electronic absorption spectra of R. sphaeroides and B. viridis cells. R. sphaeroides has RC-LH1 (~875 nm) and LH2 (~800 and ~850 nm) antenna complexes4. On the other hand, B. viridis does not have LH2 but has only RC-LH1 complex (~1015 nm)5. Membrane suspensions from aerobically-grown ΔbciA/bchZ+BvYZ cultures showed an absorption band at about 900 nm, but did not show any absorption peak over the 900-nm wavelength region (Fig. 5B). This implies that the large blue-shifted absorption band of the B. viridis RC-LH1 complex is mainly caused by polypeptide environments surrounding pigment cofactors, as well as by change of the embedded pigment (from BChl a to BChl b). Note that the ΔbciA/bchZ+BvYZ mutant was not able to grow under light anoxic conditions.

Figure 5
figure 5

Electronic absorption spectra of R. sphaeroides, B. viridis and the ΔbciA/bchZ+BvYZ mutant.

(A) Electronic absorption spectra of R. sphaeroides cells grown under light anoxic (black solid line) and dark microoxic conditions (black dashed line) and B. viridis cells grown under light anoxic conditions (red line). (B) Electronic absorption spectrum of membrane suspensions (black lines) and whole cells (gray lines) of the ΔbciA/bchZ+BvYZ strain grown under dark microoxic conditions. Cells and membrane suspensions were suspended in 10 mM Tris-HCl buffer, pH 7.5.

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Function of BchJ as an enhancer for the COR activity

BchJ has long been considered to be involved in BChl a biosynthesis, but its function is still uncertain. Previous study demonstrated that ΔbchJ mutant of the purple bacterium R. capsulatus accumulated 8V-PChlide a16 and therefore BchJ was first thought to work as DVR. Later, Chew and Bryant showed that ΔbchJ mutant of the green sulfur bacterium Chlorobaculum tepidum also accumulated 8V-PChlide a in the spent medium, but their detailed analysis found that the mutant still produced a small amount of normal (8-ethylated) BChl a and concluded that BchJ is not DVR14. Thus, the function of BchJ in BChl biosynthesis has been enigmatic. Here, we investigated the function of BchJ by using the overexpression system of exogenous COR in R. sphaeroides. We first made the deletion mutant of the bchJ gene of R. sphaeroides. HPLC elution profiles of pigments extracted from the ΔbchJ mutant demonstrated that this mutant produced a small amount of a hydrophobic BChl pigment (Fig. 6A, trace i) and a hydrophilic pigment (Fig. 6B, trace i). Compared to elution profiles of the standards of BChl a (Fig. 2A, trace i) and 8V-PChlide a (Fig. 6B, peak 4), the two pigments from the ΔbchJ mutant of R. sphaeroides are ascribable to BChl a and 8V-PChlide a, respectively, as shown in the ΔbchJ mutants previously reported14,15. We next constructed the ΔbchJ/bciA double mutant, which showed the same phenotype on pigment compositions (Figs. 6AB, traces ii) as that of the single ΔbchJ mutant (Figs. 6AB, traces i). This suggests that the intrinsic a-COR present in the double mutant works as DVR, as previously reported9,12, instead of the deleted BciA.

Figure 6
figure 6

Reverse-phase HPLC analysis of pigments extracted from the ΔbchJ-relevant strains of R. sphaeroides.

(A, traces i-iv) HPLC elution profiles of hydrophobic pigments extracted from the ΔbchJ, ΔbchJ/bciA, ΔbchJ/bciA+BvYZ, ΔbciA+BvYZ mutant strains, respectively, monitored at 770 nm (solid line) and at 797 nm (dashed line). In-line absorption spectra of peak 1 (left inset) and peak 2 (right inset) were measured with a Shimadzu photodiode-array spectrophotometer detector (SPD-M20A) equipped in a Shimadzu HPLC system. Note that the detection limit of the spectrophotometer detector for the long wavelength is 800 nm. (B, traces i-iii) HPLC elution profiles of hydrophilic pigments extracted from the ΔbchJ and ΔbchJ/bciA mutant strains and of a mixture of standards PChlide a (peak 3) and 8V-PChlide a (peak 4), respectively, monitored at 435 nm.

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The plasmid pJ7-BvYZ-Gm carrying the bchYZ genes of B. viridis was transformed into the ΔbchJ/bciA and ΔbciA mutant strains of R. sphaeroides, resulting in strains termed ΔbchJ/bciA+BvYZ and ΔbciA+BvYZ, respectively. HPLC elution profile of the ΔbchJ/bciA+BvYZ mutant exhibited two elution peaks of hydrophobic BChl pigments (Fig. 6A, trace iii). The two pigments (peaks 1 and 2 in Fig. 6A) had the same elution times as those of the BChl b (Fig. 4A, trace iv) and BChl a standards (Fig. 2A, trace i), respectively. In-line absorption spectra of peaks 1 and 2 showed λmax at 797 nm and 770 nm, respectively (Fig. 6A, insets). These results indicate that the ΔbchJ/bciA+BvYZ mutant produces both BChls a and b. This is the first example of a mutant of anoxygenic photosynthetic bacteria having both BChls a and b.

The ΔbciA+BvYZ mutant lacking BciA accumulated only BChl a and did not show the elution peak of BChl b (Fig. 6A, trace iv), although 8V-Chlide a could be potentially available as the substrate for the overexpressed B. viridis BchYZ in the mutant, indicating that the activity of the intrinsic BchYZ is much faster than that of the overexpressed B. viridis BchYZ in the mutant. On the other hand, the activities of the intrinsic and extrinsic BchYZ seemed to be almost equal in the ΔbchJ/bciA+BvYZ mutant lacking BchJ. These results from the two mutants suggest that BchJ in R. sphaeroides facilitates the catalytic activity of only the intrinsic a-COR. Further investigation is still needed to learn whether the exact function of BchJ is as a substrate carrier, a scaffold protein to form tertiary complexes, or a chaperon for pigment biosynthesis proteins. Because the amounts of BChls a and b produced in the ΔbchJ/bciA+BvYZ mutant were almost the same (Fig. 6A, trace iii), both intrinsic a-COR and exogenous b-COR catalytic components were likely to be almost equally functional. This implies that BchJ may form a tertiary complex with pigment substrates and pigment biosynthesis enzymes. Therefore, in the ΔbchJ background, the B. viridis BchYZ proteins could access pigment substrates to a degree equal to a-COR (R. sphaeroides BchYZ).

Sawicki and Willows20 suggested that BchJ might play a role as a porphyrin carrier working at the steps between BchIDH (magnesium chelatase) and BchM (Mg-protoporphyrin IX monomethyl esterase) in the early stages of BChl a biosynthesis of R. capsulatus. In addition, preliminary experiments have shown that BchJ enhances the catalytic activity of dark-operative PChlide a oxidoreductase working at the BChl a biosynthetic step followed by COR (Yamanashi, K. and Fujita, Y., personal communication). Taking these into account, it is highly likely that BchJ are involved in most of the BChl a biosynthetic pathways to facilitate enzymes working in the pigment biosynthesis.

Methods

Construction of the ΔbchZ and ΔbciA/bchZ mutants of R. sphaeroides

The wild-type strain J001 and the ΔbciA mutant of R. sphaeroides, constructed in a previous study12, were used as host strains to construct the ΔbchZ and ΔbciA/bchZ mutants, respectively. The plasmid pJSC-bchZ-Sm used for the insertional inactivation of bchZ was constructed as follows.

The aadA gene, conferring resistance to streptomycin and spectinomycin, was amplified from plasmid pHP45Ω21 by PCR using a primer set, aadA-F (Fig. S1A, shown as primer i) and aadA-R (primer ii). Primer positions and sequences are presented in Fig. S1 and Table S1, respectively. The bchZ gene and a portion of bchY were amplified from the genomic DNA of R. sphaeroides by PCR using bchZ-F (primer iii) and bchZ-R (primer iv) primers. The PCR reactions were performed with KOD-plus DNA polymerase (TOYOBO, Osaka, Japan). The DNA fragment containing bchZ was sub-cloned into pTA2 by the TA cloning method (TOYOBO), yielding pTA-bchZ (Fig. S1A). To amplify a DNA fragment from pTA-bchZ without the large inner portion of bchZ, the plasmid was used as the template for the inverse PCR with primers bchZ-inf-FI (primer v) and bchZ-inf-RI (primer vi). The DNA fragment and the above-mentioned PCR product of the aadA gene were ligated with an In-Fusion HD cloning kit (Clontech, USA), yielding pTA-bchZ-Sm (Fig. S1A). The DNA fragment containing the partial bchZ gene disrupted by the aadA gene was amplified from the plasmid pTA-bchZ-Sm by PCR using primers bchZ-inf-FII (primer vii) and bchZ-inf-RII (primer viii) and sub-cloned into the SmaI restriction sites of the pJSC vector22 by the In-Fusion technique, producing pJSC-bchZ-Sm. The plasmid pJSC is a chloramphenicol-resistant suicide vector and has the sacB gene encoding the levansucrase; the expression of sacB in the presence of sucrose is lethal for most of the Gram-negative bacteria23.

The plasmid pJSC-bchZ-Sm was transformed into the mobilizing E. coli strain S17-1 λ-pir24. By conjugation method with the E. coli S17-1 strain22, pJSC-bchZ-Sm was transferred into the wild-type strain and ΔbciA mutant of R. sphaeroides. Colonies grown in the presence of 5% sucrose, 50 μg/mL streptomycin and 100 μg/mL rifampicin were selected as double-crossover candidates and the chromosomal insertion into bchZ by the aadA gene was confirmed by analytical PCR using bchZ-comf-F (Figs. S1A, primer ix) and bchZ-comf-R (primer x) primers (see Supplementary text for details of the analytical PCR experiments). The obtained ΔbchZ and ΔbciA/bchZ mutants were grown under dark microoxic conditions in the PYS medium25 at 30°C. These strains and the wild-type strain of R. sphaeroides were used as hosts to incorporate the bchYZ genes of B. viridis.

Construction of the ΔbchJ and ΔbciA/bchJ mutants of R. sphaeroides

The wild-type and ΔbciA mutant strain of R. sphaeroides were used as host strains to construct the ΔbchJ and ΔbciA/bchJ mutants, respectively. The plasmid pJSC-bchJ-Sm for the insertional inactivation of the bchJ gene in R. sphaeroides was constructed as follows.

A 1.61-kbp DNA fragment containing bchJ was amplified from the genome of R. sphaeroides using primers, bchJ-F (Fig. S1B, primer xi) and bchJ-R (primer xii). The PCR product containing the bchJ gene was sub-cloned into the plasmid pTA2, yielding pTA-bchJ (Fig. S1B). To amplify a DNA fragment from pTA-bchJ without the inner portion of bchJ, the plasmid was used as the template for the inverse PCR with primers, bchJ-inf-FI (primer xiii) and bchJ-inf-RI (primer xiv). The resulting PCR product and the aadA gene amplified from plasmid pHP45Ω mentioned above were ligated using the In-Fusion HD Cloning Kit, creating pTA-bchJ-Sm (Fig. S1B). The plasmid pTA-bchJ-Sm was used as the template for PCR using primers, bchJ-inf-FII (primer xv) and bchJ-inf-RII (primer xvi). The amplified DNA fragment was cloned into the SmaI site of pJSC by the In-Fusion cloning technique, yielding the pJSC-bchJ-Sm plasmid. The plasmid pJSC-bchJ-Sm was transformed into E. coli S17-1 λ-pir and then into the wild-type strain and ΔbciA mutant of R. sphaeroides by the conjugation method in order to create the ΔbchJ and ΔbciA/bchJ mutants, respectively. Streptomycin-resistant colonies grown in the presence of 5% sucrose were selected as double-crossover candidates and the chromosomal insertion into bchJ by the aadA gene was confirmed by analytical PCR using bchJ-comf-F (primer xvii) and bchJ-comf-R (primer xviii) primers (see Supplementary text and Fig. S1D).

Construction of the R. sphaeroides strains expressing BchYZ of B. viridis

The broad-range host vector to overexpress exogenous gene products in R. sphaeroides under the control of the puc promoter, which we designated as pJN7, was first constructed as follows. The puc promoter region of R. capsulatus was amplified by PCR using primers, ppucf6 and pjr6 (see Table S1 for primer sequences). The PCR product was digested by the restriction enzymes SalI and BamHI and ligated into the same sites of the plasmid pBBR1MCS226 carrying the kanamycin resistance, yielding pJN6. The plasmid pJN6 was further subjected to the PCR reaction using a primer set, KOBsal-f1 and KOBsal-r1. Then, the streptomycin/spectinomycin resistant cartridge was amplified from the plasmid pJN327 by PCR using primers, Spc2f1 and Spc2r1. These two DNA fragments were digested by SacI and ligated together, yielding pJN7.

The bchY and the flanking bchZ genes of B. viridis were amplified together by PCR using primers, BvYZ-infu-F1 and BvYZ-infu-R1 (see Table S1 for primer sequences). Note that bchY and bchZ genes are usually adjacent in the genomes of phototrophic bacteria and overlapped (i.e., the start codon of bchZ comes before the stop codon of bchY in the genomes). This is the case for both R. sphaeroides and B. viridis. The amplified DNA fragment was excised from agarose gels and purified using a NucleoSpin Extract II kit (Macherey-Nagel, Duren, Germany). The purified DNA fragment containing B. viridis bchY-bchZ genes was sub-cloned into the BsaI restriction sites of the pJN7 plasmid with the In-Fusion HD cloning kit, yielding pJ7-BvYZ. In the plasmid, the KpnI restriction site was located in the region after the coding region of bchZ. The aacC1 gene, conferring resistance to gentamycin, was amplified from the plasmid pUCGM-star28 by PCR using primers, Gm-JN7-F and Gm-JN7-R. The resulting PCR product containing the aacC1 gene and the pJ7-BvYZ plasmid digested by KpnI were ligated using the In-Fusion HD cloning kit, yielding pJ7-BvYZ-Gm. The plasmid was transformed into the wild-type, ΔbciA, ΔbchZ and ΔbciA/bchZ, ΔbchJ and ΔbciA/bchJ strains of R. sphaeroides by triparental mating with Escherichia coli strain Tec5 containing the relevant plasmids29. Transconjugants were selected on PYS plates25 containing rifampicin (100 μg/ml), kanamycin (25 μg/ml) and gentamycin (10 μg/ml). Transconjugant colonies on the selective plates were grown in liquid PYS medium and we purified plasmids from cultures and confirmed them to be pJ7-BvYZ-Gm by cutting with appropriate restriction enzymes. The resultant wild-type, ΔbciA, ΔbchZ, ΔbciA/bchZ, ΔbchJ and ΔbciA/bchJ strains of R. sphaeroides expressing the B. viridis bchYZ genes under the control of the puc promoter were designated as WT+BvYZ, ΔbciA+BvYZ, ΔbchZ+BvYZ, ΔbciA/bchZ+BvYZ, ΔbchJ+BvYZ and ΔbciA/bchJ+BvYZ, respectively.

In vitro enzymatic assays for COR components

Plasmids to overexpress BchX and BchYZ of R. capsulatus and B. viridis in E. coli were constructed in the previous report9. Purification of these proteins, preparation of substrate pigments and assays for COR activities were performed according to our previous studies9,18.

HPLC conditions

The wild-type strains of R. sphaeroides and B. viridis were cultured in PYS medium25 at 30°C under light anoxic conditions. The mutant strains of R. sphaeroides were grown in PYS medium at 30°C under dark microoxic conditions. Cells were harvested by centrifugation and pigments were extracted with acetone/methanol (1:1, vol/vol) and filtered with a PVDF 0.22-μm membrane filter. To analyze hydrophobic BChl-type pigments, reverse-phase HPLC measurements were performed using an octadecylated silica gel column (Cosmosil 5C18-AR-II 4.6 φ × 150 mm, 5 μm, Nacalai Tesque, Kyoto, Japan) and the mobile phase of methanol : water = 95 : 5 with the flow rate of 1.0 mL/min. The HPLC-MS system for the analysis of hydrophilic Chlide-type pigments consisted of an octadecyl–polar group–silica gel column (Inertsil ODS-EP 3.0 φ × 150 mm, 5 μm, GL Sciences Inc., Tokyo), a photodiode-array spectrophotometer detector (SPD-M20A; Shimadzu, Kyoto) and a LCMS-2010EV quadrupole mass spectrometer equipped with an electrospray ionization (ESI) probe (Shimadzu). The mobile phase was methanol : aqueous 50 mM ammonium acetate (pH 5.25) = 70 : 30 (v/v) and the flow rate was isocratic at 0.5 mL/min. The ESI-MS settings were as follows: capillary temperature; 230°C, sheath gas (N2) pressure; 0.1 MPa and spray voltage; 1.5 kV (positive-ion ESI). For the HPLC-MS analysis of hydrophilic PChlide-type pigments, the following setting was applied: a polymeric octadecylated silica gel column (Inertsil ODS-P 3.0 φ × 150 mm, 5 μm, GL Sciences Inc.); eluent, methanol : acetonitrile : aqueous 50 mM ammonium acetate (pH 5.25) = 60 : 20 : 20 (v/v/v); flow rate, 0.75 mL/min.

Electronic absorption spectra measurements of cells and membrane suspensions

Cells of R. sphaeroides grown under light anoxic and dark microoxic conditions and B. viridis grown under light anoxic conditions were harvested and suspended in 20 mM Tris-HCl (pH 7.5). Cultures of the ΔbciA/bchZ+BvYZ mutant grown under dark microoxic conditions were harvested by centrifugation at 9,000 × g for 20 min, resuspended in 20 mM Tris-HCl (pH 7.5) and disrupted by passaging three times through a French Press at 100 MPa. Unbroken cells were removed by centrifugation at 10,000 × g for 15 min and the supernatant was used as membrane suspensions. Electronic absorption spectra were measured using a Shimadzu UV-1800 spectrophotometer (Kyoto).