Introduction

Phototrophic purple bacteria necessarily synthesize not only bacteriochlorophyll (BChl) but also some carotenoids for photosynthesis. The carotenoids in these bacteria are involved in light-harvesting complexes, photoprotection, and assembly of functional pigment-protein complexes [19]. Most of the carotenoids in the phototrophic purple bacteria, from which approximately 50 different structural types have been identified, are acyclic carotenoids, whereas monocyclic and dicyclic types occur in other anoxygenic phototrophic bacteria (i.e., green sulfur bacteria, green filamentous bacteria, and heliobacteria), cyanobacteria, algae, fungi, higher plants, and non-phototrophic bacteria [16, 18, 19]. Takaichi [18, 19] has summarized the carotenoids of the phototrophic purple bacteria and suggested the diversity of the biosynthetic pathways in these bacteria. Around the half species of phototrophic purple bacteria produce spirilloxanthin as the major carotenoid. This component is a final product of the normal spirilloxanthin pathway, which is the fundamental process of carotenogenesis in the phototrophic purple bacteria. The other half species produce a number of different carotenoids. Variations in carotenoid composition of the phototrophic bacteria might be due to the lack of one enzyme, or low or modified activity of the enzymes in the normal spirilloxanthin pathway.

Based on the 16S rRNA gene sequence information, the phototrophic purple bacteria of the genus Rhodoplanes (Rpl.) belong to the class Alphaproteobacteria, and the nearest phylogenetic neighbors are those of the genus Blastochloris in the family Hyphomicrobiaceae [2]. Cells of Rhodoplanes species are rod-shaped, motile by means of polar, subpolar, or lateral flagella, and multiply by budding and asymmetric cell division. Internal photosynthetic membranes are present as lamellar stacks parallel to the cytoplasmic membrane [3]. On the other hand, members of the genus Blastochloris contain unusual BChl b and unusual carotenes of 1,2-dihydroneurosporene and 1,2-dihydrolycopene [5, 11, 15].

A representative species of Rhodoplanes, Rhodoplanes roseus, was first described as Rhodopseudomonas rosea [7]. Later, based on 16S rRNA gene sequence and chemotaxonomic information, this species was shown to be different at the generic level from any species of the genus Rhodopseudomonas. As a result, Rhodopseudomonas rosea was transferred to a new genus Rhodoplanes as the type species Rhodoplanes roseus [4]. At the same time, Rhodoplanes elegans was described as the second species of this genus [4]. The thermotolerant species “Rhodopseudomonas cryptolactis” [17] was also transferred to this genus as “Rhodoplanes cryptolactis” [12]. Recently, two new species Rhodoplanes serenus [13] and Rhodoplanes pokkaliisoli [10] have been added to the genus Rhodoplanes.

In the genus Rhodoplanes, absorption spectra in carotenoid region of cells were somewhat different from species to species [4, 7, 10, 12, 13], suggesting that the carotenoid composition is different among the species. The major carotenoids in Rpl. roseus and “Rpl. cryptolactis” have been reported to be spirilloxanthin and rhodopin, respectively [18], while those of other species were insufficiently characterized [4, 7, 10, 12, 13]. This study was undertaken to identify the carotenoids of all established species of the genus Rhodoplanes, and to show the variation of carotenoids among them. From the information, the predicted carotenogenetic pathways and characteristics of the enzymes involved in these organisms are also discussed.

Materials and Methods

Bacterial Strains and Cultivation Conditions

Rhodoplanes pokkaliisoli strain JA415T, Rhodoplanes elegans strain AS130T, and Rhodoplanes serenus strain TUT3530T were used. Cells were cultured phototrophically as described [10, 13].

Isolation and Purification of Carotenoids

Pigments from lyophilized cells or wet cell pellets were extracted twice by sonic treatment for several seconds with a mixture of acetone and methanol (7:2, v/v). The treated samples were centrifuged, and the combined extract was then evaporated to dryness. The pigment extract was analyzed by HPLC equipped with a μBondapak C18 column (RCM type; Waters, USA) and eluted with methanol (1.8 ml/min) as described previously [20, 21].

Carotenoids were purified without saponification. Column chromatography on DEAE-Toyopearl 650 M (Tosoh, Japan) was carried out to remove BChl and polar lipids, and partial separation was done on silica gel 60 (Merck, Germany) column. The same C18-HPLC column as described above was used for final purification [21].

Spectroscopic Analysis

Absorption spectra of the pigments in the HPLC eluent of methanol were recorded with a photodiode-array detector (MCPD-3600; Otsuka Electronics, Japan) attached to the HPLC apparatus [20, 21]. For quantitative analysis, the molar extinction coefficient at the maximum wavelengths of each carotenoid in the HPLC eluent of methanol was assumed to be the same.

After purification, relative molecular masses of the carotenoids were determined by an FD-MS; M-2500 double-focusing gas chromatograph/mass spectrometer equipped with a field-desorption apparatus (Hitachi, Japan).

Results

Identification of Carotenoids in Rhodoplanes pokkaliisoli

An HPLC elution profile at 500 nm of the pigments extracted from Rpl. pokkaliisoli strain JA415T is shown in Fig. 1, which reveals detectable peaks of both BChl a and carotenoids. A component of peak 1 eluted at around 4.8 min was identified as BChl a based on its absorption spectrum and specific retention time on HPLC. All other numbered peaks detected in Fig. 1 are derived from carotenoids. Each carotenoid was purified using column chromatographs of DEAE-Toyopearl and silica gel, and HPLC with C18-column. Their absorption spectra [20] and the retention times on HPLC were compared with authentic carotenoids, and their relative molecular masses after purification were measured for identification.

Fig. 1
figure 1

HPLC elution profile of pigments extracted from Rhodoplanes pokkaliisoli strain JA415T . The peak numbers shown in the figure are referred to in the text, table and figure. 1 BChl a, 2 1,1′-dihydroxylycopene, 3 3′,4′-dihydrorhodovibrin, 4 rhodopin, 5 3,4,3′,4′-tetrahydrospirilloxanthin, and spirilloxanthin, 6 3,4-dihydroanhydrorhodovibrin, 7 lycopene

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The component of peak 4 was eluted at around 8.4 min (Fig. 1), which was compatible retention time with that of authentic rhodopin. This component had absorption maxima at 293, 361, 444, 470, and 501 nm in HPLC eluent of methanol, and the spectral fine structure (%III/II) was 73; this is the ratio of the peak height of the longest and the middle wavelength absorption bands from the trough between the two peaks [20]. The absorption spectrum corresponded to an acyclic carotenoid with 11 conjugated double bonds [20]. The relative molecular mass was 554. Based on these results, the component of peak 4 was identified as rhodopin (Fig. 2).

Fig. 2
figure 2

Structures and predicted biosynthetic pathways of carotenoids, and carotenogenesis enzymes in the genus Rhodoplanes. Numbers in parentheses indicate the peak numbers in Fig. 1

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Similarly, the components of peaks 2, 3, 6, and 7 (Fig. 1) showed similar absorption spectra with 11 conjugated double bonds, which was found also in the component of peak 4. The relative molecular masses of the peak 2, 3, 6, and 7 components were 572, 586, 568, and 536, thereby being identified as 1,1′-dihydroxylycopene, 3′,4′-dihydrorhodovibrin, 3,4-dihydroanhydrorhodovibrin and lycopene, respectively (Fig. 2). Also, trace amounts of acyclic carotenoids with 12 conjugate double bonds were detected in the tailing of peaks 3 and 6, which might be rhodovibrin and anhydrorhodovibrin, respectively (Fig. 2). Acyclic carotenoids with different number of the conjugated double bonds, such as 3′,4′-dihydrorhodovibrin (3) and rhodovibrin, have almost the same retention time on the C18-HPLC system used.

Absorption spectrum of the peak 5 component (Fig. 1) was derived from a mixture of acyclic carotenoids with 11 and 13 conjugated double bonds [20]. The retention time of peak 5 was compatible with that of authentic spirilloxanthin. The relative molecular masses of the two components corresponding to peak 5 were 600 and 596, thereby being identified as 3,4,3′,4′-tetrahydrospirilloxanthin and spirilloxanthin, respectively (Fig. 2).

Conclusively, the major carotenoid of Rpl. pokkaliisoli strain JA415T was rhodopin (4), and spirilloxanthin (5), 3,4,3′,4′-tetrahydrospirilloxanthin (5) and 1,1′-dihydroxylycopene (2) were also found. The carotenoid composition is summarized in Table 1.

Table 1 Composition of carotenoids (mol% to total carotenoids) in the genus Rhodoplanes
Full size table

Identification of Carotenoids in Other Rhodoplanes Species

The HPLC elution profiles at 500 nm of the pigments extracted from Rpl. serenus strain TUT3530T and Rpl. elegans strain AS130T were almost similar to that of Rpl. pokkaliisoli pigment (Fig. 1). However, peak 2 on HPLC was absent in both species, and the peak heights and absorption spectra of the components were different from those found in Rpl. pokkaliisoli. The pigments of Rpl. serenus and Rpl. elegans were identified based on the absorption spectra, which were typical acyclic carotenoids with 11, 12, and 13 conjugated double bonds, and the specific retention times on HPLC, which were compared with those of Rhodopseudomonas palustris pigments [20]. Consequently, they were identified as BChl a (1), lycopene (7), rhodopin (4), anhydrorhodovibrin, rhodovibrin, and spirilloxanthin (5) (Fig. 2). The major carotenoid of Rpl. serenus was spirilloxanthin, and Rpl. elegans contained substantial amounts of rhodopin, anhydrorhodovibrin, and spirilloxanthin (Table 1).

As reported previously [18], Rpl. roseus strain DSM 5909T and “Rpl. cryptolactis” strain DSM 9987T contain also the same carotenoids with different compositions, and the major carotenoids are spirilloxanthin and rhodopin, respectively (Table 1).

Discussion

At present, the genus Rhodoplanes accommodates four species with validated names, Rpl. roseus, Rpl. serenus, Rpl. elegans and Rpl. pokkaliisoli, and one species yet to be validated, “Rpl. cryptolactis”. The absorption spectra in the carotenoid region of these species have been reported to be somewhat different from each other [4, 7, 10, 12, 13], suggesting that the carotenoid composition might be different among the species. In this study, all the carotenoid components from the Rhodoplanes species were identified using the spectroscopic methods (Table 1; Fig. 2). Although any genes and enzymes for carotenogenesis in Rhodoplanes have not yet been reported, we propose the pathways of carotenogenesis and the enzymes involved in Rhodoplanes (Fig. 2) by comparing the chemical structures of the identified carotenoids and the characteristics of carotenogenesis enzymes to those in the well-studied pathways and enzymes in the genera Rhodobacter and Rubrivivax [18, 19].

As reported herein (Table 1), the major carotenoid in Rpl. roseus and Rpl. serenus was spirilloxanthin (5), which is synthesized from lycopene (7) by the combination of three enzymes: CrtC (hydroxyneurosporene synthase; acyclic carotene C-1,2 hydratase), CrtD (methoxyneurosporene desaturase; acyclic carotenoid C-3,4 desaturase), and CrtF (hydroxyneurosporene-O-methyltransferase; acyclic 1-hydroxycarotenoid methyltransferase). The consecutive biosynthetic process with these enzymes is called “the normal spirilloxanthin pathway” (Fig. 2) [18, 19]. On the other hand, the major carotenoid in “Rpl. cryptolactis” was rhodopin (4) (Table 1). Since rhodopin is the substrate of CrtD, its accumulation can be plausibly explained as due to low activity of CrtD. In this species, rhodopin might not be a suitable substrate for CrtF due to the single bond at C-3,4 thereof (Fig. 2). In the case of Rpl. elegans (Table 1), CrtC might be low activity to another side of anhydrorhodovibrin, since anhydrorhodovibrin as the substrate for CrtC was accumulated, and the content of rhodovibrin as the product was low (Fig. 2).

The carotenoid composition of Rpl. pokkaliisoli was unusual (Table 1). The high content of rhodopin (4) in this organism indicated low activity of CrtD as predicted in “Rpl. cryptolactis”. Also, the detection of substantial amounts of 3,4,3′,4′-tetrahydrospirilloxanthin (5) and its biosynthetic intermediates, 3,4-dihydroanhydrorhodovibrin (6) and 3′,4′-dihydrorhodovibrin (3), suggested that rhodopin might not be a suitable substrate for CrtD but for CrtF in spite of the single bond at C-3,4 thereof (Fig. 2). This contrasted with the case in “Rpl. cryptolactis”, in which CrtF possibly had usual substrate specificity. In this species, CrtC and CrtF similarly catalyzed the conversion of 3,4-dihydroanhydrorhodoibrin to produce 3,4,3′,4′-tetrahydrospirilloxanthin. Furthermore, the presence of 1,1′-dihydroxylycopene (2) in this species indicated CrtC could also catalyze another side of rhodopin (Fig. 2). The production of 3,4,3′,4′-tetrahydrospirilloxanthin have been reported in few species of the phototrophic proteobacteria, such as Rhodospira trueperi [14], Thiococcus pfennigii [1], and Thioflavicoccus mobilis [6], and the crtD mutants of Rhodospirillum rubrum [8] and Thiocapsa roseopersicin [9].

About half of the species of phototrophic purple bacteria accumulate spirilloxanthin as the final product of the normal spirilloxanthin pathway [18, 19], whereas variations in carotenoid composition and in characteristics of carotenogenesis enzymes at the levels of family, genus, and species levels are found in others. The phototrophic purple bacteria of the family Rhodobacteraceae, including those of the genera Rhodobacter and Rhodovulum, accumulate spheroidene and spheroidenone due to production of neurosporene and not lycopene, by CrtI (phytoenedesaturase) and the possession of CrtA (spheroidene monooxygenase). Members of some genera accumulate genus-specific unusual carotenoids; Phaeospirillum species contain hydroxylycopene glucoside, dihydroxylycopene glucoside and dihydroxylycopene di-glucosides, probably due to the absence of CrtD and the possession of glucosyltransferase [22]. Members of the genus Roseospira accumulate 3,4-didehydrorhodopin, and this is due to low activity of CrtF [22]. Rhodocyclus species accumulate carotenals, possibly because they have CrtNb-like aldehyde synthase as observed in the species of Methylomonas and Staphylococcus [23]. Members of the genera Blastochloris accumulate unusual carotenes, 1,2-dihydroneurosporene and 1,2-dihydrolycopene [5, 11, 15], although the mechanisms for 1,2-dihydroxylation are unknown.

In this study, we found variations in carotenoid composition in different species within a single genus, Rhodoplanes. It is of special interest to note whether these variations have phylogenetic implication. However, we could not find a definitive relationship between the carotenoid composition and the phylogeny of Rhodoplanes species based on 16S rRNA gene sequences. For example, although the type species Rpl. roseus clusters with Rpl. elegans and “Rpl. cryptolactis” as its sister group on the phylogenetic tree [10, 13], these three organisms differ from each other in carotenoid composition as noted above (Table 1). The carotenoid composition of Rpl. roseus is rather similar to that of Rpl. serenus, the most deeply branching species within the genus Rhodoplanes.

In conclusion, the variations in carotenoid composition found in the Rhodoplanes species (Table 1) might be due to modified substrate specificity of the carotenogenesis enzymes, CrtC, CrtD, and CrtF. This is the first report of such a possibility of different characteristics of carotenogenesis enzymes in different species within a single genus of the phototrophic bacteria. Further studies are necessary to identify genes encoding enzymes actually involved in carotenogenesis in each species of Rhodoplanes.