Novel carotenoid glucoside esters from alkaliphilic heliobacteria

Arch Microbiol (2003) 179 : 95–100 DOI 10.1007/s00203-002-0504-5

O R I G I N A L PA P E R

Shinichi Takaichi · Hirozo Oh-oka · Takashi Maoka · Deborah O. Jung · Michael T. Madigan

Novel carotenoid glucoside esters from alkaliphilic heliobacteria

Received: 18 June 2002 / Revised: 8 October 2002 / Accepted: 28 October 2002 / Published online: 14 December 2002 © Springer-Verlag 2002

Abstract Pigments of three species of alkaliphilic heliobacteria of the genus Heliorestis, H. daurensis, H. baculata and an undescribed species Heliorestis strain HH, were identified using spectroscopic methods. In these species, bacteriochlorophyll g esterified with farnesol was present, as for other heliobacteria. The carotenoids consisted of 4,4′-diaponeurosporene, also found in other heliobacteria, plus the novel pigments OH-diaponeurosporene glucoside esters (C16:0 and C16:1). In addition, trace amounts of biosynthetic intermediates, OH-diaponeurosporene and OH-diaponeurosporene glucoside, were found. Trace amounts of a carotenoid with 20 carbon atoms, 8,8′-diapo-ζ-carotene, were also found in these species as well as in the non-alkaliphilic heliobacteria. The non-alkaliphilic species Heliophilum fasciatum also contained trace amounts of the two OH-diaponeurosporene glucoside esters. The results are used to predict the pathway of carotenoid biosynthesis in heliobacteria. Keywords Carotenoid · Carotenoid glycoside esters · Diapocarotene · 4,4′-Diaponeurosporene · Bacteriochlorophyll g · Heliobacteria · Heliophilum fasciatum · Heliorestis daurensis · Heliorestis baculata Abbreviations BChl Bacteriochlorophyll · FAB-MS Fast atom bombardment-mass spectrometry

S. Takaichi (✉) Biological Laboratory, Nippon Medical School, Nakahara, Kawasaki 211–0063, Japan Fax: +81-44-7221231, e-mail: [email protected] H. Oh-oka Department of Biology, Graduate School of Science, Osaka University, Toyonaka 560–0043, Japan T. Maoka Kyoto Pharmaceutical University, Kyoto 607–8414, Japan D. O. Jung · M. T. Madigan Department of Microbiology and Center for Systematic Biology, Southern Illinois University, Carbondale, IL 62901–6508, USA

Introduction Heliobacteria are strictly anaerobic, anoxygenic phototrophic bacteria. Until recently, three genera including five species were known: Heliobacillus mobilis, Heliophilum fasciatum, Heliobacterium chlorum, Heliobacterium modesticaldum and Heliobacterium gestii (Madigan 2001). The photosynthetic system of heliobacteria resembles that of photosystem I in cyanobacteria as well as in green plants, although photosynthetic electron transfer is inhibited by oxygen and can only be measured under anoxic conditions (Oh-oka et al. 2002). Neither chlorosomes nor differentiated internal membranes are found in heliobacteria (Madigan and Ormerod 1995). The pigments of heliobacteria are unusual among anoxygenic phototrophs. Bacteriochlorophyll (BChl) g esterified with farnesol is the main chlorophyll pigment, and 81 hydroxy chlorophyll a is the primary electron acceptor (Neerken and Amesz 2001). The major carotenoid of the heliobacteria is 4,4′-diaponeurosporene, an unusual pigment containing 30 carbon atoms; trace amounts of 4,4′-diapophytoene, 4,4′-diapophytofluene, 4,4′-diapo-ζ-carotene and 4,4′-diapolycopene are also found. These carotenoids are not present in other phototrophic organisms (Takaichi et al. 1997a, Takaichi 1999). Recently, two alkaliphilic heliobacteria have been isolated from microbial mats of soda lakes and alkaline shoreline soils in Siberia, Heliorestis daurensis (Bryantseva et al. 1999) and Heliorestis baculata (Bryantseva et al. 2000). In addition, a morphologically unusual Heliorestis species, provisionally designated Heliorestis sp. strain HH, was isolated from an Egyptian soda lake (Jung et al. 2002). Like other heliobacteria, cells of Heliorestis species lack intracytoplasmic membranes or chlorosomes and an outer membrane. However, their optimum pH and temperature for growth are 9 and 30 °C, respectively, instead of the 7 and 40 °C observed for other heliobacteria. In this study, we examined the pigments of the three Heliorestis species and found that, in addition to containing BChl g and 4,4′-diaponeurosporene as for other he-

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liobacteria, the alkaliphilic species produced large amounts of carotenoid glucoside esters, carotenoids not previously observed in any other phototrophic or non-phototrophic organisms. We describe the structure of these pigments and discuss their likely route of biosynthesis.

Material and methods Bacteria and culture conditions Heliorestis daurensis strain BT-H1 (ATCC 700798T), Heliorestis baculata strain OS-H1 (DSM 13446T) and Heliorestis sp. strain HH (Jung et al. 2002) were cultured photoheterotrophically at 30 °C in the medium (pH 9) described by Bryanteseva et al. (1999, 2000). Heliophilum fasciatum (ATCC 51790T) and Heliobacterium gestii (ATCC 43375T) were cultured as described by Ormerod et al. (1996). Cells were harvested by centrifugation and washed once. Pigment extraction and analysis Pigments were extracted twice from wet cells with acetone:methanol (7:2, v/v) using an ultrasonicator for several seconds followed by centrifugation; the solvent was then evaporated to dryness. Carotenoids were purified without saponification. Column chromatography on DEAE Toyopearl 650 M (Tosoh, Japan) was used to remove BChls and polar lipids (Takaichi and Ishidsu 1992). Thin-layer chromatography (TLC) was also used for purification. KC18-TLC plates (Whatman, USA) were developed with methanol, and silica-gel-60 TLC plates (1.13748; Merck, Germany) were developed with dichloromethane:ethyl acetate (3:1, v/v). Pigments were further analyzed by HPLC containing a µBondapak C18 column (8×100 mm, RCM type; Waters, USA); pigments were eluted with methanol (2 ml/min) as described previously (Takaichi and Ishidsu 1992). The absorption spectra of the pigments were measured by a photodiode-array detector (200–800 nm, 1.3-nm interval, MCPD-3600; Otsuka Electronics, Japan) attached to the HPLC apparatus as previously described (Takaichi and Shimada 1992). The relative molecular masses of purified carotenoids were measured by field-desorption mass spectrometry (FD-MS) using a double-focusing gas chromatograph/mass spectrometer equipped with a field-desorption apparatus (M-2500; Hitachi, Japan) (Takaichi 1993). The high-resolution fast atom bombardment-mass spectrometry (FAB-MS) spectra of the carotenoids were measured by a JMS-HX/HX110A mass spectrometer (JEOL, Japan) with m-nitrobenzyl alcohol as a matrix. The 1H- (500 MHz) and 13C-NMR (125 MHz) spectra of the carotenoids in CDCl3 (24 °C) were measured in a UNITY INOVA-500 NMR system (Varian, USA). The peak assignments of the 1H- and 13C-NMR spectra were made on the basis of 1H-1H COSY (correlated spectroscopy), HSQC (heteronuclear single-quantum coherence) and HMBC (heteronuclear multiple-bond correlation) analyses and by comparison with the data from keto-myxocoxanthin glucoside ester from Roseiflexus castenholzii (Takaichi et al. 2001b).

Results Identification of photosynthetic pigments in H. daurensis When the pigments extracted from cells of Heliorestis daurensis were analyzed by HPLC, several peaks were obtained (see Fig. 1a). One component observed in H. daurensis and eluted at 3.7 min (data not shown) was identified as BChl g esterified with farnesol based on its absorption spectrum and specific retention time on HPLC

Fig. 1a, b Carotenoids from Heliorestis daurensis. a Elution profile on HPLC. The content of one component eluting at 3.4 min was too small to show. BChls were removed from crude pigment extracts by passage through a DEAE-Toyopearl column. BChl g eluted at 3.7 min. b Absorption spectrum of 4,4′-diaponeurosporene in methanol. This pigment eluted at 6.9 min as shown in a

(Takaichi et al. 1997a). BChl g esterified with phytol was not found. The pigment extract was dissolved in a small volume of n-hexane:acetone (1:1, v/v) and loaded onto a column of DEAE-Toyopearl. Carotenoids were eluted with n-hexane/acetone, while BChls and polar lipids remained on the column. On HPLC, this carotenoid fraction showed two major peaks eluting at 6.9 and 8.1 min, and three minor peaks (Fig. 1a). In the case of non-alkaliphilic heliobacteria, only one carotenoid peak, 4,4′-diaponeurosporene, eluting at 6.9 min was found (Takaichi et al. 1997a). On KC18-TLC, the carotenoids migrated as one band and were collected. The carotenoids were then resolved into two bands, carotenes and xanthophylls, by silica-gel TLC, and each band was collected. The carotenes eluted at 6.9 min on HPLC (Fig. 1a). The absorption maxima of this pigment (Fig. 1b) were also the same as those of authentic 4,4′-diaponeurosporene. The spectral fine-structure of this pigment (%III/II, the ratio of the peak heights of the longest and the middle wavelength absorption bands from the trough between the two peaks, Takaichi and Shimada

97 Fig. 2 Chemical structures of some carotenoids and postulated biosynthetic pathway of carotenoids in heliobacteria. The acyl moiety of the carotenoid glucoside esters can be either a C16:0 or a C16:1 fatty acid. In alkaliphilic heliobacteria, 4,4′-diaponeurosporene constitutes 30–40 mol% of total carotenoids, whereas in non-alkaliphilic species, this pigment constitutes more than 95 mol% of total carotenoids. The pigment 8,8´-ζ-carotene is only a minor constituent in all heliobacteria. Bold arrows of two enzymes are functionally identified in Staphylococcus (Wieland et al. 1994). Both bold and white arrows are postulated in other heliobacteria (Takaichi et al. 1997a). PPi Pyrophosphate

1992) was 88.1, and its relative molecular mass was 402. From these data, this carotene was identified as the C30 acyclic carotene 4,4′-diaponeurosporene (7,8-dihydro4,4′-diapocarotene) (Fig. 2), a pigment thus far found in all heliobacteria. As minor components, 4,4′-diapophytoene, 4,4′-diapophytofluene and 4,4′-diapo-ζ-carotene were also found. Carotenoid glycoside esters in H. daurensis The xanthophylls on silica-gel TLC described above eluted in two fractions, at 6.9 and 8.1 min on HPLC (Fig. 1a), and each fraction was collected. Both pigments appeared to be hydrophobic, as assessed by KC18-TLC, as well as polar, as determined by silica-gel TLC. These chromatographic properties are characteristics of carotenoid glycoside fatty acid esters, since the fatty acid moiety ad-

sorbs strongly on KC18-TLC, while the glycoside moiety adsorbs strongly on silica-gel TLC (Takaichi and Ishidsu 1992). The absorption spectra of the two carotenoids in methanol (data not shown) were the same as that of 4,4′diaponeurosporene (Fig. 1b), indicating that they each contained nine conjugated double bonds. The relative molecular masses of the purified carotenoids were 818 and 820, corresponding to the components eluting at 6.9 and 8.1 min, respectively, on HPLC. The high-resolution FAB-MS spectrum indicated the molecular formula of one carotenoid eluted at 6.9 min on HPLC to be C52H82O7 (observed m/z=818.6043; calculated m/z=818.6060). Table 1 shows the 1H- and 13C-NMR spectral data of this pigment dissolved in CDCl3. The spectra indicated that the carotenoid moiety was OH-diaponeurosporene, the glycosyl moiety was β-D-glucoside, and the fatty acid moiety was an unsaturated straightchain fatty acid attached to the C-6 hydroxyl group of the

98 Table 1 1H- and 13C-NMR spectral data of OH-diaponeurosporene glucoside ester in CDCl3. s Singlet, d doublet, dd doubletdoublet, t triplet, m multiplet Protons

δH (ppm)a

Carotenoid moiety 1.25 s, 1.26 s H3-4,18 1.96 s H3-19,20 H2-6 H2-7 H2-8

~ 1.51 m ~ 1.51 m 2.07 t (7)

H-10 H3-4’,18’ H3-19’,20’

5.95 d (11) 1.82 s, 1.83 s 1.96 s

H-6’

5.94 d (11)

Glucosyl moiety H-1 4.43 d (7.5) H-2 3.33 dd (9, 7.5) H-3 3.58 t (9) H-4 3.39 t (9) H-5 3.44 m H-6 4.27 dd (12, 5.5) 4.44 dd (12, 3.5)

Carbons

δC (ppm)

C-4,18 C-19,20 C-5 C-6 C-7 C-8 C-9 C-10 C-4’,18’ C-19’,20’ C-5’ C-6’

22.66 12.78, 12.90 78.42 41.53 22.35 40.47 137.3 126.1 18.59 12.78, 12.90 136.0 126.1

C-1 C-2 C-3 C-4 C-5 C-6

96.97 73.69 76.10 70.14 73.79 63.38

C-1 C-2 -(CH2)-CH= C-16

179.50 34.16 28–30 130.00 14.13

Fatty acid moiety H2-2 -(H2C)-CH= H3-16 aMultiplicity

2.34 t (7) ~ 1.25 5.35 0.88 t (7)

Minor pigments in H. daurensis In addition to the two carotenoid glucoside esters, several minor pigment components were observed in the pigment extracts of H. daurensis (Fig. 1a). The component eluting at 9.6 min by HPLC is also likely an OH-diaponeurosporene glucoside ester, since its behavior on KC18-TLC and silica-gel TLC was identical to that of the major components. However, since the retention time of this pigment on HPLC was longer than that of the major components, it likely contains a longer-chain fatty acid. Insufficient material was obtained to determine this unambiguously. Two minor components also eluted at 3.4 and 4.4 min (Fig. 1a). These are likely precursors of the major pigments, that is, unesterified OH-diaponeurosporene glucoside and OH-diaponeurosporene, based on their absorption spectra (data not shown), their retention times on HPLC, and their relative molecular masses of 582 and 420, respectively. Moreover, in the carotene fraction of silica-gel TLC, a minor component eluting at 4.8 min (Fig. 1a) showed absorption maxima at 306, 379, 400, 424 nm in methanol and a %III/II value of 87.6; these are similar to those of ζ-carotene (Takaichi and Shimada 1992). Therefore, the latter pigment could be the C20 acyclic carotene, 8,8′-diapo-ζ-carotene (8,8′-diapocarotene) (Fig. 2), although we could not determine the relative molecular mass owing to its presence in low amounts. This carotene has been found in the non-alkaliphilic species of heliobacteria as well (Takaichi et al. 1997a). Photosynthetic pigments in other alkaliphilic heliobacteria

and coupling constant in Hz

glucoside moiety. The relative molecular masses of 818 and 820 corresponded to the fatty acid moieties of C16:1 and C16:0, respectively. Thus, the chemical structure of the pigments was determined to be OH-diaponeurosporene glucoside ester (C16:0 and C16:1) (Fig. 2). The IUPAC-IUB semi-systematic name of this carotenoid glycoside ester is therefore 5-[(6-O-acyl-β-D-glucopyranosyl)oxy]-7,8-dihydro-4,4′-diapocarotene. Note that the peak eluting at 6.9 min (Fig. 1a) contains both 4,4′-diaponeurosporene and OH-diaponeurosporene glucoside C16:1 ester. During the course of this work, it was found that the heliobacterial carotenoids were relatively unstable. For example, when freeze-dried cells or evaporated pigment extracts of H. daurensis and Heliobacterium gestii were stored at room temperature in air, the contents of both 4,4′-diaponeurosporene and OH-diaponeurosporene glucoside esters decreased by about 80% per day. However, when the cells were stored at –80 °C under a nitrogen atmosphere, the pigments were stable for at least a few months. This is in contrast to the carotenoids of purple bacteria, where pigments in freeze-dried cells are stable for more than one week at room temperature, while those in evaporated extracts are stable for at least several days.

The pigments extracted from Heliorestis baculata showed the same HPLC elution profile as those of H. daurensis (Fig. 1a) and the same absorption spectrum for each pigment. The relative molecular mass of 4,4′-diaponeurosporene was 402, and those of OH-diaponeurosporene glucoside esters were 818 and 820. The pigments extracted from Heliorestis sp. strain HH also showed the same results, including the relative molecular masses. Consequently, it can be concluded that these species also contain BChl g esterified with farnesol and the same carotenoids, 4,4′-diaponeurosporene and OH-diaponeurosporene glucoside esters (C16:0 and C16:1) as in H. daurensis. Relative carotenoid contents (mol% of total carotenoids) were virtually the same in the three alkaliphilic heliobacteria. These were 30–40 mol% 4,4′-diaponeurosporene, 10–20 mol% OH-diaponeurosporene glucoside C16:1 ester, and 40–50 mol% OH-diaponeurosporene glucoside C16:0 ester. Minor carotenoids accounted for less than 1 mol% in each species. Presence of OH-diaponeurosporene glucoside esters in Heliophilum fasciatum In a reexamination of the carotenoids of Heliophilum fasciatum, the major carotenoid was found to be 4,4′-di-

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aponeurosporene, as previously determined (Takaichi et al. 1997a). However, a minor component previously detected might actually be a OH-diaponeurosporene glucoside ester, since its behavior on KC18-TLC and silica-gel TLC, absorption spectra, and the specific retention time on HPLC were the same as those of H. daurensis. However, since the content of this component was less than 2 mol% of the total carotenoids of Heliophilum fasciatum, mass spectrometric analyses could not be done. Nevertheless, this result indicates that small amounts of carotenoid glucoside esters may exist in non-alkaliphilic heliobacteria.

Discussion In this study, we found that the major carotenoids of Heliorestis species are novel glucoside ester pigments. Nonalkaliphilic heliobacteria contain 4,4′-diaponeurosporene as the major (>95 mol%) carotenoid (Takaichi et al. 1997a). Although Heliorestis species also contain this pigment, OH-diaponeurosporene glucoside esters are the predominant pigments in cells of this genus. These carotenoids have not been observed in other phototrophic organisms (Takaichi 1999). From the carotenoids identified in heliobacteria and by analogy with the diapocarotene biosynthesis pathway from Staphylococcus aureus, which also produces 4,4′-diaponeurosporene and its derivatives (Wieland et al. 1994, Raisig and Sandmann 1999; 2001), the following pathway of carotenoid biosynthesis in heliobacteria including Heliorestis is proposed (Fig. 2). Geranylgeranyl pyrophosphate synthase (CrtE) is likely absent (or inactive) in the heliobacteria, since no carotenoids derived from geranylgeranyl pyrophosphate (C20) are synthesized and the esterifying alcohol of BChl g in heliobacteria is farnesol instead of phytol. Dehydrosqualene synthase from S. aureus (CrtM, =4,4′-diapophytoene synthase), which is highly homologous to phytoene synthase (CrtB) of phototrophic bacteria, produces 4,4′-diapophytoene (C30) from two molecules of farnesyl pyrophosphate (C15). From here, dehydrosqualene desaturase (CrtN, =4,4′-diapophytoene desaturase), which is highly homologous to phytoene desaturase (CrtI) of phototrophic bacteria, desaturates 4,4′diapophytoene in a three-step reaction to yield 4,4′-diaponeurosporene (Fig. 2). In Heliorestis species, 4,4′-diaponeurosporene is likely hydrated by a hydratase enzyme, which may be a Rhodobacter-like CrtC enzyme, to produce OH-diaponeurosporene, and then glucosylated by glucosyl transferase, which may be an Erwinia-like CrtX enzyme, to produce OH-diaponeurosporene glucoside. Finally, the latter is esterified with a C16:0 or C16:1 fatty acid by a specific esterase to yield the final glucoside ester pigment (Fig. 2). Carotenoid glucoside esters have been found in only a few species of phototrophic bacteria, although their distribution from a phylogenetic standpoint is wide (Takaichi 1999). Besides the three Heliorestis (family Heliobacteriaceae) examined here, two Chloroflexus and Roseiflexus

species (Chloroflexaceae) (Takaichi et al. 1995, Takaichi 1999, Takaichi et al. 2001b), Chlorobium tepidum (Chlorobiaceae) (Takaichi et al. 1997b), and two Halorhodospira species (Ectothiorhodospiraceae) (Takaichi et al. 2001a) contain carotenoid glucoside esters. Moreover, Rhodoblastus acidophilus (formerly Rhodopseudomonas acidophila) (Bradyrhizobiaceae) contains carotenoid glucosides (Schmidt 1978, Takaichi 1999). Although perhaps only a coincidence, all of the abovementioned organisms can be considered “extremophilic” phototrophs, in that their habitats are in one way or another extreme environments (Madigan and Marrs 1997). In Chloroflexus (Tsuji et al. 1995) and Chlorobium tepidum (Takaichi and Oh-oka 1999), carotenes are the major carotenoids in the chlorosomes, while carotenoid glucoside esters are present in the cytoplasmic membranes. In purple bacteria and heliobacteria, carotenoid glucoside esters would also be present in the cytoplasmic membrane or invaginations thereof. Thus, although the functions of carotenoid glucoside esters have yet to be determined, it is possible that they may play a role in stabilizing photosynthetic membranes to environmental extremes. Finally, the observation that 4,4′-diaponeurosporene and associated glucoside esters were highly unstable compared with typical C40 carotenoids may have significance to the biology of the strictly anaerobic heliobacteria. Carotenoid instability, coupled with the fact that levels of carotenoids in heliobacteria are lower than in other anoxygenic phototrophs or oxygenic phototrophs such as spinach and cyanobacteria (Ikegami et al. 2000), may be major reasons why heliobacteria are so sensitive to molecular oxygen (Madigan and Ormerod 1995). Acknowledgements We thank Dr. N. Akimoto of Kyoto University for the analysis of the high-resolution FAB-MS spectra. Research in the laboratory of MTM is supported by grant OPP0085481 from the US National Science Foundation.

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