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16.1 Introduction

With more than 750 different molecular structures (Britton et al. 2004) the carotenoids are the most important group of pigments in nature. In contrast to phototrophic organisms for which the presence of carotenoids as photoprotectants is essential, formation of carotenoids is found only in a few heterotrophic microorganisms (Goodwin 1980). However, it is striking that carotenoids are widely distributed in extremophiles. Examples are the thermophilic bacterium Thermus thermophilus, which synthesizes zeaxanthin and β-cryptoxanthin glucoside fatty acid esters that help in membrane stabilization (Yokoyama et al. 1995, 1996a), and the psychrotrophic bacterium Arthrobacter agilis, which increases its content in C50 bacterioruberin glycosides in response to low temperature (Fong et al. 2001). Bacterioruberin is also the major carotenoid found in the radioresistant bacterium Rubrobacter radiotolerans (Saito et al. 1994) and in halophilic archaea of the family Halobacteriaceae(Oren 2002). It is striking that saltern ponds are frequently colored orange to deep red. This is due to the presence of mainly the β-carotene-rich alga Dunaliella salina (Ben-Amotz and Avron 1990), to C50 carotenoids including bacterioruberin (Straub 1987) produced by halophilic archaea, or the C40 acyl glycoside produced by the extremely halophilic bacterium Salinibacter ruber (Lutnæs et al. 2002). Interestingly, also many endospore-forming bacteria isolated from saline environments like salt marshes contain carotenoids, whereas their non-halophilic relatives do not (Turner and Jervis 1963), indicating that carotenoids may play a crucial role in salt adaptation in these organisms.

All these observations suggest an important role and ecological function of carotenoids in marine environments. In this review, we concentrate on the type and function of carotenoids produced by halophilic microorganisms with focus on Halobacillus halophilus (formerly Sporosarcina halophila). This Gram-positive bacterium is moderately halophilic (Claus et al. 1983; Spring et al. 1996) and can tolerate up to 3.0 M sodium chloride. This osmotic burden is compensated by the accumulation of compatible solutes (Roeßler and Müller 1998, 2001; Müller and Saum 2005; Saum et al. 2006; Saum and Müller 2007, 2008a, b). Like several other halophilic and halotolerant bacteria (Aasen et al. 1969; Duc et al. 2006), H. halophilus is pigmented.

16.2 Structure and Biosynthesis of Carotenoids

Carotenoids are isoprenoids containing a characteristic polyene chain of conjugated double bonds. The two general groups of pigments are the hydrocarbons (carotenes) and oxygenated derivatives (xanthophylls). Most of them consist of 40 carbon atoms, but in a few species carotenoids with a variation of the carbon atom number are formed. Especially among non-photosynthetic halophilic bacteria and archaea also carotenoids with a C30 or C50 backbone were identified (Kelly et al. 1970; Marshall and Wilmoth 1981; Takaichi et al. 1997; Krubasik et al. 2001; Köcher et al. 2009). The most prominent example of C50 carotenoids are the straight chain derivatives of α-bacterioruberin (Fig. 16.1 (I)) found in halophilic archaea (Kelly et al. 1970). Other carotenoids found in halophilic organisms, as already mentioned above, are β-carotene (Fig. 16.1 (III)) produced by the alga Dunaliella salina or the C40 acyl glycoside salinixanthin (Fig. 16.1 (II)) which is synthesized by the extremely halophilic bacterium Salinibacter ruber. But there are also new marine isolates which produce structurally novel and rare carotenoids. These pigments include 2-hydroxyastaxanthin (Fig. 16.1 (IV)) from Brevundimonas sp. SD212 (Yokoyama et al. 1996b), saproxanthin (Fig. 16.1 (V)) and myxol (Fig. 16.1 (VI)) from marine strains of the Flavobacteriaceae (Shindo et al. 2007), and deoxymyxol 1′-glucoside and 4-ketodeoxymyxol 1′glucoside from Gordonia terrae AIST-1 (Takaichi et al. 2008).

Fig. 16.1
figure 1_16

Structure of carotenoids found in halophilic microorganisms. (I) α-bacterioruberin; (II) salinixanthin; (III) β-carotene; (IV) 2-hydroxyastaxanthin; (V) saproxanthin; (VI) myxol

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Despite the structural diversity of carotenoids, a similar biosynthesis pathway is known for all carotenogenic organisms. The starting point for assembly of the carbon backbone of all carotenoids is the isomerization of the five-carbon compound isopentenyl pyrophosphate (IPP) to its allelic isomer dimethyallyl pyrophosphate (DMAPP) to generate geranyl pyrophosphate (GPP) (Fig. 16.2). This compound undergoes further condensation with IPP to form farnesyl pyrophosphate (FPP) catalyzed by FPP synthase. FPP is the main precursor for all known C30 carotenoids, which is condensed with another FPP molecule to form diapophytoene. For the synthesis of C40, C45 and C50 carotenoids one further addition of IPP to FPP to generate geranylgeranylpyrophosphate (GGPP) is catalyzed by a GGPP synthase. GGPP is then condensed to a second GGPP molecule by the action of a phytoene synthase to form the C40 precursor phytoene. Phytoene as well diapophytoene are both colorless but they represent the first step of carotenoid biosynthesis. Next a diapophytoene/phytoene desaturase introduces double bonds into the molecules to yield diapo-/neurosporene, or diapo-/lycopene.

Fig. 16.2
figure 2_16

Biosynthetic pathway of C30 and C40, C45 and C50 carotenoids. IPP, C5 (Isopentenyl pyrophosphate); DMAPP, C5 (dimethylallyl pyrophosphate); GPP, C10 (geranyl pyrophosphate); FPP, C15 (farnesyl pyrophosphate); GGPP, C20 (geranylgeranyl pyrophosphate); PP (pyrophosphate)

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16.3 Structure of the Carotenoids Produced by H. halophilus

A first indication on the nature of the H. halophilus pigments was obtained by inhibition of carotene desaturation with diphenylamine (DPA). When cells were grown in the presence of this inhibitor the biosynthesis of colored carotenoids was blocked and they showed a white pigmentation. DPA prevents the insertion of double bonds into phytoene and diapophytoene (Sandmann and Fraser 1993). Biochemical analyses revealed that a C30 carotenoid was accumulated in H. halophilus and identified as 15-cis and all-trans 4,4′-diapophytoene (Köcher et al. 2009). The spectra showed the typical maxima at 275, 285 and 295 nm. In addition to inhibitors, pigment mutants are a useful tool to elucidate a carotenogenic pathway. Several mutants of H. halophilus were generated by chemical mutagenesis with EMS (ethyl methanesulfonate) (Köcher et al. 2009). They fell into three categories according to their pigmentation: light orange (mutant OC), bright yellow (mutant YC) and white (mutant WH) (Fig. 16.3). The carotenoids which accumulated were separated and identified. In OC, the prominent carotenoid had the same retention time and spectrum as diaponeurosporenoic acid, in WH the only detectable carotenoid was apo- or diapophytoene and in YC only apo-/diaponeurosporene was detectable.

Fig. 16.3
figure 3_16

Pigment mutants of Halobacillus halophilus generated by chemical mutagenesis with EMS. WT wild type; OC light orange pigmentation; YC yellow pigmentation; WH white pigmentation

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The chemical nature of the carotenoid produced by H. halophilus was finally identified by HR-MS and NMR analyses. For this purpose the carotenoids were extracted three times with CH2Cl2:MeOH (1:1, [v/v]) and subjected to silica gel chromatography. The main compound was isolated and dissolved in CH2Cl2:MeOH (1:1, [v/v]). The carotenoid produced by H. halophilus could be identified as a C30 methyl glucosyl-3,4-dehydro-apo-8′-lycopenoate (Osawa et al. 2010) (Fig. 16.4a). This type of carotenoid is also produced by Planococcus maritimus, a marine Gram-positive bacterium (Shindo et al. 2008). To get a closer look on the biosynthesis of this new C30 carotenoid we analyzed the structure of the carotenoids of the OC mutant, which are most likely intermediates of the wild type carotenoid. And indeed two novel carotenoids, hydroxy-3,4-dehydro-apo-8′-lycopene and methyl hydroxy-3,4-dehydro-apo-8′-lycopenoate, could be identified (Fig. 16.4b).

Fig. 16.4
figure 4_16

The structure of carotenoids produced by the wild type and the mutant OC of Halobacillus halophilus

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16.4 Biosynthesis of a Unique C30 Carotenoid Produced by H. halophilus

Inspection of the genome of H. halophilus revealed two gene clusters probably involved in carotenoid biosynthesis (Fig. 16.5). Genes crtNa, crtNc, and crtM are arranged in the same orientation. crtNa and crtNc show an overlap of 3 bp, and crtNc and crtM are separated by only 3 bp. 429 bp downstream of crtM crtNb, orf-GT, and orf-AT build a second cluster in the opposite orientation. They are separated by 21 bp between orf-GT and orf-AT and overlap 117 bp in the case of crtNb and orf-GT.

Fig. 16.5
figure 5_16

Organization of carotenogenic genes in Halobacillus halophilus. crtNa: diapophytoene desaturase; crtNc: putative diapopyhtoene dehydrogenase; crtM: diapopyhtoene synthase; crtNb: putative diapolycopine oxidase; orf-GT: glucosyl transferase; and orf-AT: acyl transferase

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The arrangement of the putative carotenoid biosynthesis genes is indicative of their organization in transcriptional units. To prove the existence of such a polycistronic messenger, mRNA was isolated from H. halophilus cells and transcribed into cDNA. This cDNA was then used as a template in a PCR using primers that bridge the intergenic regions between crtNa and crtNc, between crtNc and crtM, between crtNb and orf-GT and between orf-GT and orf-AT. Products with the expected fragment sizes were obtained indicating that the cDNA originated from two different polycistronic mRNA. This result demonstrates that crtNa, crtNc, and crtM as well as crtNb, orf-GT and orf-AT are part of an operon, respectively.

Three of the genes products, designated CrtNa, CrtNb, and CrtNc, are similar to bacterial carotene desaturases. CrtNa has 48% amino acid identity (similarity 71%) with that of a CrtN homologue in Methylomonas sp 16a. CrtNb had 31% amino acid identity (similarity 54%) with that of a 4,4′-diapolycopene oxidase in Methylomonas sp. 16a and 31% amino acid identity (similarity 54%) with the diapophytoene desaturase in Psychroflexus torquis ATCC 700755. We could also identify a third homologue of a diapophytoene desaturase. CrtNc is highly similar to the diapophytoene dehydrogenase of Heliobacillus mobilis (AAC84034) (55%). CrtM compares best to a putative phytoene/diapophytoene synthase that catalyses the condensation of two molecules of farnesyl-pyrophosphate. The protein of the latter has a length of 301 amino acids and is 55 and 44% identical (similarities, 71% and 61%) to CrtM from Bacillus sp. NRRL B-14911 and Oceanobacillus iheyensis, respectively. Besides these typical proteins for carotenoid biosynthesis, we could identify two additional proteins that appear to be involved in pigment biosynthesis in H. halophilus. The product of orf-GT with a deduced length of 369 amino acids is similar to a glycosyl transferase from Chlorobaculum tepidum TLS (57% similarity, 35% identity) involved in carotenoid synthesis. orf-AT is a putative acyl-transferase with a deduced length of 236 amino acids. It is 57% similar (35% identity) to the protein found in Exiguobacterium sibiricum 255-15 (Köcher et al. 2009).

The biochemical and genetic data allowed us to postulate a biosynthesis pathway for the carotenoids in H. halophilus. It should be pointed out that the identified intermediates as well as methyl glucosyl-3,4-dehydro-apo-8′-lycopenoate are 8′-apo derivatives with asymmetrical arrangement of the methyl groups, unlike the 4,4′-diapo derivatives, which are typically synthesized with two molecules farnesyl diphosphate (Tao et al. 2005; Köcher et al. 2009). In addition, the identification of hydroxyl-3,4-dehydro-apo-8′lycopene without terminal end group oxidation excludes the possibility that apo-8′ products are derived by cleavage of C40 carotenoids at position 8′. Therefore, we propose that the biosynthesis pathway in H. halophilus initially starts with apo-8′-phytoene by the condensation of C20 geranylgeranyl pyrophosphate and C10 geranyl pyrophosphate, catalyzed by an apophytoene synthase, encoded by crtM (Fig. 16.6). In the next step, apo-8′-lycopene is produced by the action of CrtNa, an apophytoene desaturase. In the later biosynthesis steps towards methyl glucosyl-3,4-dehydro-apo-8′-lycopenoate hydroxyl-3,4-dehydro-apo-8′lycopene is glycosylated and oxidized to the corresponding aldehyde propably catalyzed by an CrtNb-like enzyme as it is described by Tao et al. (2005), and then oxidized with another oxidase, which may be encoded by crtNc.

Fig. 16.6
figure 6_16

Proposed carotenoid biosynthetic pathway in Halobacillus halophilus. Gene products catalyzing the individual reactions are indicated next to the arrow

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16.5 Function of Carotenoids in Halophilic Microorganisms

Carotenoids play an important biological role not only in phototrophic but also in heterotrophic organisms. Especially for halophilic microorganisms the diversity of these functions which involve interactions with and without light is essential for surviving in such extreme habitats.

16.5.1 Carotenoids and Light Harvesting

The most prominent function of carotenoids is their contribution to the harvesting of light energy by absorbing light and passing excitation energy on to (bacterio)chlorophyll, thereby extending the wavelength range of the light that can be harvested. Much simpler retinal-based energy transducers are found in halophilic archaea, bacteriorhodopsin and archaerhodopsin. Absorption of light and the proton transport take place within one protein molecule containing a single retinal chromophore. This protein undergoes a cycle of reactions, resulting in the translocation of a proton from inside the cell to the outside and the generation of a transmembrane potential that is usable for ATP synthesis, ion transport, and cell motility. The reaction is initiated by light-induced isomerization of the chromophore from all-trans to 13-cis (Mathies et al. 1991) and involves changes in the pK a values of the buried carboxyl groups (Balashov 2000), and small scale (Lanyi and Schobert 2004) and large-scale (Subramaniam et al. 2002) conformational changes of the protein. The early events involve spectral evolution, which has been interpreted as relaxation through a series of excited states and photoproducts (denoted as H, I, and J) (Sharkov et al. 1985; Gai et al. 1998; Kobayashi et al. 2001; Herbst et al. 2002; Kahan et al. 2007) that leads to the formation of the 13-cis photoproduct (Kochendoerfer and Mathies 1995). Another retinal protein, xanthorhodopsin (Balashov et al. 2005), from the cell membrane of the extremely halophilic eubacterium S. ruber, showed an unexpected association with a C40 carotenoid, salinixanthin (Lutnæs et al. 2002). This carotenoid was found to undergo large reversible absorption changes when the retinal chromophore was removed and replaced in the protein (Balashov et al. 2005). The carotenoid is bound to the protein in a 1:1 ratio. The action spectrum for proton transport indicated that light absorbed not only by the retinal but also by the carotenoid is utilized for proton transport, with 40% efficiency (Balashov et al. 2005). Subsequent studies using steady-state fluorescence measurements demonstrated that there indeed exists an efficient energy-transfer channel between salinixanthin and retinal (Balashov et al. 2008), making xanthorhodopsin the simplest antenna protein known so far.

16.5.2 Carotenoids as Photoprotectants

High on the list of functions attributed to carotenoids is “photoprotection” against photoxidative damage by quenching singlet oxygen as well as other harmful radicals that are formed when cells are illuminated (Demmig-Adams and Adams 2002). Light energy especially in combination with oxygen, can be very harmful, causing damage to cells via singlet oxygen (1O2) and oxidizing free radicals. Living organisms have evolved defenses to prevent or minimize this damage. Carotenoids are a major part of this defense and can be effective in a number of ways. Not only photosynthetic organisms have to prevent the formation and damaging effects of 1O2, also in non-photosynthetic organisms, exogenous or endogenous sensitizers (e.g., porphyrin such as protoporphyrin IX and heme) can be excited and cause photooxidative stress by damaging DNA, protein, lipids and other cell components (Sies and Mehlhorn 1986). When cells are illuminated photosensitizer can form the longer lived, lower energy triplet state. This in turn can undergo energy transfer to oxygen to form the highly reactive singlet oxygen. The triplet energy level of carotenoids is comparatively low, so that these pigments have the ability to accept energy from a triplet state sensitizer or from 1O2, thereby preventing or minimizing damage. The triplet excited state of the carotenoid can then return to the ground state dissipating the energy excess as heat. Thus, the carotenoid acts as a catalyst in the deactivation of 1O2 (Becker et al. 1991). It is known from many studies that light is the environmental factor with the most influence on carotenoid biosynthesis. The best understood examples for non-photosynthetic prokaryotes are the blue light induced carotenoid biosynthesis in Myxococcus xanthus (Burchard and Dworkin 1966) and the light induced pigment production in Streptomyces coelicolor (Takano et al. 2005). But also for halophilic organisms there is some evidence for the function of carotenoids to prevent photooxidative damage. For Halobacterium salinarum it could be shown that white mutants lacking bacterioruberins grown under high light intensities approaching those of full sunlight were outcompeted by the pigmented parent strain (Dundas and Larsen 1963). It has also been shown that bacterioruberin offers protection against ionizing radiation and UV both in vitro, reducing the occurrence of DNA strand breaks (Kottemann et al. 2005) and thymidine degradation (Saito et al. 1997), and in vivo with a decrease in survival of colorless H. salinarum mutants lacking bacterioruberin (Shahmohammadi et al. 1998). Also for H. halophilus the C30 carotenoid is essential for growth under oxidative stress (Köcher et al. 2009). Growth of non-stressed cells at conditions where the synthesis of colored carotenoids was inhibited was comparable to the non-inhibited culture. However, when oxidative stress was applied by the addition of duroquinone, the culture devoid of colored carotenoids did not grow. This result indicates that the carotenoids produced by H. halophilus cope with oxidative stress. This is in accordance with the fact that the main carotenoid produced by H. halophilus (methyl glucosyl-3,4-dehydro-apo-8′-lycopenoate) is a very potent antioxidant (Shindo et al. 2008). Also hydroxy-3,4-dehydro-apo-8′-lycopene and methyl hydroxy-3,4-dehydro-apo-8′-lycopenoate, the main carotenoids in the OC mutant, possess a potent antioxidative activity. But they were not as potent as the wild type carotenoid. This may be explained by their reduced hydrophobicity (Shimidzu et al. 1996). Further studies on the antioxidative activities of these carotenoids are in progress.

16.5.3 Carotenoids as Membrane Stabilizers

Membranes in extremophilic prokaryotes often contain polar carotenoids (Anwar et al. 1977; Yokoyama et al. 1995; Chattopadhyay et al. 1997; Jagannadham et al. 2000; Fong et al. 2001). All these organisms live under extreme conditions, and therefore, are dependent of solid membranes as an intact barrier to prevent an uncontrolled penetration of small molecules and ions.

Because of their lipophilic nature carotenoids are located within the hydrophobic inner membrane. The rod-like structure, the presence of polar end groups, and the molecular dimensions of a typical carotenoid, which match the thickness of the bilayer, are directly responsible for the localization and orientation of the molecule within the membrane and for effects on the membrane properties (Gruszecki 1999; Okulski et al. 2000; Sujak et al. 2000). Unpolar carotenoids like β-carotene are entirely lipophilic and remain within the hydrocarbon inner part of the bilayer. Resonance Raman spectroscopy or linear dichroism have indicated an orientation of these pigments with their long axis roughly parallel to the membrane surface (Johanson 1981). In contrast, polar dihydroxycarotenoids like zeaxanthin span across the bilayer with one polar end group associated with each polar surface (Gruszecki and Sielewiesiuk 1990, 1991). By EPR and NMR studies the effect of carotenoids on the structure and dynamics of the lipid membranes was determined (Gruszecki et al. 1999). For the polar carotenoids lutein and zeaxanthin it could be shown that they restrict the molecular motion of lipids and increase the rigidity of the membrane in its fluid state (Subczynski et al. 1992, 1993; Strzalka and Gruszecki 1994). It is concluded, therefore, that the carotenoid acts as a “rivet” mechanically reinforcing and strengthening the bilayer. This idea is supported by studies with Acholeplasma laidlawii whose membranes normally contain cholesterol, obtained from his host (Rottem and Markowitz 1979). When no cholesterol is available, the organism begins to synthesize polar carotenoids which take the place of cholesterol in the membranes. As mentioned above the carotenoid molecule is of suitable length to span across the membrane with polar substituents in the end groups associated with the polar outer faces of the bilayer and the hydrocarbon polyene chain in the hydrophobic lipid core. The length of carotenoids produced by the organisms is dependent in the thickness of the membrane of each organism, since it was shown that the incorporation ratio is higher when the molecular length of the carotenoid corresponds to the thickness of the phospholipid bilayer (Lazrak et al. 1987). Glycosylation of carotenoid end groups strengthens the association with the polar head groups of the bilayer. A good example is the acyclic xanthophylls bacterioruberin of Halobacterium species, which fits perfectly in the thick diphytanyl lipid membrane of these organisms. The incorporation of bacterioruberins in reconstituted Halobacterium lipid membranes was found to greatly increase their rigidity and to decrease their water permeability (Lazrak et al. 1987). For Haloferax mediterranei, which grows best in the presence of 1.5–2.5 M NaCl (Rodriguez-Valera et al. 1983), it was shown that with decreasing the NaCl concentrations to 1.0 M in the medium the carotenoid content increased ninefold compared to cells grown in the presence of 3.4 M NaCl (D’Souza et al. 1997). It was suggested, that carotenoids stabilize the membranes, and therefore, prevent the lysis of the cell at such low salt concentrations.