Journal of Basic Microbiology 2011, 51, 325 – 329 325 Short Communication The relationship between carotenoids and sunlight response in members of the family Micrococcaceae Magdalena Pezzoni, Cristina S. Costa, Ramón A. Pizarro and Oscar J. Oppezzo Comisión Nacional de Energía Atómica, Departamento de Radiobiología, Argentina The aim of this study was to compare the photoprotective effect of carotenoids in phylogentically related bacteria, which synthesize structurally different pigments. Two organisms were isolated from the same environment. Their 16S rDNA sequences and phenotypic characteristics identified them as members of the family Micrococcaceae. Reverse phase HPLC and absorption spectroscopy revealed that one of them, designated RMB40, synthesized 3 carotenoids with 9 conjugated double bonds, whilst the other, designated RMB42, synthesized a single and more hydrophobic pigment carrying 11 conjugated double bonds. Survival curves were obtained during sunlight exposure for both organisms and for carotenoid deficient mutants derived from them. Increased sunlight sensitivity was found in the carotenoidless mutant derived from RMB42. In contrast, pigment depletion had no appreciable effect on the sunlight response of RMB40. It is concluded that the structure of bacterial carotenoid probably exert an important influence on the effectiveness of these compounds to provide photoprotection in vivo. Keywords: Carotenoids / Photoprotection / Sunlight Received: June 09, 2010; accepted: October 17, 2010 DOI 10.1002/jobm.201000223 Introduction* Solar radiation challenges survival of bacteria in natural environments [1], and the carotenoid accumulation is generally accepted as a widespread mechanism to prevent photodynamic effects induced by sunlight in these organisms [2]. Nevertheless, the photoprotective functions exerted by carotenoids in non photosynthetic bacteria have been sparingly studied [3 – 7], and comparisons of the sunlight responses of pigmented and non pigmented bacteria provided arguments in favor of [8, 9] and against [10] the notion that pigments protect bacteria from sunlight damage. Moreover, functions other than photoprotection have been proposed for bacterial carotenoids [11 – 13]. Within this context, further studies are required to establish the actual role of pigmentation in the sunlight response of non photosynthetic bacteria. Correspondence: Dr. Oscar J. Oppezzo, Comisión Nacional de Energía Atómica, Departamento de Radiobiología, Avenida General Paz 1499, B1650KNA General San Martín, Buenos Aires, Argentina E-mail: oppezzo@cnea.gov.ar Phone: 54-11-6772-7013 Fax: 54-11-6772-7188 © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim In vitro the antioxidant activity of carotenoids depends on their structure [14], polarity, and ability to modify the physical properties of lipid membranes [15]. These facts led us to propose that the structure of the carotenoids accumulated in a bacterium could influence the effectiveness of such pigments in preventing damage when the bacterium is exposed to sunlight. To test this hypothesis, we compared the effect of carotenoid depletion on the sunlight sensitivity of two bacterial species which share many characteristics but synthesize structurally different carotenoids. Materials and methods Bacterial strains The organisms used were selected among airborne bacteria isolated on Nutrient Agar plates kept open on the roof of the laboratory. One of them, forming yellow colonies, was designated RMB40 and the other, exhibiting orange pigmentation, was designated RMB42. In order to identify these bacteria their genomic DNA was isolated and 16S rRNA gene was amplified using primers fD1bis (5′ aga gtt tga tcc tgg ctc ag 3′) and rD1bis (5′ www.jbm-journal.com 326 M. Pezzoni et al. Journal of Basic Microbiology 2011, 51, 325 – 329 Table 1. Characteristics of RMB40 and RMB42 strains. Characteristic Colony pigmentation Gram stain Cell morphology Growth on nutrient agar with up to 10% NaCl Relation to oxygen Catalase test Nitrate reduction to nitrite Acid production from glucose Acid production from xylose Acid production from mannose Acid production from maltose Acid production from fructose Hydrolysis of gelatin Hydrolysis of esculin Hydrolysis of Tween 80 Hydrolysis of starch Oxidase test Utilizationa of citrate Utilizationa of fructose Utilizationa of lactose Utilizationa of mannitol Antibiotic sensitivity a Strain RMB40 RMB42 yellow + cocci in pairs or tetrades + orange + cocci in pairs or tetrades + strictly aerobic + – strictly aerobic + – – – – (NR_025310.1) and Kocuria rosea (DQ060382.1), respectively. Therefore, RMB42 is related to the genera Rothia and Kocuria, which belong to the family Micrococcaceae. None of the organisms included in the genus Rothia produces carotenoids, but this is a common feature in the genus Kocuria [19]. The 66 mol% guanine plus cytosine content determined in RMB42 DNA by thermal denaturation, the presence of glycolipids detectable by thin layer chromatography, and pigmentation suggest that RMB42 could be tentatively considered as a member of the genus Kocuria. Carotenoidless mutants derived from RMB40 and RMB42, designated RMB41 and RMB43, respectively, were induced irradiating the parental strains with a germicidal lamp (3 min at 3.5 W m–2) and isolated taking advantage of the pale color presented by their colonies compared to the original strains. – + + – – – – – – + – – + – – kanamycin kanamycin chloramphenicol chloramphenicol erytromycin tetracycline novobiocin methicilin penicillin neomycin vancomycin polymixin B streptomycin as sole carbon source aag gag gtg atc cag cc 3′), based on the universal set of primers fD1+rD1 [16]. PCR products (about 1500 bp) were purified and sequenced using the same primers. Sequences were compared to entries of the NCBI nucleotide sequence database using BLASTN algorithm [17], and the phenotypes of both organisms were characterized [18] (Table 1). The 16S rRNA gene sequence of RMB40 (EMBL Nucleotide Sequence Database accession number FN551251) showed 99% similarity with that of Micrococcus luteus (FJ189776.1). This result and the phenotype of RMB40 identified the strain as belonging to the genus Microccocus. The 16S rDNA sequence of RMB42 (accession number FN551252) showed 98% and 96% similarity with those of Rothia nasimurium © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Results and discussion Pigments present in RMB40 and RMB42 HPLC analysis of carotenoids synthesized by RMB40 and RMB42 demonstrated remarkable differences in pigmentation. The material absorbing visible light extracted from RMB40 eluted as three peaks at 14.4, 31.5 and 39.3 min (Fig. 1a). The pigments in these peaks exhibited the same absorption spectrum, with sharp maxima at 414, 438 and 469 nm, and the presence of carotenoids with 9 conjugated double bonds in a linear chain was inferred from these data [20]. Hydrophilic carotenoids, characterized by their short elution times, were absent in the extract from RMB42 (Fig. 1b). Most of the pigment accumulated by this organism eluted as a single peak at 44.8 min, and the absorption spectrum recorded during the elution of this peak has a shoulder at 426 nm and maxima at 450 and 479 nm. The wavelength of maximum absorption and the loss of fine structure in the spectrum are compatible with chromophores containing 11 conjugated double bonds and cyclic structures containing some of these bonds at the ends [20]. Protective effect of carotenoids The influence of pigmentation on the sunlight responses of RMB40 and RMB42 was evaluated by comparing survival curves for pigmented and carotenoidless cells for each organism. Carotenoid depletion had negligible effects on the sunlight response of RMB41 (Fig. 2a), but the irradiation time required to observe lethal effects in the carotenoid deficient mutant RMB43 was shorter than that found for the parenwww.jbm-journal.com Journal of Basic Microbiology 2011, 51, 325 – 329 Photoprotection by carotenoids in Micrococcaceae a Abs. Abs. 327 0.01 0.01 0.00 300 400 500 (nm) b Abs. Abs. 0.00 0.2 0.2 0.1 0.1 0.0 300 400 500 (nm) 0.0 0 10 20 30 40 50 Elution time (min) Figure 1. Reverse phase HPLC elution profiles of crude extracts of RMB40 and RMB41 (panel a), RMB42 and RMB43 (panel b). Carotenoids were extracted from lysozime treated bacteria by the successive addition of methanol (2.5 vol.), chloroform (2.5 vol.) and water (1.25 vol.). Pigments contained in the chloroform phases were analyzed in an Ultrasphere ODS column eluted with acetonitrile/water (9 : 1) (A) and isopropanol (B) applying a gradient from A/B (90 : 10) to A/B (45 : 55). Absorbance was monitored at 440 or 450 nm. Absorption spectra recorded during elution of peaks are shown in the inserts (corresponding to elution times 14.2, 31.5 and 39.3 min in panel a, and 44.8 min in panel b). In both panels solid lines correspond to wild type strains and dashed lines to carotenoid deficient mutants. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 2. Survival curves for bacteria exposed to natural sunlight. Cultures were grown to stationary phase in Nutrient Broth, with the –4 addition of 10 M diphenylamine when required, and cells were irradiated according to a procedure described elsewhere [21]. Panel a: RMB40 and RMB41 irradiated (circles) (temperature 29 °C, irradiance 924 W m–2), or maintained in the dark under similar conditions (squares). Panel b: RMB42 and RMB43 irradiated (triangles) –2 (temperature 25 °C, irradiance 925 W m ), or maintained in the dark under similar conditions (pentagons). In both panels solid symbols and solid lines correspond to wild type bacteria, whilst empty symbols and dashed lines to correspond to carotenoid deficient mutants. Each assay was performed three times in independent experiments, and comparable results were obtained. tal strain RMB42 (Fig. 2b), indicating an increased sensitivity in this mutant. Beyond the shoulder of the survival curves, kinetics of cell death seems to be similar for RMB42 and RMB43, suggesting that carotenoids exert their photoprotective functions during the accumulation of damage before there is a loss of bacterial viability. In order to exclude the presence of undetected mutations as a reason for the increased sensitivity found in RMB43, an alternative procedure was employed to produce carotenoid depletion in RMB42. Wild type cells were grown in the presence or absence of diphenylamine, an inhibitor of carotenoid synthesis. Irradiation times required to induce lethal effects in diphenylamine treated cells were shorter than those www.jbm-journal.com 328 M. Pezzoni et al. observed for untreated controls (data not shown), resembling the effect found in the carotenoid deficient mutant. An accidental coincidence of the effects produced by undetected mutations and a side effect of the enzymatic inhibitor is unlikely, and the increased sensitivity should be ascribed to pigment depletion. The greater effectiveness of photoprotection in RMB42 is in concordance with the well known relationship between reactivity and length of the polyene chromophore in carotenoids [14]. In model membranes, polar carotenoids were shown to protect lipids against peroxidation while apolar carotenoids had a weaker activity or even a pro-oxidant effect [22, 23], and carotenes exhibit a higher rate constant for quenching of singlet oxygen than xanthophylls [24]. In this sense, it should be noted that the pigment responsible for photoprotection in RMB42 is the most hydrophobic carotenoid detected by reverse phase chromatography in the present study (Fig. 1). The lack of photoprotection in RMB40 was unexpected, but is consistent with the previously available information. Radiation effects observed in Micrococcus using toluidine blue as an exogenous photosensitizer are not comparable with the results presented here [25]. In Myxococcus xanthus carotenoid functions are related with an unusual accumulation of porphyrin [5], and in Micrococcus roseus ATCC 516 [6], at present classified as Kocuria rosea [19], photoprotection is provided by canthaxanthin, echinenone, 4-hydroxyechinenone, and other pigments with a β,β-carotene structure [26], which are structurally different from those found in RMB40. Sarcina lutea ATCC 9341 [3, 4] was classified as Micrococcus luteus in 1977, but it was re-classified as Kocuria rhizophila in 2003 [27] and its pigments have not been characterized. Since ATCC 9341 and RMB40 are not the same organism they could exhibit different sunlight responses. The pigments present in RMB40 could be involved in functions other than photoprotection, as it has been proposed for carotenoids in related bacteria [13, 29 – 31]. Since menaquinones have been identified as the photosensitive compounds protected by carotenoids in Micrococcus [31], the presence of different menaquinones in Micrococcus and related genera [19] could be relevant for the photosensitivity of these bacteria, and eventually for the need of protective pigments. Our results indicate that carotenoids with different structure could be found in phylogenetically related bacteria, isolated from the same environment. In keeping with predictions from in vitro studies, the effectiveness of these pigments to prevent light induced damage in vivo is different. Since the generally accepted notion © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Journal of Basic Microbiology 2011, 51, 325 – 329 that bacterial carotenoids are always photoprotectors raised from studies in only four species of non photosynthetic bacteria [3 – 7] this subject should be reconsidered. Acknowledgement This work has been supported in part by a grant from Agencia Nacional de Promoción Científica y Tecnológica (PICT 38241). References [1] Rozen, Y., Belkin, S., 2001. Survival of enteric bacteria in seawater. FEMS Microbiol. Rev., 25, 513 – 519. [2] Mathews-Roth, M.M., 1997. Carotenoids and photoprotection. Photochem. Photobiol., 65S, 148S – 151S. [3] Mathews, M.M., Sistrom, W.R., 1959. Functions of carotenoid pigments in non-photosynthetic bacteria. Nature, 184, 1892 – 1893. [4] Mathews, M.M., Sistrom, W.R., 1960. 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