Photochromogenesis I. Photochromogenicity in Genus Nocardia, Corynebacterium, Arthrobacter, Brevibacterium, and Flavobacterium Y

Japan. J. Microbiol. Vol. 18(1), 49-56, 1974

Photochromogenesis

I. Photochromogenicity in Genus Nocardia, Corynebacterium, Arthrobacter,Brevibacterium, and Flavobacterium

YasumasaKOYAMA, Yoshitaka YAZAWA1, Saburo YAMAGISHI, and Tadashi ARAI Divisionof MicrobialChemistry, Faculty ofPharmaceutical Sciences, Chiba Universily,Chiba-shi, and Departmentof Antibiotics, Institute ofFood Microbiology, Chiba University,Narashino-shi (Receivedfor publication, August 10, 1973)

ABSTRACT Theeffect of lighton the pigmentationof various strains of Nocardia,Corynebacterium, Arthro- bacter,Brevibacterium, and Flavobacterium wasinvestigated. It wasrevealed that thirtyout of fifty- sevenstrains of Nocardia,two out of fifteen strains of Corynebacterium, twostrains of Arthrobacter,sixout of thirteenstrains of Brevibacterium andtwo out of fourteen strains of Flavobacterium werephotochromo- genic;i. e., thesestrains produced yellow or orangepigments when grown under illumination but entirelyunpigmented in total darkness. From these results, it maybe concluded that photochromo- genicityis nota particularphenomenon limited to specific species, but a common,widely distributed phenomenonin nonphotosynthetic bacteria.

During the course of an investigation of has been unpublished to date. Therefore, the metabolites of Noc rdia brasiliensis IFM83, it appeared of interest to investigate the we observed that colonies of this strain effect of light on the pigmentation of a large developed a rich orange pigments when collection of Nocardia in our laboratory and grown under diffused sunlight, but entirely the other bacteria available for this experi- unpigmented in the dark. Among non- ment, most of which are pigment-forming photosynthetic microorganisms, it has been strains. The present paper deals with known that a certain species of Mycobacterium photochromogenicity in genus Nocardia, [1, 6, 8], Flavobacterium dehydrogenans [12], Corynebacterium, Arthrobacter, Brevibacterium, Myxococcus xanthus [3, 4], Neurospora crassa and Flavobacterium. [13, 14], and Fusarium aquaeductuum [5, 7] MATERIALS AND METHODS exhibited such a specific action toward light which was referred to as photochro- Microorganisms. All of the strains of mogenicity [10] or photoinduced caro- Nocardia, Brevibacterium, and Flavobacterium tenogenicity. On the intracellular pigmen- as well as strains ME24, GB9, SB14, SB24, tation of Nocardia and the other bacteria, and SD15 of the Corynebacterium sp. were however, any reference to such a phenomenon selected from the stock cultures of the Insti- tute of Food Microbiology, Chiba University. Requests for reprints should be addressed to Dr. The strains of Arthrobacter and Corynebacterium Yasumasa Koyama, Division of Microbial Chemi- stry, Faculty of Pharmaceutical Sciences, Chiba numbered in the series of IAM were kindly University, 33, 1-chome Yayoi-cho, Chiba 280, supplied from the Institute of Applied Japan. Microbiology, University of Tokyo, and 1 Present address: Eiken Chemical Co., Ltd., the strains of Corynebacterium numbered in 7-2, 4-chome Higashi-Shinkoiwa, Katsushika-ku, Tokyo, Japan. the series of IFO from the Institute for

49 50 Y. KOYAMA, Y. YAZAWA, S. YAMAGISHI AND T. ARAI

Fermentation, Osaka. Stock cultures of dark-grown and light-exposed bacterial cells the test organisms were maintained on nutri- were harvested by filtration after their ent agar except for those of Nocardia which maximal growth had been attained and were maintained on Kelner-Morton agar. extracted twice with acetone-methanol (1:3 Media and cultivation of organisms. Glycerol v/v). Extracts of the dark-grown and the Kelner-Morton medium (glycerol 60 g, photoinduced bacteria were concentrated starch 10 g, polypeptone 5 g, meat extracts in vacuo, respectively. Each of the concen- 3 g, K2HPO4 0.8 g, FeSO4 0.1 g, 1000 ml trated extracts was dissolved in a small of potato infusion prepared by simmering amount of chloroform and submitted to 200 g of potato slices for 40 min in distilled thin-layer chromatography using silica gel water, pH adjusted at 7.0) and glycerol (layer thickness 0.25 mm, precoated, Merck) Kelner-Morton agar (adding 20 g of agar for the adsorbent and benzene-methyl- to the above medium) were used for the ethylketone (5:1 v/v) for the solvent system. experiments with Nocardia. Nutrient broth RESULTS and agar were used for the other bacteria. The cultivation of organisms was carried On the basis of the results shown in Tables out at 27 C with rigid light exclusion unless 1-5, the test strains have been classified otherwise specified. When organisms were into photochromogenic, scotochromogenic, transplanted, a photographic red lamp was and nonphotochromogenic strains which unavoidably used for illumination. After were characterized as follows. two cultivations on slants in darkness, the bacteria from each slope were inoculated PhotochromogenicStrains onto four plates each containing 20 ml of These strains produce yellow or orange glycerol Kelner-Morton agar or nutrient pigmented colonies if exposed to light but agar. they remain colorless, whitish or beige when Procedure of photoinduction. Two of the grown in total darkness. Pigmentation four agar plates thus inoculated were placed does not occur immediately after illumina- at a distance of 20 cm under two 30 W white tion, but maximal pigmentation is only fluorescent lamps (Toshiba) and exposed to achieved after a relatively long period of light during the incubation period at 27 C. incubation. If the photochromogenic The remaining two agar plates were incubated strains are exposed to light at 0 C and then in total darkness. In another experiment allowed to stand at low temperatures (0-5 C), with Nocardia, photoinduction was carried little or no pigmentation is observed. But out by exposure to sunlight through a window following incubation in the dark and higher for 1 hr out of every 24 hr incubation of the temperatures, pigmentation results. Al- initial 4 days of growth. The color of the though no attempt has been made to measure dark-grown and light-exposed organisms the quality or intensity of the light sources, was observed after maximal growth was only a brief moment of exposure to light attained. is enough to photoinduce pigmentation of Extraction of pigments produced by photoinduced most photochromogenic strains. and dark-grown organisms. Following pro- cedures were devised for every test organisms. ScotochromogenicStrains Each test organism was shake-cultured in These strains produce various types of 100 ml of the medium placed in 500 ml shake pigments (yellow, orange, coral, carrot etc.) flask in the dark at 27 C. Ten milliliters even in complete darkness. As far as tested of the preincubated bacterial suspension was the pigments produced by dark-grown inoculated into each of two 500 ml Erlenmeyer bacteria are identical with those produced by flasks containing 100 ml of the liquid medium light-exposed bacteria. and a suitable amount of glass wool to provide maximal aerobic conditions. One NonphotochromogenicStrains of the two flasks inoculated was incubated Colonies of these strains are colorless, under illumination with white fluorescent whitish or beige. The pigmentation of lamps and the other in total darkness. The cells, if any, is scant and not light-conditioned. PHOTOCHROMOGENESIS. I. 51

Table 1. The pigmentation of various test strains of genus Nocardia when grown under illumination and in the dark

a) photo-=photochromogenic strain, scoto-=scotochromogenic strain, nonphoto-=nonphotochromogenic strain. 52 Y. KOYAMA, Y. YAZAWA, S. YAMAGISHI AND T. ARAI

Photochromogenicityin Genus Nocardia strains were nonphotochromogenic. It was Fifty-seven strains of 21 species of Nocardia of interest that photochromogenic corynebac- including eight strains of Nocardia madurae teria fully grown in the dark were entirely (Actinomadura madurae) and two strains of No- unpigmented by exposure to light, even under cardia pelletieri (Actinomadura pelletieri) were sunlight. This is also the case with pigmen- tested on glycerol Kelner-Morton agar. tation of photochromogenic brevibacteria Colors of the colonies of the test strains grown and flavobacteria described below. under illumination and in the dark are shown in Table 1. Photochromogenicity in Genus Brevibacterium As indicated in the Table, 30 strains of Thirteen strains of seven species of Brevi- ten species were photochromogenic, 15 bacterium were tested. As shown in Table 3, strains of nine species were scotochromogenic, six strains of two species of Brevibacterium. and 12 strains of seven species were non- were photochromogenic, four strains of two photochromogenic. It may be of impor- species were scotochromogenic, and three tance to explain that colonies of some photo- strains of three species were nonphotochro- chromogenic strains, e.g., Nocardia aster- mogenic. oides IFM45, were intensely colored with carrot red pigments upon exposure to sunlight Photochromogenicity in Genus Arthrobacter during its growth but not so under white Two species of Arthrobacter were tested. As fluorescent lamps. shown in Table 4, both test strains were observed to be photochromogenic. Photochromogenicityin Genus Corynebacterium Ten strains of eight species of Corynebacterium Photochromogenicity in Genus Flavobacterium and five strains of unidentified Corynebacterium Fourteen strains of unidentified species were tested. of Flavobacterium were tested. As shown in As shown in Table 2, it was observed that Table 5, two strains of the bacteria were two strains of two species of Corynebacterium photochromogenic, five strains were scoto- were photochromogenic, eight strains of six chromogenic, and seven strains were non- species and an unidentified strain were photochromogenic. scotochromogenic, and four unidentified

Table 2. The pigmentation of various test strains of genus Corynebacterium when grown under illumination and in the dark

a) See legend for Table 1. PHO TOCHROMOGENESIS. I. 53

Table 3. The pigmentation of various test strains of genus Brevibacterium when grown under illumination and in the dark

a) See legend for Table 1.

Table 4. The pigmentation of test strains of genus Arthrobacter when grown under illumination and in the dark

a) photo-=photochromogenic strain .

Table 5. The pigmentation of various test strains of genus Flavobacterium when grown under illumination and in the dark

a) See legend for Table 1. 54 Y. KOYAMA , Y. YAZAWA, S. YAMAGISHI AND T . ARAI

Fig. 2. Visible light absorption spectra of cell extracts and the fractionated pigments A, B, and C of photoinduced Brevibacterium sulfureum AU-31, The pigments A B, and C were obtained by frac- tionation of the methanol-acetone extracts from cells of B. sulfureum AU-31 grown under illumi- Fig. 1. Diagramatic representation of thin-layer nation by means of thin-layer chromatography chromatograms of the bacterial pigments by one dimensional separation. The detail of the method (see Fig. 1). Extract: extracts of the bacterial cells with methanol-acetone (3: 1). was described in the text. Adsorbent and solvent system were silica gel (layer thickness 0.25 mm, precoated, Merck) and benzene-methylethyl- of dark-grown cells of scotochromogenic ketone (5: 1 v/v,) respectively. Numbers, 1-7, indicate pigments of light-ex- bacteria agreed with those of light-exposed posed cells of the following strains, and numbers, cells of the bacteria (4, 4', 5, 5' of Fig . 1). 1'. 2', 3', 4', 5', 7' show pigments of dark-grown Visible light absorption spectra of the cells of their respective strains. 1 and 1': N. aste- methanol-acetone extract from a photo- roides IFM 1. 2 and 2': N. brasiliensis IFM82. chromogenic strain, Brevibacterium sulfureum 3 and 3': N. madurae IFM73. 4 and 4': N, asteroides IFM3Y. 5 and 5': N. polychromogenes IFM17. AU-31, and pigments A, B, and C fraction- 6: C. paurometabolum IFO12160. 7 and 7': B. sut- ated by preparative thin-layer chromato- fureum AU31. graphy are indicated in Fig. 2. From these results it could be expected Thin-Layer Chromatograms of the Pigments Pro- that the production of carotenoid-like pig- duced by Photochromogenicand Scotochromo- ments was induced by exposure to light in genic Bacteria the photochromogenic bacteria . Thin-layer chromatograms of the methanol-acetone extracts of cells of the photochro- DISCUSSION mogenic and scotochromogenic strains by As pointed out in the beginning of this one dimensional separation are illustrated in paper the previous investigations of photo- Fig. 1. As shown there, the three pigments chromogenicity in bacteria have dealt ex- with their respective RF values given symbols clusively with certain species of Mycobac- A (RF 0.68), B (RF 0.56), and C (RF 0.36- terium, F. dehydrogenans,and M. xanthus and no 0.38) were observed in all the chromatograms attention has been paid to other bacteria . of the extracts from the test photochromogenic However, it was revealed through this strains grown under illumination whereas investigation that numbers of strains of these spots were found as extremely weak various genera of bacteria exhibited photo- spots or not observed at all on chromatograms chromogenicity whose descriptions were of the extracts from photochromogenic virtually identical with that of Runyon's cells grown in the dark ( 1, 1', 2, 2', 3, 3', definition [10] described for the classification 6, 7, 7' of Fig. 1). of Mycobacterium. In view of the above On the other hand, the patterns of chroma- facts it may be concluded that photochro- tograms of the methanol-acetone extracts mogenicity is not a specific phenomenon PHOTOCHROMOGENESIS. I. 55 in a particular species of bacteria , but a genie. Although three out of the 21 strains common, widely distributed phenomenon were scotochromogenic, N. brasiliensis may in various species of bacteria. be said to be a homogeneous species from It has been well known that the non- or the standpoint of photochromogenicity. One poorly diffusible color of bacteria varies of the exceptional strains, N. brasiliensis according to the cultural conditions. Even IFM76, is apparently deviated from the under the same cultural condition , e.g., other strains of this species in gross ap- medium, temperature or aeration , the color pearance of morphology as well as its color of bacterial cells is occasionally different . and the other two strains, N. brasiliensis Pigmentation of bacteria is so variable that IFM71 and IFM75, are atypical scoto- this description is not rigid and takes a chromogenic strains which may require certain range of colors observed by different further examination. experiments; for instance, pale yellow to orange. Now we can point out the effect ACKNOWLEDGEMENT of light on pigmentation of bacteria as one The authors are grateful to Dr. Ushio Shimizu, the Institute of Food Microbiology, Chiba University, of the causes for fluctuation in the color of for kindly supplying some of the strains used in this bacterial cultures. study and his helpful discussions, and to the Institute In photochromogenic strains of mycobac- of Applied Microbiology, University of Tokyo, and teria [2, 6, 8, 9], F. dehydrogenans [11], M. to the Institute of Fermentation, Osaka, for relinqui- shing their valuable strains. xanthus [3, 4], and F. aquaeductuum [5, 7], it has been presumed that the photoinduced REFERENCES formation of pigments consists of two steps: [1] Baker, J. A. 1938. Light as a factor in the pro- 1) photochemical reaction requiring both O2 duction of pigment by certain bacteria. J. Bac- and light, and 2) a series of temperature- teriol. 35: 625-630. dependent dark metabolic reactions which [2] Batra, P. 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