FEMS MicrobiologyLetters 84 (1991) 301-306 © 1991 Federation of European MicrobiologicalSocieties 0378-1097/91/$03.50 Published by Elsevier

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FEMSLE 04705

Antibiotic production by the marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: localization of activity to the chromatophores J. G r a n t Burgess, H i d e a k i Miyashita, H i r o a k i S u d o a n d T a d a s h i M a t s u n a g a Department of Biotechnology, Tokyo Uniuersityof Agriculture and Technology, KoganeL Tokyo, Japan

Received 8 August 1991 Revision received 16 September 1991 Accepted 17 September 1991 Key words: Antibiotic; Photosynthetic bacteria; Chromatium; Chromatophore; Purple sulfur photosynthetic bacteria; Marine bacteria

1. S U M M A R Y Over 200 strains of marine purple photosynthetic bacteria were isolated. Two strains showed antibiotic activity towards Saccharomyces cereuisiae and were tentatively identified as Chromatium purpuratum. Crude antibiotic, prepared by solvent extraction, showed a broad antimicrobial spectrum. The highest activity was found in the chromatophore fraction. Chromatographic separation of purified light harvesting complex from one strain, NKPB 031704, showed the presence of two separate pigmented compounds which were responsible for antimicrobial activity. Our findings reveal the unexpected ability of photosynthetic bacteria to produce broad spectrum antibiotics. In addition, this is the first example of

Correspondence to: T. Matsunaga, Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan.

intracellular localization of antibiotic activity in a marine bacterium.

2. I N T R O D U C T I O N The production of antibiotics by marine bacteria is well documented [1-9] although, until now, no studies on marine photosynthetic bacteria have been performed despite their widespread occurrence [10]. A current and widely applicable definition of an antibiotic is a compound which in small quantities can inhibit the growth of, or kill, microbes. Thus, antibiotics may be intermediates or the end products of metabolism or may be waste products or other compounds which have antibiotic properties. Recently, there has been growing interest in the field of marine biotechnology [11]. In addition, improved optical fiber photobioreactors [12], combined with the availability of recombinant DNA technology for marine phototrophic bacteria [13], make photosynthetic pro-

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duction of medically important compounds an attractive goal. We describe the discovery of two strains of marine Chromatium which produce antimicrobial compounds with a broad activity spectrum. The anti-yeast activity of these extracts was investigated and attempts made to find out where in the cell these compounds are located. We have found that the light harvesting complexes contain the highest level of antibiotic activity.

3. M A T E R I A L S A N D M E T H O D S

3.1. Isolation of strains and antibiotic screening Photosynthetic bacteria were collected and purified according to established techniques [14]. Cultures were grown in R C V B N medium which is RCVB medium supplemented with 3% NaCi [13,15]. Cells were harvested by centrifugation, resuspended in water and placed in a boiling water bath for 1 h before the cleared supernatant was recovered and used for bioassay against Saccharomyces cereuisiae, Lactobacillus acidophilus and Escherichia coli. This was carried out by using the p a p e r disc method [20]. Crude antibiotic (50 pA) was added to a sterile filter p a p e r disc (diameter 8 mm) placed on an agar plate which had been inoculated with the test organism. Positive inhibition was indicated by the appearance of a clear zone after 15 h incubation at 37°C. 3.2. Bioassay of extracted antibiotic The two strains which were found to have antimicrobial activity, Chromatium NKPB 031704 and NKPB 002108 were used to prepare antibiotic by solvent extraction. Total lipids were extracted with a c h l o r o f o r m : m e t h a n o l :water mixture ( 1 : 2 : 0 . 8 vol). The organic phase was dried in a vacuum desiccator and the lipid residue was redissolved in ethanol to a final concentration of 5 m g / m l . This crude organic extract was added to cultures of S. cere~'isiae to a final concentration of 100 p~g/ml to determine anti-yeast activity. Yeast was grown in Y P G medium containing per liter: glucose 20 g, peptone 20 g, yeast extract 10 g, p H 6.5. C h r o m a t o p h o r e membranes, intracytoplasmic m e m b r a n e s which contain the photo-

synthetic apparatus, were prepared from strain NKPB 031704 by the method of Niederman and Gibson [16]. Cells were disrupted using a French press and the resulting extracts separated by sucrose step centrifugation. A cytoplasmic membrane fraction was removed from the sucrose gradient cushion of 60% (wt : wt) at the bottom of the tube. Cytoplasmic material was recovered from the aqueous phase at the top of the gradient. Light harvesting complexes (LHC) were prepared from NKPB 031704 by the method of Schwerzmann and Bachofen [17] and were extracted with a c e t o n e : m e t h a n o l (7:2) and fractionated on a sephadex LH-20 column with ethanol as the mobile phase. The activity of each fraction was assayed by the paper disc method using 8-ram diameter p a p e r discs and adding 50 /xg of pigment to each disc. Incubation zones were measured after a 24-h growth period at 37°C for each organism tested. Incubation zones for the fungi, Trichophyton sp. were measured after 3 days growth at room temperature. Mucor

racemosus, Pyricularia oryzae, Candida albicans and Saccharomyces sake were grown on YPG medium containing 1.5% agar. Bacillus subtilis was grown in medium which contained per liter: glucose 10 g, yeast extract 5 g, bacto tryptone 5 g, agar 15 g, malt extract 10 g, pH 7.2. Staphylococcus aureus and Micrococcus luteus were grown in medium which contained per liter: beef extract 7 g, peptone 10 g, sodium chloride 3 g, agar 15 g, p H 7.0. Escherichia coli was grown in Y T medium which contained per liter: bacto tryptone 8 g, yeast extract 5 g, sodium chloride 2.5 g, pH 7.0. Candida utilis and the three species of Trichophyton tested were grown on medium which contained per liter; peptone 5 g, yeast extract 3 g, malt extract 3 g, glucose 10 g, agar 15 g, pH 6.0.

4. R E S U L T S A N D D I S C U S S I O N Inhibition of yeast growth by extracts prepared from the two Chromatium strains is shown in Fig. 1. Anti-yeast activity has previously been observed in marine species of the genera: Pseu-

domonas, Aeromonas, Alcaligenes, Chrornobacterium, FlaL~obacterium and Vibrio [2,18,19]. How-

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Fig. 1. Anti-yeast activity of extracts from two strains of marine Chromatium. Growth of Saccharomyces cerevisiae in YPG medium (©) is inhibited by the addition of hot water extracts from Chromatium NKPB031704 (e) and strain NKPB002108 (~). These extracts were prepared by heating the Chromatium cells in a boiling water bath for 60 rain. 1 ml of extract was added to each 9 ml S. cereuisiae culture.

ever, the basis for antibiosis in these experiments remains unclear. Extract from Chromatium NKPB031704 was the subject of further investigation. The appearance of antibiotic activity in the supernatant was time-dependent (Fig. 2) suggesting an intracellular location rather than being present on the surface of the cells, as has been found for a number of antibiotics from marine bacteria [20]. Total lipid was found to contain almost all of the antibiotic activity (Fig. 3) since no growth inhibition of yeast could be detected when the aqueous phase of acetone:methanol (7:2, v / v ) extracted cells was added to yeast cultures (data not shown). Total inhibition of yeast growth was obtained after adding the crude antibiotic fraction (lipid fraction). This experiment also demonstrated that the antibiotic effect originally found during screening of hot water extracts was not a heat induced artefact. Hydrophobic antibiotics have also been purified using similar methods [3]. Chromatographic separation of solvent extracted LHC from NKPB031704 revealed that the antibiotic activity was due to two pigmented components, a red fraction and a

e'o

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Fig. 2. Effect of boiling time on the antibiotic properties of the hot water extract prepared from Chromatium NKPB031704. Almost total inhibition of growth ( > 90%) was obtained after addition of extract prepared by incubating cells for 90 min at 100°C. A relatively small amount of antibiotic was r e l e a s e d during the first 20 min. Percentage growth was m e a s u r e d relative to a control which did not contain extract.

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Fig. 3. Localization of antibiotic activity to the lipid fraction of whole cells. Growth of S. cerevisiae in YPG medium is shown (o). Extracted lipids were dissolved in ethanol (5 m g / m l ) and added to the growth medium to give a final concentration of 100 /xg/ml. Lipid was extracted from Chromatium NKPB031704 ( • ) and from NKPB002108 ( v ). A control containing no lipid (ethanol) was also performed (e). Total inhibition of growth is evident.

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yellow fraction. The antimicrobial spectra of these fractions are shown in Table 1. These compounds were found to be broad-spectrum antibiotics antagonistic towards filamentous fungi, yeast and Gram-negative as well as Gram-positive bacteria. The antibiotic polysaccharides described by Gauthier and Flateau [20] markedly inhibited Grampositive bacteria but with lesser activity against Gram-negative bacteria. In addition, although many antibiotic producing marine bacteria were isolated and described by the pioneering work of Rosenfeld and Zobell [1] inhibition of Staphylococcus aureus and Bacillus subtilis was not recorded for the most actively antagonistic genera. It therefore appears that the antibiotic substances described in this report are unique, firstly in having such a broad antibiotic spectrum, and secondly in being isolated from a phototrophic bacterium which is regarded as being an indigenous marine species [10]. Assay of separated cellular components for antibiotic properties revealed that purified chromatophores possessed the greatest antibiotic activity (Fig. 4). Cytoplasmic material showed a little activity, while cells incubated in the presence of air showed a greatly reduced ability to inhibit the growth of S. cereL,&iae. We have been

Table 1 Antimicrobial spectrum of substances extracted from Chrom a t i u m p u r p u r a t u m NKPB031704

Test organisms

M u c o r racemosus Candida albicans S a c c h a r o m y c e s sake Bacillus subtilis Staphylococcus aureus Micrococcus luteus Escherichia coli Pyricularia oryzae Candida utilis Trichophyton asteroides Trichophyton r u b r u m Trichophyton interdigitale

Antimicrobial activity Yellow fraction

Red fraction

+_ + + + + + + + + + + +_ +_ + + -

+ + + + + + + + + + + +

+ + + + + + + + + + + +

+ + + +

+ +

+ + + Inhibition zone > 25 mm; + + Inhibition zone > 10 mm; + Inhibition zone > 5 ram; + Inhibition zone 1-2 mm; - No effect.

Fig. 4. Localization of antibiotic activity to the chromatophore membranes. Inhibitory activity of cellular fractions is shown. 1: Chromatophore membranes from NKPB031704 resuspended in YPG medium; 2: cytoplasm; 3: cytoplasmic membrane; 4: viable cells incubated in air for 48 h; 5: control, no added material. Test organism is S. ceretqsiae.

unable to find similar attempts at subcellular localization of antibiotic activity in other marine bacteria. It is unlikely that these compounds function as antagonistic chemicals in the marine ecosystem since they were found not to be excreted from the cell in extensive amounts. Thus it appears that the substances responsible are natural components of the cell. The demonstration that the antimicrobial activity can be purified by chromatography into distinct pigmented fractions suggests that the antibiotic activity is not simply the result of metal ion toxicity or the combined toxicity of a number of different substances. In addition antibiotic could not be purified from other marine isolates of C h r o m a t i u m so that this effect is not due to common cellular constituents. We suggest that the active fractions may be photosynthetic pigments or pigment derivatives, intermediates or degradation products which would explain the colour of the active fraction and could suggest a possible reason as to why the activity is located in the photosynthetic chromatophores.

305 ACKNOWLEDGEMENTS T h i s w o r k was f u n d e d by T h e C h e m i c a l M a t e rials R e s e a r c h a n d D e v e l o p m e n t F o u n d a t i o n . J . G . B . t h a n k s t h e J a p a n S o c i e t y for P r o m o t i o n o f S c i e n c e a n d T h e M i n i s t r y of E d u c a t i o n , S c i e n c e a n d C u l t u r e for s u p p o r t . W e also t h a n k T. Koya n o , T O N E N Co. Ltd., for a s s i s t a n c e in d e t e r mining the antimicrobial spectrum.

REFERENCES [1] Rosenfeld, W.D. and Zobell, C.E. (1947) J. Bacteriol. 54, 393-398. [2] Krassil'nikova, E.N. (1961) Mikrobiologiya 30, 545-550. [3] Burkholder, P.R., Pfister, R.M. and Leitz, F.H. (1966) Appl. Microbiol. 14, 649-653. [4] Anderson, R.J., Wolfe, M.S. and Faulkner, D.J. (1974) Marine Biology 27, 281-285. [5] Lemos, M.L., Toranzo, A.E. and Barja, J.L. (1985) Microbial Ecology 11, 149-163. [6] Baam, B.R., Gandhi, N.M. and Freitas, Y.M. (1966) Helg. wiss. Meeresunters 13, 181-185. [7] Fujioka, R.J., Loh, P.C. and Lau, L.J. (1980) Appl. Er~viron. Microbiol. 6, 1105-1110.

[8] Wratten, S.J., Wolfe, M.S., Andersen, R.J. and Faulkner, D.J. (1977) Antimicrob. Agents Chemother. 11,411-414. [9] Austin, B. (1989) J. Appl. Bacteriol. 67, 461-470. [10] Imhoff, J.F. (1988) In: The Halophilic Bacteria, Vol. I. (Rodriguez-Valera, Ed.), pp. 85-108. CRC Press, Boca Raton, FL. [11] Matsunaga, T., Matsunaga, N., Tsubaki, K. and Tanaka, T. (1986) J. Bacteriol. 168, 460-465. [12] Matsunaga, T., Takeyama, H., Sudo, H., Oyama, N., Ariura, S., Takano, H., Hirano, M., Burgess, J.G., Sudo, K. and Nakamura, N. (1991) Appl. Biochem. Biotechnol. 28/29, 157-167. [13] Matsunaga, T., Tsubaki, K., Miyashita, H. and Burgess, J.G. (1990) Plasmid 24, 90-99. [14] Truper, H.G. (1970) Helgolander wiss. Meeresunters. 20, 6-16. [15] Beatty, J.T. and Gest, H. (1981) Arch. Microbiol. 129, 335 -340. [16] Niederman, R.A. and Gibson, K.D. (1978) In: The Photosynthetic Bacteria (Clayton, R.K. and Sistrom, W.R., Eds.) p. 82. Plenum Press, New York. [17] Schwerzmann, R.U. and Bachofen, R. (1989) Plant Cell Physiol. 30, 497-504. [18] Buck, J.D., Meyers, S.P. and Kamp, K.M. (1962) Science (NY) 138, 1339-1340. [19] Buck, J.D., Ahearn, D.G., Roth Jr, F.J. and Meyers, S.P. (1963) J. Bacteriol. 85, 1132-1135. [20] Gauthier, M.J. and Flateau, G.N. (1976) Can. J, Microbiol. 22, 1612-1619.

Antibiotic production by the marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: localization of activity to the chromatophores.

Over 200 strains of marine purple photosynthetic bacteria were isolated. Two strains showed antibiotic activity towards Saccharomyces cerevisiae and w...
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