Melanin and novel melanin precursors from Aeromonas media

FEMS Microbiology Letters 169 (1998) 261^268

Melanin and novel melanin precursors from Aeromonas media Lewis F. Gibson, Anthony M. George * Department of Cell and Molecular Biology, University of Technology Sydney, P.O. Box 123, Broadway, NSW 2007, Australia Received 6 July 1998; received in revised form 8 September 1998; accepted 17 September 1998

Abstract Many bacteria produce reddish brown to black pigments and some of these have been characterised. This report describes the isolation and characterisation of a diffusible brown melanin-like pigment from the bacterium Aeromonas media. Physicochemical testing suggested that the pigment is a true melanin. New butanol-soluble yellow, red and brown pigments were isolated from the A. media strain under reducing conditions during melanogenesis and these pigments were shown to be unstable precursors of the polymeric brown melanin product. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Melanin ; Melanin precursor; Aeromonas media

1. Introduction Melanins are polyphenolic heteropolymers and are ubiquitous in nature. Their distribution and chemistry have been described in many excellent reviews [1^ 4]. Melanin biosynthesis occurs by the process of melanogenesis which proceeds most commonly via the classic Mason-Raper pathway in which tyrosinase converts tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and then to dopaquinone which undergoes auto-oxidisation and polymerisation to brown or black pigmented eumelanins. Bacteria are known to produce DOPA-melanins, the pheomelanins or eumelanins [5^10]; and melanin-like pigments that are derived from non-nitrogenous phenols, the allomelanins or pyomelanins [4,11,12]. Thus, not all bacterial brown and black pigments are true melanins, and * Corresponding author. Tel.: +61 (2) 9514 4158; Fax: +61 (2) 9514 4003; E-mail: [email protected]

dark, insoluble pigments are often referred to as melanins without regard to their chemistry. Aeromonas media was ¢rst isolated from river water and identi¢ed as a new species of Aeromonas [13]. The type strain is ATCC 33907. Some isolates of A. media are known to produce a di¡usible brown pigment [13]. This study is concerned with the melanin-producing A. media ATCC 23309 (UTSA199). In this study, A. media UTSA199 was examined for all three product types. The need for such a complete characterisation was prompted by our development of the strain as a potential probiotic for the control of bacterial pathogens in aquaculture [14].

2. Materials and methods 2.1. Strains and media A. media UTSA199 is ATCC 23309. A. media

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 4 9 5 - 9

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UTSA173 is a river water isolate whose identity was con¢rmed by diagnostic testing [15]. Trypticase soy agar (TSA) or broth (TSB) were used as the growth medium. Specialised agars are described below. 2.2. Isolation of melanin TSA soft (0.7% agar) plates were seeded with an exponential-phase culture of A. media UTSA199. Plates were incubated at 35³C for 48 h by which time there were luxuriant lawns of bacteria over the agar surfaces and the plates were a very dark brown colour from the melanin produced. The plates were frozen at 320³C (usually overnight) then thawed to room temperature. The agar was scraped into centrifuge bottles to which chloroform (0.2 vol) was added to kill the bacteria and deproteinise the melanin pigment. The homogenised mixtures were centrifuged (6000Ug; 1 h; room temperature) to remove agar, bacteria and other debris. The deep brown liquor was ¢ltered through a 0.45-Wm membrane to ensure sterility, adjusted to pH 13 with NaOH to ensure complete polymerisation, then to pH 2 with HCl. The precipitated melanin was centrifuged as above, washed with 100% methanol (0.1 vol), 70% ethanol (0.1 vol), air-dried, then resuspended in 100 mM Tris-HCl (pH 7.5) and stored at 4³C. The concentration of crude melanin was estimated from a standard curve of synthetic melanin (Sigma Chemical Co.) at OD400 . In a variation of this scheme, the melanin could be processed further after the ¢ltration step by charging the ¢ltrate to DEAE-cellulose (Sigma Chemical Co.) according to [16]. The dark brown pigment was bound strongly to the resin but the £ow-through liquor was a reddish colour. This red pigment was investigated further (see below). The brown pigment was recovered from the resin by elution in 1.0 N NaOH. 2.3. Properties of melanin from A. media UTSA199 Chemical and physical tests that typically identify melanin pigments were carried out as described previously [11,17,18]. 2.4. HPLC Late exponential phase TSB cultures of A. media

UTSA199 (test) and UTSA173 (control) were used to produce cell-free ¢ltrates. These were acidi¢ed to pH 3.5 with 0.1 M ammonium acetate, and 5-Wl samples were injected through a rheodyne valve with a 20-Wl sample loop onto an ODS C18 reverse-phase column in a Waters Associates model 450 liquid chromatograph and UV detector. An LKB model 2150 pump maintained a £ow rate of 1 ml min31 of the eluting solvent (0.01 M ammonium acetate, pH 3.5). L-3,4-Dihydroxyphenylalanine (L-DOPA) or homogentisic acid (HGA) were detected at 260 and 280 nm, respectively. L-DOPA and HGA (both from Sigma Chemical Co.) were used as standards. 2.5. Isolation of yellow, red and brown butanol-soluble pigments TSA plates (0.7% agar) were supplemented with ascorbic acid at 0.05^0.25%, seeded with a lawn of an overnight A. media UTSA199 culture and incubated for 24^48 h at 32³C. Pigmented agar was acidi¢ed and processed following the appearance of the three pigments in the various ascorbate-agar plates. Yellow, red and brown pigments were isolated from the ascorbate-supplemented TSA plates as described above for crude melanin except that following the ¢ltration step, the pigments were extracted directly into butanol then concentrated or dried in a stream of nitrogen. The coloured pigments were then puri¢ed further as detailed below. 2.6. Gel ¢ltration chromatography, absorption spectrophotometry and mass spectrometry Crude melanin or precursor coloured pigments were chromatographed through Sephadex G-50 (Pharmacia) in 50 mM Tris-HCl (pH 7.0) containing 1% NaCl at a £ow rate of 1 ml min31 . The column dimensions were 1.5U50 cm. Fractions (1 ml) were monitored at OD400 . Ultraviolet-visible absorption spectra of puri¢ed column fractions of yellow, red, or brown pigments were recorded on a Varian Cary 1E UV-Visible Spectrophotometer. Pigment peak fractions were concentrated by extraction into butanol, dried in a stream of nitrogen and redissolved in the matrix p-nitrobenzyl alcohol for FAB mass spectrometry in a Jeol JMS-DX303 instrument using ar-

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gon as the carrier gas and p-nitrobenzyl alcohol as the matrix.

3. Results 3.1. Properties of melanin from A. media Since melanins are normally complexed with protein [4], the crude melanin isolated from A. media was checked following the deproteinisation step. Standard synthetic melanin (Sigma Chemical Co.) and A. media melanin were adjusted to a concentration of 400 Wg ml31 and the UV absorbances measured. The OD254 and OD280 were 0.813 and 1.719 (Sigma melanin) and 0.818 and 1.749 (A. media melanin), respectively. These absorbances indicated that crude melanin was essentially free of protein and equivalent in concentration to the synthetic melanin. Crude melanin from A. media UTSA199 showed typical properties of melanins [11,18]. It was insoluble in chloroform, isoamyl alcohol, butanol, ether and ethyl acetate; was precipitated below pH 3 and redissolved only in alkali above pH 10; was precipitated in alkaline FeCl3 , bleached in H2 O2 and produced a blue colour in FeSO4 /ferricyanide; pigment formation was delayed or prevented by ascorbic acid in the growth medium; and was overproduced in

Fig. 1. Melanin pigment precursors produced by A. media UTSA199 in broth culture. Growth of the culture was monitored at OD600 (a) and the formation of pigment at OD400 (b). The ¢rst arrow represents the appearance of yellow pigment in the culture, changing to red (second arrow) and ¢nally brown pigment (third arrow).

Fig. 2. Gel ¢ltration elution pro¢les of melanins. The pro¢les on a Sephadex G-50 column denote brown (a), red (O) and yellow (E) pigment precursors. Calibration compounds on the same column (not shown) with elution volumes (Ve , ml) and molecular sizes (Mr , Da) were : blue dextran (29 ; 2U106 ); vitamin B12 (63; 1357) ; bromophenol blue (114.5; 670); and malachite green (122 ; 659).

medium supplemented with tyrosine. Its absorption spectrum was typical for melanins [6,11] with a featureless decreasing pro¢le over the range 200^500 nm with minor shoulders and no peaks (Fig. 3A). Synthetic melanins (Sigma melanin or oxidised LDOPA) produced similar absorption pro¢les (not shown). A. media UTSA199 readily produced melanin in enriched agar media after 24^48 h (Table 1). The intensity of the dark brown pigment increased with time and in the presence of melanin precursor amino acids, especially tyrosine. The use of cysteine as a supplement resulted in a blackish pigment which could indicate the incorporation of sulfur into the melanin polymer. The use of ascorbic acid as a strong reducing agent moderated the onset of melanin synthesis and pigmentation, manifested initially by a slight yellow tinge in the medium which became more intense with time and then changed gradually to a reddish colour. When added to the medium, tyrosine caused an increase in dark brown pigment, cysteine produced a blackish pigment, and ascorbate produced red and yellow pigments with the red pigment turning to brown after several days of incubation. Although not included, a TSA plate containing

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Table 1 Melanin production by A. media UTSA199 in agar bases in the presence of di¡erent supplements

non-melanin-producing strain, did not exhibit an LDOPA peak (Table 2).

Plate typea

3.3. Isolation of yellow, red and brown precursor pigments

MA MA+Tyr 0.1% NA TSA TSA+Cys 0.1% TSA+Phe 0.1% TSA+Tyr 0.1% TSA+Asc 0.1% TSA+Asc 0.2% TSA+Asc 0.3%

Pigment production at 30³Cb 24 h

48 h

3 3 + +++ 3 +++ ++++ + (y) 3 3

3 + ++ ++++ +++ (b) +++++ ++++++ +++ (r) ++ (y) 3

a MA = minimal agar ; NA = nutrient agar; TSA = trypticase soy agar. `Tyr', `Cys', `Phe' and `Asc' are tyrosine, cysteine, phenylalanine and ascorbate, respectively. Plates were seeded with equivalent inocula of cells. b A minus sign indicates growth but no visible pigment in the medium. Plus signs indicate the intensity of the pigmentation which was dark brown except for the cysteine-containing medium which deposited black (b) pigment, and the ascorbate-containing medium which was yellow (y) or red (r) as indicated.

0.3% ascorbic acid showed the normal bu¡-coloured ^ but less con£uent ^ growth of the strain without any visible pigmentation in the agar. 3.2. L-DOPA is a precursor of melanin produced by A. media The major pathway to melanin formation begins with the tyrosinase-catalysed conversion of tyrosine to L-DOPA [1]. Previous studies have detected phenolic precursors such as L-DOPA, catechol or HGA in cell-free culture ¢ltrates of various bacterial species [7,12]. Using HPLC, L-DOPA was detected in a late exponential-phase culture ¢ltrate of A. media UTSA199. The retention time and OD260 /OD280 ratio of one of the A. media UTSA199 culture ¢ltrate peaks were identical to that of a standard L-DOPA sample and di¡erent from that of a standard HGA sample (Table 2). Whereas a standard addition of LDOPA to a ¢ltrate sample produced a higher peak at the same retention time point, an HGA addition produced an additional peak with a longer retention time (data not shown). Moreover, an HPLC trace of a culture ¢ltrate sample from A. media UTSA173, a

In the DEAE-cellulose ¢ltration procedure for the binding of the dark brown melanin pigment, it was noticed that the £ow-through fraction contained a distinctively red coloured pigment which seemed to be a precursor since it changed gradually to dark brown when left at room temperature, or instantly to brown when adjusted to a pH greater than 10. The absorption spectrum of the red-to-brown converted pigment matched that of the original melanin (data not shown), and also could now be bound to DEAE-cellulose. The red pigment was stable for several days at 4³C at a pH less than 3. Like brown melanin, it was precipitated in alkaline FeCl3 and was insoluble in ether and chloroform, but atypically was not precipitated in acid and was partially soluble in ethyl acetate and completely soluble in amyl alcohol or butanol. The ready convertibility of the red pigment to brown melanin suggested that it was a major precursor, yet it was isolated as a minor component. Thus its potential role as a true precursor and not a minor by-product was investigated more fully. In earlier experiments, serendipitous observation had shown that yellow and red pigments were produced by A. media UTSA199 on TSA supplemented with ascorbic acid (Table 1). This experiment was repeated to obtain several soft agar plates each containing yellow, red or brown pigments, which were isolated subsequently from the acidi¢ed, centrifuged and ¢ltered agar. The clear yellow, red or brown liquors were extracted into butanol and stored at 4³C inde¢nitely with no deterioration in colour. Unlike the red pigment transition to brown melanin described above, the yellow pigment was stable in ascorbate agar even after 2 weeks at room temperature. Moreover, the extracted yellow pigment did not change to red or brown but only to an amber colour when the pH was adjusted to 11. It seemed, therefore, that the molecular stage of polymerisation of the yellow pigment was insu¤cient to enable its ready conversion to dark brown melanin, as occurred for the red pigment. When all three coloured

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Fig. 3. Absorption spectra of melanin and precursors from A. media UTSA199. The plots represent puri¢ed melanin (A), butanol-soluble yellow (y), red (r) and brown (b) precursor pigments (B), red precursor spectra at pH 2, 5, 8 and 12 (C) and yellow precursor spectra at pH 2, 4, 6, 9 and 12 (D). For plots C and D, the curves are in decreasing order of pH from top to bottom.

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Table 2 Identi¢cation of L-DOPA in a cell-free culture ¢ltrate of A. media UTSA199 Sample

Retention time (min)

OD260 /OD280

L-DOPA HGA UTSA199 ¢ltrate UTSA199 ¢ltrate+L-DOPA UTSA173 ¢ltrate

3.2 5.1 3.2 3.2 no peak

1.41 2.38 1.45 1.44 ^

pigments were recovered from ascorbate agar and extracted with butanol, colour was partitioned into the aqueous and organic phases. That is, there were butanol-soluble and -insoluble yellow, red and brown pigment components. The butanol-insoluble brown pigment exhibited similar properties to the crude melanin described earlier. The red and brown butanol-soluble pigments were readily converted to butanol-insoluble dark brown melanin by the addition of alkali. When the coloured pigments were dried down from butanol and made into aqueous solutions, the alkali lability or auto-oxidising property of the pigments meant that they had to be stabilised at lower pH values, viz. pH 2 (yellow), pH 4 (red) and pH 6 (brown). The yellow and red pigments were also produced in a nutrient broth culture of A. media grown at 37³C and aerated by shaking at 225 rpm. An overnight culture was diluted with fresh medium to an OD600 of 0.1. Pigments appeared in this second culture after approximately 16 (yellow), 19 (red) and 26 h (brown) within the stationary phase and were detected visually or by the measurement of the absorbance at 400 nm (Fig. 1). In this instance, nutrient broth was selected as the growth medium since its faint yellow colour did not mask the appearance of the darker yellow pigment. Whereas the yellow pigment was only transiently produced and close in colour to the medium, the red pigment was longer lasting and could be recovered from the broth culture as described above for the soft agar extraction. It was also noted that the pH of the stationary phase culture medium had increased from 7.4 to 9.0 upon the appearance of the red pigment. The increasingly alkaline pH was probably responsible for the conversion of the red pigment to the brown melanin endproduct. The appearance of the red and brown pigments did not increase the optical density readings at

600 nm for the growth curve, a not unexpected observation since the visible absorption of melanin pigments is very low ([11]; also see Fig. 3). 3.4. Gel ¢ltration, mass spectrometry and absorption spectra of melanins Crude melanin from A. media had two major elution peaks in a G-50 column (not shown). The ¢rst eluted very close to blue dextran and probably contained some melanin complexed with high molecular mass polysaccharide or protein. The second, major melanin peak eluted a little before commercial Sigma melanin and therefore probably has a slightly higher average molecular mass ( s 2000 Da). This is not dissimilar from averaged estimates for eumelanin of 1100^6000 Da [19] and pheomelanin of 2365 Da [20]. The three coloured pigment precursors (Fig. 2) had elution volumes that were lower than vitamin B12 (MW 1357 Da) but much higher than bromophenol blue (670 Da) or malachite green (657 Da). Although these estimations were only approximate, they suggested that the brown, red and yellow precursor pigments had molecular sizes of about 1300 Da, and this was con¢rmed by FAB mass spectrometry data (see below) which gave the highest detectible m/z values in the range of 1200^1400. A second experiment (data not shown) in which the brown, red and yellow pigments were isolated and chromatographed as before, gave similar results, which indicated the acid-stable homogeneity of the pigments. FAB mass spectra of the yellow, red or brown melanin precursor pigments produced at least eight major signature ions with m/z values of 290, 308, 391, 409, 628, 767, 808 and 890. These data indicated that the fragmentation pathway of the coloured polymers produced common molecular ion fragments for all three pigments; and many other major m/z

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ions common to pairs of pigments, or else speci¢c to single pigments (not shown). The highest common fragment that could be resolved was 890. No FAB fragments were detected beyond an m/z value of 1330. This suggested that the average molecular sizes of these pigments might not be as large as expected for melanin polymers ( s 2000 Da), which would tend to con¢rm their precursor designation. The ultraviolet-visible absorption spectra of butanol soluble yellow, red and brown pigments from A. media UTSA199 were quite varied. Unlike the featureless spectrum of crude melanin (Fig. 3A), all had absorption maxima between 250 to 300 nm (Fig. 3B; spectra are labelled `y', `r' and `b' for yellow, red and brown pigments, respectively). The yellow pigment had an absorption maximum at 290 nm (in aqueous solution at pH 2), red at 254 nm (pH 4) and brown at 275 nm (pH 6). When the pH of the red (Fig. 3C) or yellow (Fig. 3D) solutions was adjusted by increments into the high alkali range, the spectra began to resemble the melanin spectral plot more closely. Whereas the alkaline red pigment changed to the typically dark brown colour of melanin, the alkaline yellow pigment changed only to a dark amber colour. The brown precursor spectrum converted very rapidly to one resembling melanin (not shown).

4. Discussion In this study, we have isolated and characterised the di¡usible dark brown pigment produced by A. media, showing it to be a true melanin by a number of physical and chemical tests. It is likely to be a DOPA-derived pheomelanin, similar to that isolated from other bacteria such as the closely related Aeromonas salmonicida and Aeromonas hydrophila [5] and to the pigment from the unrelated Alteromonas nigrifaciens [10], Proteus mirabilis [8], Azospirillum brasilense [6], Streptomyces castaneoglobisporus [9] and Bacillus thuringiensis [7]. We have also reported ^ for the ¢rst time ^ the isolation and partial characterisation of butanolsoluble yellow, red and brown melanin precursors from A. media. These were ¢rst noticed in ascorbate agar but could also be detected during the late stationary phase of an aerated broth culture of A. media UTSA199 (Fig. 1). Polymer production in the sta-

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tionary phase broth culture has been observed previously for Shewanella colwelliana [12], for which HGA ^ rather than DOPA ^ is the major precursor. The polymerisation of melanin is predominantly an oxidative process. The timing of pigmentation was coincident with a rise in oxygen levels and in the pH of the S. colwelliana culture medium [12]. In the present study, a rise in pH from 7.4 to 8.8 was also noted as the red pigment converted to brown melanin. The precursor red pigment, with a molecular mass of at least 1000 Da and a Vmax of 254 nm, is not red dopachrome which has a lower molecular mass (194 Da) and higher Vmax (475). Nor are the yellow and red precursor pigments coloured trichochromes which, like dopachrome, have lower masses and higher V maxima. Also, trichochromes are only minor components of pheomelanins and are insoluble in butanol or neutral water [4]. The isolation and characterisation of the ascorbic acid agar-derived coloured pigments was repeated in order to test the reproducibility of the data. A second batch of yellow, red and brown butanol soluble pigments gave identical results to the ¢rst batch, as determined by gel ¢ltration, mass spectrometry and absorption spectrophotometry. The reddish brown colour of the crude melanin produced from A. media suggests ^ circumstantially at least ^ that it is more likely to be an alkali-soluble pheomelanin than a less soluble brown-black eumelanin. However, this tentative identi¢cation must await a careful analysis of degradation products, as outlined elsewhere [4,21]. Finally, the stability of the yellow, red and brown precursors under various conditions should allow for a more detailed evaluation of their structures (in progress in this laboratory), and may lead ultimately to a clearer understanding of the complex chemistry of melanins. An important follow-up to this study would be to utilise the ascorbate-agar method for the potential isolation of coloured precursor pigments from eumelanin-producing microorganisms.

Acknowledgments This work was supported by a University of Technology Sydney Internal Research Grant.

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