Review

Melanins form a diverse group of pigments synthesized in living organisms in the course of hydroxylation and polymerization of organic compounds. Melanin production is observed in all large taxa from both Pro- and Eukaryota. The basic functions of melanins are still a matter of controversy and speculation, even though their adaptative importance has been proved. Melanogenesis has probably evolved parallel in various groups of free living organisms to provide protection from environmental stress conditions, but in pathogenic microorganisms it correlates with an increased virulence. The genes responsible for melanization are collected in some cases within operons which find a versatile application in genetic engineering. This review summarizes current views on melanogenesis in Pro- and Eukaryotic microorganisms in terms of their biotechnological and biomedical importance.


InTroducTIon
Production of melanin is one of the most universal, but at the same time enigmatic adaptations of living organisms to the variable conditions of the Earth.The presence of various kinds of melanins in representatives of almost every large taxon suggests an evolutionary importance of melanogenesis.Up to now, no agreement has been reached concerning the primary or basic function of this process and its product, or even concerning its character as a primary or side effect.Just the opposite -it is the matter of a permanent discussion and argument (Wood et al., 1999).Numerous reviews are available on the biology, genetics, and medical implications of melanogenesis in higher animals and in human (Zecca et al., 2001;Halaban, 2002;Slominski et al., 2005).Much less is known about melanogenic pathways in microorganisms.Practical implications of these phe-nomena prompted us to focus on this topic and to present at least the most important examples.This may shed new light on the evolution of melanogenesis in higher organisms as well.
In general, pheomelanins contain sulphur, whereas most types of allomelanins do not contain nitrogen.The second important difference concerns configuration of quinone or quinonimine residues in melanins: orto-configuration is present in eu-and pheomelanins, and para-(polymers of γ-glutaminyl-4-hydroxybenzene) or meta-(polymers of DHN) in allomelanins.

EnzyMEs of MIcrobIAl MElAnogEnEsIs In rElATIon To ThEIr AnIMAl counTErPArTs
Tyrosinases (EC 1.14.18.1), which catalyze oxidation of monophenols to o-dihydroxyphenols and further to o-quinones, have a putative common ancestor with oxygen-binding and oxygen-transporting proteins (Dursewitz & Terwilliger, 1997;Decker & Terwilliger, 2000).They appeared on the Earth in consequence of the change in the chemical character of the atmosphere from reducing to oxidizing one, which was a result of photosynthesis.They originate from primitive metalloproteins, which bind dioxygen through the prosthetic group that contains a coordinated transient metal cation.
Melanin synthesis in microorganisms mester & Scheller, 1996).Only the latter one is conserved in all taxa.The CuA domain from arthropod hemocyanin differs greatly from the molluscan counterpart, which is similar to the CuA domain present in various tyrosinases (including Prokaryotic ones).This is a possible consequence of evolutionary parallellism (Lieb et al., 2001).Arthropod hemocyanins form a monophyletic group with prophenoloxidases.These two families of proteins are closely related, whereas the homology to molluscan hemocyanins and tyrosinases from other animal taxa, which form a sister group, remains distant (Fujimoto et al., 1995).It is accepted that the structure of the CuB domain is ancient and was established very early in evolution.It is particularly characteristic for tyrosinases, which are present in both Pro-and Eukaryota: a homology between Streptomyces sp. and mouse tyrosinases can still be noticed (Lerch, 1988).Molluscan hemocyanins and tyrosinases most probably originate from a single polypeptide forming two domains -CuA and CuB, while arthropod hemocyanins are the product of a duplication and subsequent fusion of CuB-encoding genes (Kawabata et al., 1995;Immesberger & Burmester, 2004).The unique common feature of molluscan hemocyanins and fungal tyrosinases is the thioether bridge between cysteine and histidine residues conservatively located in the CuA domain (Lerch, 1983;1988;van Gelder et al., 1997).The similarity of molluscan hemocyanins and tyrosinases is also manifested by their ability to oxidize o-diphenols to o-quinones under physiological conditions.Conversely, with arthropod hemocyanins it can be achieved only after a limited proteolysis or SDS denaturation (Decker & Rimke, 1998;Salvato et al., 1998;Decker et al., 2001).
laccases (oxidoreductases p-diphenol : dioxygen, EC 1.10.3.2) are also metalloproteins containing one to four copper atoms in their active site (Valderrama et al., 2003).Not related to tyrosinases, they belong to the family of blue copper-containing oxidases, together with ascorbate oxidase and ceruloplasmin.They form a strongly diversified group of low homology, and very little is known about their evolution -much less than in the case of tyrosinases.Both types of these melanogenic enzymes can be found in Eukaryota and in Prokaryota, which confirms their ancient evolutionary origin (Messerschmidt & Huber, 1990;Valderrama et al., 2003).
Polyketide synthases (PKS) produce DHN melanins, and belong to an old family of multidomain proteins related to the animal fatty acid synthases (Kroken et al., 2003).The PKS family is diversified and plentiful.Numerous microorganisms employ these enzymes to produce pigments, antibiotics, toxins and other products of intermediate metabolism (Hutchinson, 2003;Snyder et al., 2003).There are only few DHN-melanin-producing PKS enzymes, which belong to the PKS-type-I group producing aromatic, not reduced polyketides (Kroken et al., 2003).
Some species of bacteria produce and secrete pyomelanin, a product of homogentisic acid polymerization.One of the main enzymes engaged in pyomelanin production is p-hydroxyphenylpyruvate oxidase (HPPD, also known as p-hydroxyphenylpyruvate hydroxylase -HPPH; EC 1.13.11.27), which catalyses homogentisinic acid (alkapton) formation (Kotob et al., 1995).This enzyme, which belongs to the phenylalanine and tyrosine degradation pathway, is ubiquitous among living organisms.Deregulation of this metabolic pathway may lead to the retention of potentially toxic alkapton in the cells and tissues (Menon et al., 1991;Phornphutkul et al., 2002).
Another melanogenic enzyme, 4-hydroxyphenylacetic acid hydroxylase (HPA,EC 1.14.13.3), belongs to a separate family of hydroxylases (Gibello et al., 1997).Besides its main substrate, 4-phenylacetic acid it catalyzes hydroxylation of other aromatic compounds, which leads to the formation of dibenzoquinone and other o-quinone derivatives, which then polymerize spontaneously to allomelanin-like polymers (Gibello et al., 1995).Tyrosine is also a substrate of HPA, but (unlike tyrosinase) this enzyme does not contain copper, which does not increase its enzymatic activity.

MElAnIns As ElEcTron AccEPTors In dIssIMIlATIng bAcTErIA
In the process of anaerobic respiration, reducing, dissimilating bacteria use a wide spectrum of electron acceptors replacing dioxygen in the last step of the respiratory chain.Among the substances utilized as alternative electron acceptors, there are mainly hydrated ferrous oxide, nitrates, sulphates, as well as organic compounds, in particular the ones containing quinone groups, e.g.related to melanin humic substances (Coates et al., 2002).
Melanin, similarily to humic substances, is a polymer of various groups able to donate or to accept an electron.Therefore it can act as a final acceptor or a shuttle in the electron exchange with insoluble compounds of iron (Menter & Willis, 1997).The facultatively anaerobic bacterium Shewanella algae produces pyomelanin and reduces it simultaneously with the oxidation of gaseous hydrogen (Turick et al., 2002).Having accepted numerous electrons, such "reduced' melanin serves the bacteria as a reductor of insoluble ferric (III) oxides to the ferrous (II) state (Fig. 4).As S. algae is unable to carry out fermentation, its survival in the conditions of variable oxygen concentration strongly depends on the presence of 432 2006 P. M. Plonka and M. Grabacka appropriate electron acceptors (Turick et al., 2002).In the mineralized marine deposits the availability of such soluble compounds is limited, therefore production of melanin is an important evolutionary adaptation.Moreover, like other organisms, S. algae produces melanin also for the protection from ultraviolet irradiation.
A similar, still poorly understood phenomenon concerns another marine bacterium -Marinomonas mediterranea, which has been discovered to contain two melanin-generating enzymes, namely tyrosinase and laccase with a wide spectrum of activity (Sanchez-Amat et al., 2001;Lopez-Serrano et al., 2002).As it was shown by mutation analysis, only tyrosinase is indispensable for the production of the dark pigment and laccase has other physiological functions (Sanchez-Amat et al., 2001;Lopez-Serrano et al., 2002).The co-expression of both enzymes is unique to Prokaryota.The natural substrate for M. mediterranea melanogenesis in the oligotrophic sea water remains unknown.The existence of microenvironments rich in organic compounds cannot be excluded.An example of such a niche are surfaces biofilms, where concentration of amino acids may reach 10-30 nM (Lucas-Elio et al., 2002).Some aromatic compounds and polyphenols can be oxidized both by tyrosinase and by laccase.Many species of Fungi employ laccase to decompose lignins and other organic compounds (Leonowicz et al., 2001).Therefore another function of laccase, evolutionarily beneficial to marine bacteria, would be decomposition of deposits of poorly accessible organic matter.

MElAnIn In ATMosPhErIc nITrogEn fIxATIon by bAcTErIA
The soil aerobic bacterium Azotobacter chroococcum contains an active polyphenol oxidase and can produce melanin from catechol (Shivprasad & Page, 1989).This microorganism produces particu-larly large amounts of melanin when cultured under aerobic conditions.This is easy to explain, as oxygen is one of the substrates for polyphenol oxidase, but it turns out that this process is intensified in the absence of a nitrogen source in the medium (Shivprasad & Page, 1989).Although the intensity of melanogenesis does not seem to be directly correlated with the activity of nitrogenase (the key enzyme of atmospheric nitrogen fixation), it is possible that Azotobacter employs melanogenesis to enhance utilization of oxygen and to maintain reducing conditions necessary for binding atmospheric nitrogen.The presence of iron and copper ions in the medium significantly increases the rate of Azotobacter melanization.This is not only because polyphenol oxidase requires the presence of Cu ions in the active site.Enhanced melanogenesis also confers a protective role against hydroxyl radicals (OH .) produced in the Fenton reaction which takes place in the presence of Cu and Fe ions (Shivprasad & Page, 1989;Sutton & Winterbourn, 1989).This protection involves providing catechol and binding metal ions in the structure of melanin.Catechol, an intermediate in the melanogenic pathway, is a potential target of the oxidizing hydroxyl radical, and a potential "sweeper" of this extraordinally active form of oxygen (Nasr et al., 2005).
In the symbiotic Rhizobium bacteria living in the root nodules of Papillionaceae plants, genes encoding melanogenic enzymes, including tyrosinase, are located in plasmids required for the development of the symbiosis (Hawkins & Johnston, 1988).Surprisingly, these genes do not seem to be obligatory for the development of the nodules or for the nitrogen fixation itself.The function of these genes in Rhizobium is still unclear, but one of postulated explanations suggests that tyrosinase may play a role in detoxication by oxidation of phenolic compounds deposited in ageing root nodules (Hynes et al., 1988).

MElAnIn As A fAcTor of vIrulEncE In PAThogEnIc bAcTErIA
The ability of pathogenic bacteria to produce melanin seems to originate from the evolutionary achievements of free-living bacteria.In some genera containing both free-living and parasitic strains (e.g.Vibrio sp.), there are both pyomelanogenetic and eu/pheomelanogenetic pathways, sometimes simultaneously active in one organism.Free-living strains of Vibrio cholerae are usually amelanotic or they produce pyomelanin (Kotob et al., 1995).Meanwhile, under stress (hyperthermia, hyperosmotic medium, starvation) they induce synthesis of eumelanin, and a pathogenic mutant HTX-3 -ph- Melanin synthesis in microorganisms eomelanin (Ivins & Holmes, 1981;1980;Coyne & Al-Harthi, 1992).
Production of pyomelanin has been reported in many species of bacteria, like Pseudomonas aeruginosa, Hypomonas sp., Shewanelia colwelliana, some of which consist of both free-living (marine) and pathogenic strains (Shivprasad & Page, 1989).Legionella pneumophila may serve as another example of the latter group.It causes the so called Legionnaire's disease (LD) and Pontiac fever (Wintermeyer et al., 1994;Rathore & Alvarez, 2006).One of the factors responsible for the pathogenicity of this microorganism is legiolysin (Lly), a protein responsible for the fluorescent properties, pigment production and hemolytic ability of the pathogen (Wintermeyer et al., 1991).It reveals over 80% similarity with HPPD (the product of MelA gene -see later), crucial for melanin production in Schewanella colwelliana and in Pseudomonas sp.(Fuqua et al., 1991;Fuqua & Weiner, 1993;Wintermeyer et al., 1994;Steinert et al., 2001).This key enzyme for pyomelanogenesis confers hemolytic activity as well (Croxatto et al., 2002).The expression of HPPD is regulated in a similar way to other genes responsible for the virulence factors, such as production of metalloproteinases and extracellular lipopolysaccharides, used to create biofilms and necessary for adhesion to biological surfaces like fish skin (Croxatto et al., 2002).
The pathogenic bacterium Burkholderia cepacia serves as an example of how melanin production increases virulence.This microorganism causes dangerous lung infections, which often develop to sepsis, mainly due to the presence of lipopolysaccharide (LPS) which strongly enhances production and release of proinflammatory cytokines (Zughaier et al., 1999).Although LPS does not trigger an oxygen burst directly, it stimulates the immunological system to an accelerated oxidative response to other stimuli.Melanin isolated from B. cepacia reveals a dose-dependent ability to sweep O 2 -. produced by leukocytes during oxygen burst.However, melanin does not influence the release or kinetics of the production of reactive oxygen species (ROS) (Zughaier et al., 1999).The ability to remove superoxide anion allows B. cepacia to survive phagocytosis, so the host phagocytes are not able to eliminate the pathogen.They remain in the state of a permanent stimulation by the bacterial LPS, which causes a chronic inflammation.Similarly to Legionella pneumoniae, B. cepacia is able not only to survive in the phagocytes (alveolar macrophages), but also to proliferate intracellularly, which leads to cell destruction and to the infection of secondary macrophages (Saini et al., 1999;Abu-Zant et al., 2005).
In Proteus mirabilis, the second (after E. coli) important cause of community-acquired infections of the urinary tract, tyrosinase was identified as the enzyme responsible for melanization.As it has been shown in this species, melanin decreases the level of ROS, which probably makes the pathogen more resistant to the oxygen burst connected with the immunological response of the host (Agodi et al., 1996).
Klebsiella pneumoniae, responsible for a particularly dangerous form of pneumonia, produces melanin from 4-hydroxyphenylacetic acid using HPA, which is an unusual pathway of melanogenesis (Gibello et al., 1995;1997).However, the importance of HPA and melanin for the virulence of this pathogen remains to be determined.
In a separate study, the sequence of fungal laccase was used to search for homologous genes in Prokaryota (Alexandre & Zhulin, 2000).Similar sequences were found in 14 bacterial species, including such important human pathogens as E. coli, Bordetella pertusis, P. aeruginosa, Campylobacter jejuni, Yersinia pestis, or mycobacteria, including Mycobacterium leprae.In all these pathogens the production of melanin or laccase activity should be suspected as a potential mechanism of virulence.M. leprae has an ability, unique among mycobacteria, to oxidize diphenols to o-quinones, and in consequence oxidation of l-DOPA has become a convenient diagnostic feature for M. leprae (Prabhakaran et al., 1977;Prabhakaran & Harris, 1985).

MolEculAr gEnETIcs of bAcTErIAl MElAnogEnEsIs
The genetic background of melanogenesis in prokaryotic microorganisms has been elucidated only in several cases, even though a large number of examples of melanin production have been described.
The actual role of MelC1 has been revealed only recently.First of all, it has been shown that this protein forms a heterodimer with tyrosinase, acting as its trans-activator (Chen et al., 1993).Next, it was demonstrated that this interaction is necessary to incorporate two copper ions crucial for the enzymatic activity of the enzyme.Incubation of apotyrosinase 2006 P. M. Plonka and M. Grabacka with copper ions did not lead to the full restoration of enzymatic activity, if it was not supplemented with MelC1.This protein seems, therefore, to possess a chaperone-like function (Chen et al., 1992).Even more interestingly, it is not only responsible for the proper folding and incorporation (proper folding by incorporation?) of the metal ions into the active site, but also enables the secretion of native tyrosinase by S. antibioticus (Leu et al., 1992).Tyrosinase is secreted in complex with MelC1.Recently, a deeper biological sense of this phenomenon has been discovered.The complex is secreted using one of the three known bacterial mechanisms of protein secretion, namelyvia interaction with Tat (Twin-arginine translocation) proteins (Schaerlaekens et al., 2001).The mechanism of their action is related structurally and functionally to the mechanism generating the pH gradient in tylakoids of chloroplasts.It turned out that only MelC1 contains the signal peptide responsible for translocation of the dimer.This is the reason for which tyrosinase is secreted only when bound to the MelC1 protein, as a ready, natively-folded protein.This mechanism (the so-called "hitchhiker" mechanism) based on the interactions with Tat proteins is also responsible for the secretion of the product of a two-gene operon of hydrogenase 2 in E. coli.However, the primary biological meaning of the presence of tyrosinase in the secretomes of some Streptomyces species remains unknown.
The genome of Marinomonas mediterranea also contains a two-cistron operon, ppoB, responsible for melanogenesis (Lopez-Serrano et al., 2004).The ppoB1 gene encodes apotyrosinase and ppoB2 a chaperone incorporating copper ions to the active site of the enzyme.While there are known ortologues of ppoB1 in Eucaryota and some bacteria, including Streptomyces sp., the ppoB2 product does not reveal any significant homology either with MelC1 of Streptomyces antibioticus, or with other proteins checked in this context.Interestingly, PpoB2 contains six transmembrane helices and is not secreted, unlike its functional counterpart in streptomycetes (Lopez-Serrano et al., 2004).
M. mediterranea contains also another melanogenetic enzyme -laccase (ppoA gene) (Sanchez-Amat et al., 2001).Regulation of ppoA and ppoB expression proves the evolutionary importance and strongly adaptative character of melanin production for this species.It is regulated via a two-component system of signal transduction, which consists of a membrane-anchored sensor histidinyl kinase and its intracellular response regulator (Lucas-Elio et al., 2002).After receiving an environmental stimulus, the extracellular, N-terminal domain of the kinase undergoes a conformational transformation leading to the autophosphorylation of the histidine residues in the C-terminal, intracellular domain of the pro-tein.The phosphate group is then transferred onto an aspartyl residue of the response-regulating intracellular protein, which finally leads to the cellular response.The M. mediterranea sensor kinase (PpoS -polyphenol oxidase sensor kinase) has a structure similar to the BvgS kinase regulating the virulence of Bordetella pertusis (Lucas-Elio et al., 2002).It has been shown that a mutation of ppoS down-regulates the expression of tyrosinase and laccase in bacteria under stress conditions, including, e.g., the stationary growth phase when the resources of the medium begin to dwindle.
In free-living Shewanella colwelliana with Mel+ phenotype, a monocistronic, 1.3 kb melA operon has been identified.It contains an ORF with the transcription initiation site 115 nucleotides upstream of the putative translation initiation site.The melAencoded protein (39.5 kDa, 346 amino acids) was identified as HPPD found also in Vibrio cholerae, Hypomonas sp., or Pseudomonas sp.(Fuqua et al., 1991).
The Klebsiella pneumoniae 4-phenyloacetic acid hydroxylase activity is determined by two proteins encoded by separate cistrons -hpaA and hpaH.Their expression in E. coli resulted in production of a brown pigment in the medium.hpaA encodes a flavoprotein (58 781 Da) with the enzymatic activity, while the hpaH product (18 680 Da) is a "helper" protein, necessary to achieve a high catalytic efficiency of HPA (Gibello et al., 1997).

bAcTErIAl MElAnogEnEsIs In bIoTEchnology And gEnETIc EngInEErIng
The enzymatic activity of tyrosinase is manifested in bacteria by extracellular melanin production, and by easily noticeable darkening of the medium.Therefore it is easy to quantify the expression of a tyrosinase gene or the whole melC operon, e.g. by a spectrophotometric assay of the medium.This phenomenon has been widely employed in genetic ingeneering, and the melC operon or its constituents (the structural part devoid of the promoter or melC2 alone) are parts of various gene constructs serving as reporter genes (Paget et al., 1994).A successful transfer of melC genes of S. antibioticus has been reported for various expression systems, including other streptomycetes (e.g. S. lividans) and E. coli (Katz et al., 1983;della Cioppa et al., 1990;Tseng et al., 1990).A remarkable example of the utilization of this operon in molecular investigations concerns the studies on transcription factors of the highly pathogenic Streptococcus thermophilus.Particularly promising are the results of a study on the structural genes of the melC operon from Streptomyces antibioticus under the control of streptococcal promoters expressed in E. coli (Solaiman & Somkuti, 1995;1997)

. Even
Melanin synthesis in microorganisms more spectacular is the successful transfection of amelanotic murine skin cells with melC, leading to the restoration of the pigmented phenotype of the murine cells (Zhao et al., 2000).Such experimental approach may in future lead to new methods of investigation of animal melanogenesis, but also to new therapeutic strategies against skin and hair pigmentation disorders.
An opposite approach, where the expected effect was the lack of melanin synthesis, was used in the construction of combinatorial libraries of cyclic peptides and proteins (Scott et al., 1999).Peptides encode five times more information than nucleic acids of the same number of mers.The maintenance of a satisfactory level of stable products which easily undergo catabolic decomposition is highly problematic.One of the solutions is cyclization of peptides and oligopeptides using inteins -fragments of polypeptides or proteins appearing as intermediate conformeres and revealing their own catalytic activity.Such an activity is transient, and is usually connected with a specific excision and/or ligation of other fragments of oligopeptide chain during posttranslational modification.The authors used an inhibitor of tyrosinase -pseudostellarin F, a cyclic octapeptide, as a product of intein activity.The proof for the presence and activity of the whole gene construct, i.e. for the actual appearance of the intein and its expected ligase activity is the lack of tyrosinase activity, manifested by the lack of medium darkening.Such a system may be used to stabilize other oligopeptides, besides pseudostellarin F. It should be noticed that the intein system originated from a cyanophyta -Synechocystis sp.(PCC6803 Ssp), the tyrosinase and melC1 gene from Streptomyces antibioticus, and the library was expressed in E. coli.
Besides pseudostellarin F, bacteria are also a source of other specific, efficient and often nontoxic inhibitors of tyrosinase, or of melanogenesis in general.Such inhibitors have been isolated e.g. from the culture medium of Streptomyces sp. or S. clavifer (Ishihara et al., 1991;Komiyama et al., 1993;Lee et al., 1997).Finally -it must be mentioned that various authors suggest the possibility to employ bacteria directly to produce various kinds of melanins for further use in industry (della Cioppa et al., 1990).

ProTozoA
This group is probably the least explored one in respect to melanogenesis and melanin function among eukaryotic organisms.Melanin is usually noticed in the context of the response of invertebrates to microbial parasites, e.g in the case of mosquitos (Anopheles) being invaded by Plasmodium (Chun et al., 2000;Cui et al., 2000;Kumar et al., 2003;Volz et al., 2006).The host usually reacts with encapsulation of the parasite and melanization of the surrounding tissues.This basic type of invertebral defense depends in mosquitos on melanization-regulating genetic module or network encompassing at least 14 genes (Volz et al., 2006).However, melanin is here produced by the host, not by the protozoal parasite (Nappi & Ottaviani, 2000;Kumar et al., 2003).
Many protozoal species are able to synthesize melanin precursors which may spontaneously polymerize to form the pigment.An interesting example is the "pig" (which stands for "pigmented") mutant of Tetrahymena thermophila, able to secrete huge amounts of allomelanin precursors to the culture medium (Kaney & Knox, 1980;Martin Gonzalez et al., 1997).These compounds, mostly products of catecholamine oxidation, spontaneously polymerize to melanin, which is likely to be a result of hyperactivity of aromatic amino-acids decarboxylase (l-DOPA decarboxylase, EC 4.1.1.26).Activity of this enzyme, as in the case of bacteria, can be easily observed and quantified in the culture.Therefore, the "pig" mutant has been employed to test the effectiveness of some eukaryotic antibiotics and mycotoxins (Martin Gonzalez et al., 1997).
The marine unicellular algae Dinoflagellata have been shown to express their own PKS-type-I enzymes encoded by dinoflagellate-resident genes (Snyder et al., 2005) responsible for the production of various neurotoxins and potential anti-cancer agents (Rein & Borrone, 1999;Snyder et al., 2003), but also substances toxic for fish (Berry et al., 2002).Although melanin synthesis in these organisms has not been reported yet, this phenomenon seems to be worth noticing for several reasons -it concerns a potentially melanogenic pathway, and serves as another example of the existence of such pathways in Protista, it documents a quite unusual localization and employment of PKS, and finally, it has clear practical (medical and biotechnological) aspects.fungI Melanin biosynthesis is a common feature in the kingdom of Fungi, but for the purpose of this paper only examples relevant from the biotechnological and biomedical point of view have been chosen.

cEllulAr locAlIzATIon And MEchAnIsMs of MElAnIn synThEsIs In PArAsITIc fungI
The ascomycete fungus Cryptococcus neoformans is a parasite of the central nervous system, responsible for 5-10% lethal infections in patients with immunodeficiency syndromes (Curie & Casadevall, 436 2006 P. M. Plonka and M. Grabacka 1994;Mitchell & Perfect, 1995).C. neoformans expresses laccase which produces melanin precursors from tyrosine, DOPA, and catecholamines, abundant in the host organism (Wang et al., 1995;1996).However, the nature of the pigment produced in vivo under such conditions (eumelanin or other products of catecholamine oxidation) remains controversial (Liu et al., 1999).C. neoformans laccase is localized mainly in the outer layer of the cell wall, and this is also the place of melanin deposition.Such a localization is beneficial for the fungus for two reasons (Wang et al., 1995;1996).First, it prevents the potentially toxic side-products and intermediates of melanogenesis from penetration to the cytoplasm.In higher animals this is achieved by localization of the whole process of melanogenesis in separate organella -melanosomes, within the pigment-generating cells, melanocytes (Nordlund et al., 1998).Secondly -the cellular wall deposition of melanin makes it a convenient barrier and a defence against toxins (e.g.drugs, antibiotics) and other harmful factors which melanin may neutralize.And indeed, melanotic strains of C. neoformans are much more resistant to fungicides and antibiotics (amphotericin B, fluconazol, caspofungin) than non-pigmented ones (Ikeda et al., 2003).Caspofungin and amfotericin B are bound in the outer layer of the cell wall of the fungus, so only a limited amount is able to penetrate to the cytoplasm (Ikeda et al., 2003).
Melanized cell wall of C. neoformans is unusually durable.It maintains its paramagnetic properties, structure, shape and size of the parasitic cell even after a drastic treatment destructive to amelanotic cells (boiling in 6 M HCl, treatment with 4 M guanidine isothiocyanate), whereafter fungal cells can still be observed as the so called "melanin ghosts" (Wang et al., 1996).It raises the intriguing question about the existence and the mechanisms of fungal melanin degradation in vivo.Such a phenomenon seems to occur for endogenous melanins (Plonka et al., 2005).
Melanin production correlates with enhanced virulence of other facultatively parasitic Ascomycetes.Paracoccidioides brasiliensis, Sporothrix schenckii and Exophiala (Wangiella) dermatitidis can serve as examples (Schnitzler et al., 1999;Romero-Martinez et al., 2000;Gomez et al., 2001).These fungi reside in soil where they grow in the form of free-living mycelium.After rising the temperature to 37°C or in vivo in the host organism they form conidia covered by a melanized cell wall.This phase is called the "yeast" form, in contrast to the free-living mycelium, and the whole phenomenon is described as thermodimorphism.Due to the presence of pigment such fungi are often called "black yeast".
Paracoccidioides brasiliensis causes dangerous, sometimes fatal systemic paracoccidiomycoses, mainly in the South America (Gomez et al., 2001).It contains laccase localized probably in the cell wall.In the presence of DOPA or other catechol substrates in the environment it synthesizes DOPA-melanin, however, in the absence of phenolic compounds it is able to produce DHN-melanin from tetrahydroxynaphthalene using the pentaketide pathway.In Sporothrix schenckii this pathway leads to melanization of conidia with the cell wall covered from the outside with melanin-like granules (Romero-Martinez et al., 2000).This fungus is often found on rose prickles and causes sporothrichosis called "Rose-picker's disease".In Europe it is occasionally diagnosed in flower shop employees selling roses planted in tropical countries.The presence of melanin in the cell wall protects the conidia against digestion by proteases and hydrolases secreted by competitive microorganisms or against bacterio-and fungicidal proteins of animal origin, such as defensins, magainins or protegrins (Doering et al., 1999;Rosas & Casadevall, 2001).The protective role of melanin is mainly based on the inactivation of these proteins on the cell surface or on the prevention of their contact with intracellular substrates.
The DHN pathway of melanin biosynthesis is very common in the Fungi kingdom, utilized, among others, by many fungal parasites of higher plants (Jacobson, 2000;Langfelder et al., 2003;Thomma, 2003).It should be recalled that members of the PKS family, the key enzyme in this pathway, have been identified not only in fungi, but also in bacteria, and even in protozoa (Kroken et al., 2003;Snyder et al., 2003).

bIochEMIcAl MEchAnIsMs of vIrulEncE of MElAnoTIc PArAsITIc fungI
The ability to produce melanin as an important factor of virulence is well documented and confirmed in vivo for C. neoformans.Mice infected with the parasite devoid of laccase and therefore unpigmented, survived significantly longer than the ones infected with the melanized fungus (Williamson, 1997;Barluzzi et al., 2000).The virulence could be easily restored by transfection with the CNlac1 plasmid containing the laccase gene (Williamson, 1997).Melanin was found to protect the parasite from ROS and RNS (reactive nitrogen species) produced during the oxygen burst by activated host macrophages (Wang & Casadevall, 1994b;Jacobson et al., 1995;Wang et al., 1995).
Due to its electrochemical properties, melanin may act both as an electron donor and electron acceptor (McGinness et al., 1974;Menter & Willis, 1997).Therefore, it is not only an effective free radical "sweeper", but also an ion-exchange resin able Melanin synthesis in microorganisms to bind iron ions (Pilas et al., 1988).The limited iron ion pool decreases the possibility of Fenton reaction and the formation of extremely reactive hydroxyl radical from superoxide radical anion and hydrogen peroxide (Pilas et al., 1988).It has also been shown that melanin imitates action of superoxide dismutase (SOD), thus additionally limiting oxidative stress (Korytowski et al., 1986).More importantly, it has recently been shown that through oxidation of DHI-CA and DHI residues to respective semiquinones, DOPA-melanin scavenges also RNS, mainly the extremely toxic radical of nitrogen dioxide ( .NO 2 ) produced by disproportionation of nitrite (NO 2 -) in low pH (Reszka et al., 1998).This mechanism is probably responsible for the survival of melanotic C. neoformans strains in acidic solutions of NaNO 2 (Wang & Casadevall, 1994b).
Melanotic Sporothrix schenckii strains are more resistant to UV, NO and H 2 O 2 than amelanotic ones (Romero-Martinez et al., 2000).Non-pigmented cells are also more prone to killing by murine macrophages and human monocytes because of their high vulnerability to oxygen burst and activation of nitric oxide synthase (Romero-Martinez et al., 2000).
Exophiala dermatitidis, another representative of "black yeast" is responsible for pheohypomycoses (e.g. the ones related to diabetes) (Schnitzler et al., 1999).It also preferentially infects the nervous system of immunodeficient patients and exploits the protective function of DHN-melanin as well as carotenoids -torulene and torulorodyne -against strong oxidants (hypochlorite, permanganate) and free radicals.Carotenoids reveal mainly a screening activity while melanin can actively neutralize the oxidants (Schnitzler et al., 1999).Melanized strains of E. dermatitidis are more resistant to killing by neutrophils than the amelanotic strains.Melanin protects the cells against ROS and lysis in phagosomes, although it does not affect the efficiency of phagocytosis, nor the kinetics of oxygen burst (Schnitzler et al., 1999).

AcTIon of MElAnIn on ThE IMMunologIcAl sysTEM of ThE hosT
The present examples illustrate an extremely intriguing aspect of the general biological implications of melanin production and activity.To get the whole picture, it should be supplemented with the fact that melanization is one of the effector mechanisms of invertebrate immunity (Nappi & Ottaviani, 2000).It can be argued whether melanization of the infected tissue is just a side-effect or the protection against the on-going inflammatory processes and the corresponding production of various active oxygen or nitrogen species, or an active defence against the pathogen, e.g.leading to the isolation of the invader from the rest of the organism or to inactivation (adsorption) of harmful and toxic products secreted by the parasite (Marmaras et al., 1996).Whatever is said, melanization is a sword of two edges, which in some cases may limit the parasite invasiveness, and in some others -the immunological mechanisms of the defence.
Melanins turned out to be immunogenic.Mice immunized with melanin extracts from the cells of C. neoformans produced specific anti-melanin antibodies (Nosanchuk et al., 1998;Rosas et al., 2001).The authors claim that melanins belong to the T-independent antigens, which can induce an immunological response by binding directly to the immunoglobulin-like receptors on the surface of Blymphocytes (Nosanchuk et al., 1998).
Various kinds of melanins turned out to reveal immunomodulatory activity through the inhibition of pro-inflammatory cytokine production by T-lymphocytes and monocytes, as well as fibroblasts and endothelial cells (Huffnagle et al., 1995;Arramidis et al., 1998;Barluzzi et al., 2000;Mohagheghpour et al., 2000).
The presence of melanin in the cell wall is correlated with less efficient phagocytosis, both in the case of C. neoformans, and S. schenckii (Nosanchuk & Casadevall, 1997;Romero-Martinez et al., 2000).A likely explanation is that phagocytosis is impaired by the decrease of the negative electric charge of the cell wall, which is caused by melanin deposition (Wang et al., 1995;Nosanchuk & Casadevall, 1997).
Finally, melanins of the cryptococcal cell wall activate supplement via the alternative way (Rosas et al., 2002).Deposition of the C3 fragments on the surface of the polysaccharide envelope of C. neoformans is obligatory for the effective phagocytosis and removal of the fungus by phagocytes.Melanization, however, does not influence the kinetics of this deposition, nor the final amount of the deposited C3 fragments (Rosas et al., 2002).

MElAnIn In ThE ProTEcTIon AgAInsT PrEdATory ProTozoA And nEMATodEs
Development of such an intricate mechanism of virulence as the one in C. neoformans, an organism which is only a facultative parasite, and which does not need an animal host to successfully complete its life cycle, is puzzling.It most probably evolved as an adaptation of a free-living organism to environmental stress conditions.Melanin production offers protection from UV light and ionizing radiation, and resistance to heat or cold and activity of inorganic antimicrobial compounds, such as silver nitrate (Wang & Casadevall, 1994a;Rosas 2006 P. M. Plonka and M. Grabacka & Casadevall, 1997;Garcia-Rivera & Casadevall, 2001;Nybakken et al., 2004).Such a phenomenon was also discovered in an arctic lichene Cetraria islandica, where fungal melanin produced in the sun highly lowers the cortical transmittance for UV-B (Nybakken et al., 2004).
One of the most probable and interesting hypotheses concerning the evolutionary transformation of melanin from such an environmental protector to a virulence factor supposes that the virulent properties appeared as a means of protection against predatory amoebae of the genus Acanthamoeba, for which such fungi as C. neoformans are a natural prey in the soil environment (Steenbergen et al., 2001).Amoebae, like macrophages, phagocyte fungal cells and digest them in their phagosomes using lysosomal enzymes.The presence of a polysaccharide envelope decreases the phagocytic effectiveness, most probably due to its negative charge (Nosanchuk & Casadevall, 1997).Cells devoid of the envelope are more prone to phagocytosis, but among them, the melanized ones survive longer after incubation with amoebae (Steenbergen et al., 2001).
The strains of C. neoformans most virulent for mice, i.e. the ones with the polysaccharide envelope, active laccase and phospholipase, are able to survive and proliferate inside phagosomes and cause death of the amoeba or macrophage which ingested the fungus (Steenbergen et al., 2001).Moreover, the same mechanisms of virulence observed during cryptococcal infections in mammals, including melanin production, are also responsible for the death of the predatory nematode Caenorhabditis elegans feeding on Cryptococcus sp.fungi (Mylonakis et al., 2002).Melanized strains of cryptococci are much more effective in infection and killing the nematodes in DOPA-containing media than the ones devoid of laccase (Mylonakis et al., 2002).

MElAnIn In ThE PEnETrATIon of ThE hosT cEll wAll by PArAsITIc fungI
Pathogenic fungal parasites of plants often develop melanized conidia or appressoria -specialized organs which faciliate adhesion to the plant surface and penetration of the hypha into the host tissues (Howard & Valent, 1996;Money, 1999).DHN is synthesized in the cytoplasm and transported to the cell wall, where it is polymerized by laccase (Thomma, 2003).The thickest melanin deposition is found in the outer layer of the wall, similarly to the situation in C. neoformans.Cochliobolus sp. and Alternaria sp.produce pigmented conidia and small, nonpigmented appressoria, whereas Colletotrichium sp. and Magnaporthe sp.-non-pigmented conidia and melanized appressoria (Takano et al., 1997).In the latter genus, albinotic mutants lose virulence and the ability to penetrate the plant cell wall -thus the ability to produce melanin appears an important adaptation to the parasitism (Howard & Valent, 1996).Takano et al., (1997) showed that albinotic mutants of Colletotrichum lagenarium, devoid of the PKS gene (Pks-), in contrast to the wild type, were not able to infect cucumber leaves and to penetrate their cellulose wall.This ability was, however, restored by transfection of the parasite with the gene of PKS from Alternaria alternata, which restored appressorium pigmentation (and in the species of origin caused pigmentation of conidia).Although such "rescued" mutants were still less effective in the infection of cucumber leaves, most probably due to the lower melanin content than in the wild type, this experiment confirmed the key role of the pigment in the parasitic properties of the fungus (Takano et al., 1997).
Appressorial cells reveal unusual turgor and mechanical resistance.Their osmotic pressure was estimated to reach 8 MPa.In Magnaporthe grisea the force which acts on the plant cell wall in the apical part of the appressorium equals 8 µN/µm 2 , and in Colletotrichum graminicola -about 17 µN/µm 2 , which is roughly equivalent to 8 tonnes on the surface of a human palm (Bechinger et al., 1999).Such a high osmotic pressure is reached thanks to the extraordinary concentration of glycerol (over 3 M) in the appressorium (de Jong et al., 1997;Latunde-Dada, 2001).To maintain such a high intracellular concentration, an unusual density of cell membranes and walls must be achieved.Howard et al. (1991) showed that melanin present in the cell wall of M. grisea decreases the pore diameter below 1 nm, whereas water-permeability remains unaffected.At the same time, the mechanical resistance of the cell wall profoundly increases due to melanization.Fungi from the genus Magnaporthe or Colletotrichum which attack rice, coffee plants, legumous plants and other valuable species, are able to penetrate even intact cuticula using their melanized appressoria (Howard & Valent, 1996;Latunde-Dada, 2001).Cochliobolus or Alternaria -fungi with non-pigmented appressoria -do not achieve high enough turgor to penetrate tissue, therefore they need a specialized enzymatic apparatus degrading the plant cell wall (Bechinger et al., 1999).These species, however, produce pigmented conidia, which improves their resistance to various dangerous factors of the environment (Deising et al., 2000).

MyxoMycETEs
These organisms, including especially the true (acellular) slime moulds, combine features of Melanin synthesis in microorganisms both protozoa and fungi (McCormick et al., 1970;Lenaers et al., 1988;Roger et al., 1996).On the one hand, as relatives of primitive fungi, they are able to produce strongly melanized spores (McCormick et al., 1970;Rakoczy & Panz, 1994;Chet & Hutterman, 1977;Loganathan & Kalyanasundaram, 1999).But on the other hand, accumulation of melanin or melanin-like pigments may also occur in the vegetative form of their life cycle, i.e. in the plasmodium, when the slime mould is similar to a multinuclear amoeba.This feature has so far been reported only in one species, Physarum nudum, which produces the plasmodial melanin when exposed to visible light (Plonka & Rakoczy, 1997).This seems to be a primordial reaction to light, which might have evolved to sun-tanning observed, e.g., for humans.Therefore, these microorganisms may in fact be closer to the animal melanogenetic phenomena than any other mentioned in the current review.

conclusIons
The ability to produce melanin is widespread among microorganisms.From the chemical point of view the only common feature of microorganismal melanins is being a product of oxidative polymerization of various phenolic substances, thus melanins form a quite heterogenous group of biopolymers.As a consequence, melanogenesis can serve as an example for evolutionary convergence − the final product can be achieved on various metabolic pathways, even in one taxon.As mutations in genes determining melanin production are rarely lethal, at least under in vitro conditions, this feature seems not to be indispensable for life, although it creates a powerful adaptative advantage.The possible scenario of evolution might include, first, extracellular autooxidation of some phenolic compounds and amino acids due to the appearance of oxygen in the atmosphere, then, to improve the environmental conditions, active secretion of the substrates of melanogenesis and of enzymes supporting this process, followed by a gradual adaptation to the intracellular control of the process, to export or storage of the product.In parallel "secondary" biological functions of melanins might have developed.
Besides mimicry, signalling, as well as protection against UV and visible light, free radicals, ex-treme temperatures, and maintaining a proper balance of metal ions, there are three important functions of melanins particularly attributed to microbial organisms: 1.The function of alternative electron acceptors or carriers, making it possible to produce energy in processes analogical to oxidative phosphorylation, but under anaerobic conditions.
2. The panenvironmental function of soil melanins and humic substances, products of enzymes contained in secretomes of various soil microorganisms (e.g.streptomycetes).The most important aspect of this function is lowering the vulnerability of the soil microecosystems to UV irradiation, but also maintaining the proper ion balance in the soil.It is hardly possible to imagine a natural ecosystem which supports the existence of higher plants and is devoid of humic substances.
3. The function of a factor of virulence in pathogenic microorganisms lowering the vulnerability to the defence mechanisms of the hosts.Important for both Pro-and Eukaryotic pathogens, it draws one's attention in quest of specific targeting of individual microbial virulence factors as an alternative antimicrobial strategy (Dubin et al., 2005).
In contrast to bacteria, whose melanogenic enzymes are usually secreted to the environment, in the case of eukaryotic microbial enzymes melanin production is usually connected with modification of the cellular wall.The molecular mechanisms of exploitation of melanin by parasitic fungi are refined and they must be a result of a long co-evolution.Consequently, these mechanisms are not good candidates for the primary phenomena from which the mechanisms of melanogenesis in higher Eukaryota evolved.As myxomycetes are able to both deposit melanin in the cell wall of the spores, and to produce melanin in the plasmodium after exposure to light, they seem to be closer to the primordial melanogenic Eukaryota.A closer examination of protozoa will surely reveal some new aspects of animal melanogenesis, and, perhaps, shed new light on the evolutionary origin of this phenomenon.

figure 4 .
figure 4. Possible function of melanin as an electron acceptor in the respiratory chain of marine bacteria, in comparison to the aerobic variant of the process.HS, humic substances.