The Mechanism Of Melanin Synthesis In Aeromonas Media WS

Melanin is a kind of negatively charged hydrophobic pigment which is formed by the oxidation and polymerization of phenolic and/or indolic compounds. Production of melanin has been described to occur in almost every taxon of living organisms, including animals, plants and microorganisms.The ability of organisms to produce melanin plays an important role in enhancement to compete and survive under certain environmental stress conditions. Since melanin has been reported to serve as antioxidant, photoprotectant (against UV and visible light), antiviral, cation exchangers and drugs carriers, it is generally accepted that this pigment holds important biological function and has the potential to be developed in multiple biotechnological applications.Most bacteria produce eumelanins or pyomelanins. Eumelanins are derived from L-3,4-dihydroxyphenylalanine (L-DOPA), and pyomelanin are produced from catabolism of tyrosine or phenylalanine via 4-hydroxyphenylpvruvate and homogentisate (HGA).Aeromonas media WS was isolated from water samples collected from East Lake, Wuhan, China, and exhibits a significantly high yield of melanin, which has been shown potential to act as a photoprotectant agent for biological pesticides and sunscreen cream. The whole genome sequence of A. media WS has been completed by our lab. Our previous studies identified the intermediate L-DOPA in the supernatant of A. media WS by HPLC and isolated a distinct gene tyrA encoding tyrosinase. However, subsequent research indicated that this gene is not a key factor responsible for melanin formation in A. media WS, since the deletion of tyrA from the bacterium does not bring any obvious change regarding the pigment production. In this study, we used a transposon mutagenesis to identify the molecular determinants of melanin production in A. media WS.Here, we used Tn5 transposon which has no patiality for a specific target DNA sequence for the mutagenesis. The antibiotic resistance cassettes of plasposons pUTKm2, pTnMod-OGm based on transposon Tn5 were not suitable for A. media WS. We constructed new plasposons, pTnCm and pTnMod-O-Cm, replacing the kanamycin, gentamicin cassettes with chloramphenicol cassette and deleting the ampicillin cassette. By parental mating, pTnCm and pTnMod-O-Cm were introduced into A media WS, respectively. Since pTnMod-O-Cm could replicate in A. media WS, the plasposon pTnCm was chosen for further study.In order to identify the possible genes involved in melanogenesis, the transposon mutants were picked and inoculated into 96-well plates containing 200 ?L LB-ampicillin-chloramphenicol agar medium. From 20,000 mutants screened,14 mutants exhibited reduced or abolished melanin production compared to the level of the parent strain, called M1-M14. By monitoring the general growth,12 mutants were screened as potential candidates for the identification of genes affecting melanin synthesis.DNA sequences flanking the transposon sites were determined by cloning chromosomal restriction fragments and thermal asymmetric interlaced PCR (Tail-PCR), which displayed the insertions mapped to genes predicted to encode different kinds of proteins. Unfortunately, transposon-flanking sequences for two non-pigment-producing mutants (M10, M13) were not obtained. Among the identified genes, there are two genes (phhA, tyrB) involved in pyomelanin synthesis.Comparison of extracellular intermediates in the supernatants of wild-type A. media WS and the unpigmented mutants (M10, M13) revealed HGA was the main intermediate product in melanin synthesis. This is the first report of HGA as an intermediate in melanization in Aeromonas.Analysis of the genome resulted in identification of other genes, aspC and hppD, which may be involved in pyomelnain synthesis besides phhA, tyrB. Deletion and complementation analysis showed:1) phhA encodes the enzyme PhhA which would help promote melanin production by providing an additional source of tyrosine through conversion of phenylalanine to tyrosine in the medium.2) The products of tyrB cooperated with aspC were responsible to synthesize 4-hydroxyphenylpyruvate and the product of tyrB played the more important role.3) hppD is the central gene in melanization and encodes a HppD enzyme that catalyzes the conversion of 4-hydroxyphenylpyruvate to HGA.A genomic search in microbial databases revealed the existence of similar genes organization encoding the pyomelanin synthesis pathway in Aeromonas species whose genomes are totally known. When the key genes (hppD) in pigmentation were cloned from the pigment-producing Aeromonas strains (A. salmonicida_AB98041,A. salmonicida KACC14791) and the non-pigment-producing Aeromonas strain (A. hydrophila_XS91-4-1) and expressed in Escherichia coli, the phenotypes of the E. coli strains were transformed to melanogenic phenotypes. To our knowledge, the hppD gene cloned from non-pigment-producing A. hydrophila_XS91-4-1 mediating melanin production in E. coli has not been reported in bacteria. Transcriptional analysis of the phhA, phhB, tyrB, aspC, hppD genes in the Aeromonas strains (A. salmonicida_AB98041, A. salmonicida KACC14791, A. media WS, A. hydrophila_XS91-4-1) revealed all the genes can transcribe in the strains. So we hypothesized the pyomelanin production pathway is ubiquitous in Aeromonas and there are other regulatory mechanism except the key genes involved in pyomelanin production pathway. HPLC analysis of the intermediates in A. salmonicida_AB98041, A. salmonicida KACC14791, A. hydrophila_XS91-4-1 also supported this hypothesis. Moreover, the hppD genes cloned from A. salmonicida (A. salmonicida_AB98041,A. salmonicida KACC14791) mediated melanin synthesis at 22?. It appears that the temperature dependence of the activity of HppD corresponds well with the growth temperature for A. salmonicida and the enzyme is temperature sensitive.Aeromonas is widely distributed in terrestrial and aquatic environments. The genus comprises psychrophiles and mesophiles and causes different kinds of diseases to many cold-blooded and warm animals, such as melanized A. salmonicida and unpigmented A. hydrophila. Our study revealed the molecular mechanism of melanin synthesis in A. media WS, extended the understanding of diversity of melanin synthesis mechanism in bacteria, and provided a theoretical basis for improving bacterial melanin production by molecular methods.