Identification and molecular characterization of the homogentisate pathway responsible for pyomelanin production, the major melanin constituents in Aeromonas media WS

Abstract

The pigmentation of many Aeromonas species has been thought to be due to the production of a L-DOPA (L-3,4-dihydroxyphenylalanine) based melanin. However, in this study we found that although L-DOPA synthesis occurs in the high-melanin-yielding Aeromonas media strain WS, it plays a minor, if any, role in pigmentation. Instead, the pigmentation of A. media strain WS is due to the production of pyomelanin through HGA (homogentisate). Gene products of phhA (encodes phenylalanine hydroxylase), tyrB and aspC (both encode aromatic amino acid aminotransferase), and hppD (encodes 4-hydroxyphenylpyruvate dioxygenase) constitute a linear pathway of converting phenylalanine to HGA and disruption of any one of these genes impairs or blocks pigmentation of A. media strain WS. This HGA biosynthesis pathway is widely distributed in Aeromonas, but HGA is only detectable in the cultures of pigmented Aeromonas species. Heterologous expression of HppD from both pigmented and non-pigmented Aeromonas species in E. coli leads to the production of pyomelanin and thus pigmentation, suggesting that most Aeromonas species have the critical enzymes to produce pyomelanin through HGA. Taken together, we have identified a widely conserved biosynthesis pathway of HGA based pyomelanin in Aeromonas that may be responsible for pigmentation of many Aeromonas species.

Fig 1. Locations of the genes in pyomealnin-synthesis pathway.

The transposon insertion sites of five mutants in the genome of A. media strain WS are marked with small black arrows. The numbers after the mutant names stand for the nucleotide (nt) site just following the transposon in the genome of A. media strain WS. Locus tags, putative protein function, and the number of amino acids of the deduced proteins are listed.

Fig 2. HPLC analysis.

HPLC analysis of the supernatants of wild-type A. media strain WS and non-pigmented mutants WS-M10 and WS-M13. Samples were taken after 24 h of growth at 30°C in LB. (A) Analysis of HGA. (B) Analysis of L-DOPA. HGA Standard: commercial HGA (Sigma); DOPA Standard: commercial L-DOPA (Sigma). The asterisk indicates the peak of L-DOPA.

Fig 3. The function of phhA in pigmentation in A. media strain WS.

(A) Wild-type A. media strain WS, phhA mutants WS-M5, WS-M6 and WSΔphhA were inoculated into LB or LB with 1mg ml^-1 tyrosine, and then at 72 and 96 h post-inoculation, the OD₄₀₀ of the cultures were determined. (B) Wild-type A. media strain WS was inoculated into LB by addition of different amounts of tyrosine (0, 0.5, 1, 2 mg ml^-1), and then at 72 and 96 h post-inoculation, the OD₄₀₀ of the cultures were determined. (C) A. media strain WS, WSΔphhA (pBBR1MCS-5), WSΔphhA (pBBR1MCS-5-phhA) and WSΔphhA (pBBR1MCS-5-phh(A+B)) were cultured in LB, and then at 72 h and 96 h post-inoculation, the OD₄₀₀ of the cultures were determined. (D) Photographs of 72 h LB cultures of A. media strain WS, WSΔphhA, WSΔphhA (pBBR1MCS-5), WSΔphhA (pBBR1MCS-5-phhA) and WSΔphhA (pBBR1MCS-5-phh(A+B)). The data presented are the means and standard deviations from triplicate cultures.

Fig 4. The function of tyrB and aspC in pigmentation in A. media strain WS.

(A) Wild-type A. media strain WS, WSΔtyrB, WSΔaspC, WSΔtyrBΔaspC, WSΔtyrB (pBBR1MCS-5), WSΔtyrB (pBBR1MCS-5-tyrB), WSΔtyrBΔaspC (pBBR1MCS-5), WSΔtyrBΔaspC (pBBR1MCS-5-tyrB), WSΔtyrBΔaspC (pBBR1MCS-5-aspC) were cultured in LB, and then at 72 h and 96h post-inoculation, the OD₄₀₀ of the cultures were determined. (B) Photographs of cultures from 72 h LB cultures of the strains. (C) Pigment production can be restored to WSΔtyrB and WSΔtyrBΔaspC by the addition of 5 mM 4-hydroxyphenylpyruvate to the LB.

Fig 5. The function of hppD in pigmentation in A. media strain WS.

(A) Wild-type A. media strain WS, WSΔhppD, WSΔhppD (pBBR1MCS-5) and WSΔhppD (pBBR1MCS-5-hppD) were cultured in LB, and then at 72 h and 96 h post-inoculation, the OD₄₀₀ of the cultures were determined. (B) Photographs of 72 h LB cultures of the strains. (C) HPLC chromatograms of culture supernatants of wild-type A. media strain WS, WSΔhppD and WSΔhppD (pBBR1MCS-5-hppD). Samples were taken after 24 h of growth at 30°C in LB. (D) MS analysis of the sample from wild-type A. media strain WS and WSΔhppD (pBBR1MCS-5-hppD), the arrow indicates the molecular ion peaks at 167, the same to that of HGA.

Fig 6. Gene organization of those genes encoding the pyomelanin synthesis pathway in Aeromonas strains.

Genes are represented by arrows as follows: phhA, gene encoding the phenylalanine hydroxylase; phhB, gene encoding the 4a-carbinolamine dehydratase; tyrB, gene encoding the aromatic amino acid aminotransferase; aspC, gene encoding the aromatic amino acid aminotransferase; hppD, gene encoding the 4-hydroxyphenylpyruvate dioxygenase. The numbers beneath the arrows indicate the levels of amino acid sequence identity (expressed as percentages) between the encoded gene products and the equivalent products from A. media strain WS.

Fig 7. Expression of hppD genes from multiple Aeromonas sp. in E. coli BL21 results in pigmentation.

(A) Photographs of E. coli BL21 (pET26b(+)), E. coli BL21 (pET26b(+)-hppD-AS), E. coli BL21 (pET26b(+)-hppD-AH) and E. coli BL21 (pET26b(+)-hppD-WS), incubated at 30°C and 22°C, respectively. (B) HPLC analysis of the supernatants of BL21 (pET26b(+)), BL21 (pET26b(+)-hppD-AS), BL21 (pET26b(+)-hppD-AH), BL21 (pET26b(+)-hppD-WS), incubated at 22°C.

Fig 8. HPLC analysis.

Detection of the intermediates from cultures of A. salmonicida_AB98041, A. salmonicida KACC14791 and A. hydrophila_XS91-4-1. Samples were taken after 36 h of growth in LB. (A) Analysis of HGA. (B) Analysis of L-DOPA. HGA Standard: commercial HGA (Sigma); DOPA Standard: commercial L-DOPA (Sigma).

Fig 9. Pathway for pyomelanin synthesis and phenylalanine/tyrosine catabolism in A. media strain WS (modified from reference 49).

The intermediates of the pathway are indicated. PhhA, phenylalanine hydroxylase; PhhB, 4a-carbinolamine dehydratase; AspC, aromatic amino acid aminotransferase; TyrB, aromatic amino acid aminotransferase; HppD, 4-hydroxyphenylpyruvate dioxygenase; HmgA, homogentisate dioxygenase; HmgB, fumarylacetoacetate hydrolase; HmgC, maleylacetoacetate isomerase. The asterisk indicates the location of the related genes mutated in this study.