AbstractAeromonas salmonicidasubsp.salmonicida(A.s.s) is a major pathogen affecting fisheries worldwide. It is a well-known member of the pigmentedAeromonasspecies, which produces melanin at ≤ 22 °C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30-35 °C while bacterial growth is not affected. The mechanism and biological significance of this temperature-dependent melanogenesis are not clear. Heterologous expression of anA.s.s.4-hydroxyphenylpyruvate dioxygenase (HppD), the most crucial enzyme in the HGA-melanin synthesis pathway, results in thermosensitive pigmentation inEscherichia coli, suggesting that HppD plays a key role in this process. In the current study, we demonstrated that the extreme thermolability of HppD is responsible for the temperature-dependent melanization ofA.s.s.Substitutions in three residues, Ser18, Pro103, or Leu119 of HppD fromA.s.sincreases the thermolability of this enzyme and results in temperature-independent melanogenesis. Moreover, replacing the corresponding residues of HppD fromAeromonasmedia strain WS, which forms pigment independent of temperature, with those ofA.s.sHppD leads to thermosensitive melanogenesis. Structural analysis suggested that mutations at these sites, especially at position P103, can strengthen the secondary structure of HppD and greatly improve its thermal stability. In addition, we found that HppD sequences of allA.s.sisolates are identical and that two of the three residues are completely conserved withinA.s.sisolates, which clearly distinguishes these from otherAeromonasstrains. We suggest that this property represents an adaptive strategy to the psychrophilic lifestyle ofA.s.s.ImportanceAeromonas salmonicidasubsp.salmonicida(A.s.s) is the causative agent of furunculosis, a bacterial septicemia of cold water fish of theSalmonidaefamily. As it has a well-defined host range,A.s.shas become an ideal model to investigate the co-evolution of host and pathogen. For many pathogens, melanin production is associated with virulence. Although other species ofAeromonascan produce melanin,A.s.sis the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here we demonstrate that thermosensitive melanogenesis inA.s.sstrains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). The strictly conservedhppDsequences amongA.s.sand the exclusive thermosensitive pigmentation of these strains might provide insight into the role of melanin in the adaptation to a particular host, and offer a novel molecular marker to readily differentiateA.s.sstrains from otherA. salmonicidasubspecies andAeromonasspecies.
4-hydroxyphenylpyruvate dioxygenase thermolability is responsible for temperature-dependent melanogenesis inAeromonas salmonicidasubsp.salmonicida