Metabolism Of Melanin Biosynthesis In Aeromonas Media WS Strain

Aeromonas are ubiquitous opportunistic pathogenic bacteria of poikilothermic animals,including fish,reptiles and amphibians.Melanin has been suspected as a factor of pathogenic virulence in pathogenic microorganisms and production of melanin has been described in many Aeromonas species.However,the molecular determinant involved in melanin metabolic pathway has not been characterized in Aeromonas.Aeromonas media strain WS was isolated from water samples collected from East Lake,Wuhan,China,and exhibits a significantly high yield of melanin.In previous work,L-3,4-dihydroxyphenylalanine(L-DOPA)was confirmed as one important intermediate product from cell-free culture filtrates of WS and it indicated the major pathway of melanin formation in this strain is with the tyrosinase-catalysed conversion of L-DOPA to DOPA melanin.However,no effective method of tyrosinase activity assay was found except native-PAGE using L-dopa as substrate and no melanin formation could be observed when overexpression the tyrosinase designated TyrA isolated from WS strain using N-terminal seqencing and adaptor PCR technical strategy in Escherichia coli.In order to ascertain whether TyrA belongs to tyrosinase,firstly we charactered the tyrosinase activity of TyrA using expression system in pET-26b(+)vector in subsequent research.Tyrosinase from Streptomyces antibioticus(MelC)and Bacillus cereus(MelBc)are two different models for bacterial tyrosinase and were used as controls.Unlike TyrA,both of MelC and MelBc exhibit significant tyrosinase activity and could be determined in native-PAGE using 3,4-dihydroxyphenylalanine as a substrate.In addition,melanin formation could be examined when MelC and MelBc were expressed in E.coli but not of TyrA.Furthermore,we investigated the role of histidine residues in the function of TyrA which were required for the tyrosinase activity of other bacterial tyrosinase by examing a series of substitution mutants based on megaprimer PCR.Howerer,the tyrosinase activity determination shows no significant difference between TyrA and substitution mutants.Finally,an in-frame deletion of melA,the gene encoding TyrA,was made using suicide vector pDM4 and we found the deletion of tyrA from the bacterium does not bring either obvious change regarding the pigment production nor the polyphenel oxidase activity in native-PAGE between WS wild type and mutant strain WS A ?melA.Taken together,the results indicated that TyrA is not a key factor responsible for melanin formation in WS strain.In subsequent research,we sequenced the complete genome sequence of WS strain for the melanin research purpose because of the difficults in the identification of genes associated with this metabolic process.The whole genome of strain WS was sequenced using a combination approach of Illumina/Solexa sequencing and PacBio sequencing technology.The complete genome of A.media strain WS contains a circular chromosome of 4,777,154 bp(60.7%G+C content)and one circular plasmid(named pWSY)of 11,274 bp in size.Mummer comparison of the A.media and A.hydrophila chromosomes shows as expected for two chromosomes of nearly the same size,but large inversion occurred in the WS chromosome relative to the A.hydrophila chromosome,accounting for the regions of forward strand alignment.Analysis of DOPA-melanin metabolism on the WS genome shows no typical bacterial tyrosinase was found by searching the whole genome against HMM PF00264 yet,but only a laccase-like protein YfiH.In contrast,the genome of strain WS carries mostly genes responsible for HGA-melanin synthesis.It was an interesting founding since HGA as a melanin precursor in A.media has not been reported before,but all these genes were found in other Aeromonas strain including non-pigmented strains.Furthermore,functional categories of proteins on WS genome using Web Gene Ontology Annotation Plot shows nearly three hundred proteins are involved in pigmentation process,include proteins related to nucleotide biosynthesis,transcription and regulation which also been found in Pseudomonas aeruginosa PA14 strain before.In addition,the analysis result of 64 signal transduction related proteins also regulated the pigmentation process infers melanin formation process is a response to variational environments.Although the major melanin metabolism was performed via HGA pathway in WS strain in a separate study,L-3,4-dihydroxyphenylalanine(L-DOPA)was confirmed as one important intermediate product from cell-free culture filtrates of WS in reverse-phase high-performance liquid chromatography(HPLC)experiment as described before.In order to learn whether DOPA melanin was one part of WS melanin,the purified melanin from WS strain was charactered based on infrared spectroscopy(IR),electrospray ionization(CE)and mass spectrometry(ESI-MS).In the results,Indole-5,6-quinone,the monomer of DOPA melanin could be found and it indicated the WS melanin seems to be the mixture of melanins derived from L-DOPA and HGA.Meanwhile,two polyphenol oxidases with physiological function involvement in DOPA melanin formation were found in WS strain using native-PAGE method and we speculated that DOPA melanin metabolic process is exsited in WS strain and catalyzed by two polyphenol oxisases.In order to identify these polyphenol oxisases,the mutant strain of laccase-like protein YfiH(a member of polyphenol oxisases family)from genome analysis was made.One staining band was disappeared in the sample of WS ?yfiH in native-PAGE experiment,and it indicated YfiH was one of the polyphenol oxidases(PPO1)in WS strain.Another polyphenol oxidase(PP02)was charactered using LC-MS/MS technology and one protein annotated catalase(CAT,product of katE)was the favorite candidate protein among 55 proteins of MASCOT search results.The PP02 activity of CAT was proved for the disappearence of another staining band in native-PAGE experiment of WS ?katE mutant strain.In addition,no staining band exhibited polyphenol oxidase activity could be obseverd in native-PAGE using the double mutant strain WS AyfiHAkatE.Laccases with polyphenol oxidase activity were reported in many organisms,but no catalase exhibited polyohenol oxidase activity has been reported in prokaryote before.Firstly,we tested the catalase activity of CAT and found WS wild type strain exhibited high catalase activity(nearly 3.68 U/mL)using catalase activity assay kit,but none of WS ?katE mutant strain.The result was consistent with the KatE domain of CAT in the bioinformatic analysis.Catalases with KatE domain were also exsited in other organisms,such as Hpii(one of three catalases)in E.coli BL21(DE3)strain.Loss function of Hpii repressed 80%catalase activity in this strain,and Hpii may also exhibit both activities of catalase and polyphenol oxidase since the comprision of CAT and Hpii amino acid sequence showed 47%similarity.CAT and Hpii were both overexpressed in vitro,and the active His6-tagged CAT and Hpii were purified and both exihibted catalase and polyphenol oxidase activities.The result proposed the situation of CAT exhibiting polyphenol oxidase activity such as in A.media WS strain may abundantly distribut in bacterium as an evolutionary strategy.Based on the enzymatic activity assay of PPO1 and PP02,we studied the contribution to melanin formation of these polyphenol oxidases in WS strain.The loss function of YfiH seems slightly influence(10%decline)to melanin formation and it indictated little significance of YfiH to melanin production in WS strain.However,the loss function of CAT causes a half decline of melanin production in WS strain and melanin formation process also advanced distinctly(from period of 24 h to 15 h).These phenomenons consisted with the HGA pathway research not very well,which showed HGA melanin was the mostly composition of WS melanin.The regulation of reactive oxygen species(ROS)in melanin formation was taken into account.In ROS assay,we found a 5.5-fold increase of ROS level was occurred in the process of melanin formation and reached to a stable level(ROS-B)finally in WS wild-type strain.It indicated the generation of ROS during pigmentation in WS strain as reported in other researchs.The loss function of CAT caused the increase of oxidative stress in WSAkatE mutant strain which was found in ROS assay and the excellular pigment formation process was repressed when ROS level reached to a stable level(ROS-B).Meanwhile,incellular melanin operates as a ROS scavenger and the melanin formation process is started up when ROS level is higher than ROS-B in WS?katE mutant strain with 8 hours earlier than normal stuation.The WS ?katE-pBBRlMCS-5-katE which contain an intact katE in pBBR1MCS-5 vector recovered the level of melanin synthesis and PP02 activity in native-PAGE.In conclusion,based on the sequencing and analysis of whole genome of A.media WS strain,the research results of melanin formation in WS strain will be helpful to expand our knowledge on the biological significance of the pigment in this strain during variational environments.