The Mechanism Of The Impact Of Catalase On The Growth And Melanogenesis Of Aeromonas Media WS

Melanin formed by polymerization of phenols or indoles is a type of natural pigments and widely dispersed in nature.Although it's not essential for organisms,it has various functions such as light absorption,radiation protection and free radical scavenging,which can provide survival advantages for themselves.Recent studies have found that melanin is closely related to serious diseases such as Alzheimer's disease and albinism.Therefore,studies on melanin synthesis mechanism and physiological function have always been concerned.As for catalase,it's an important antioxidant enzyme and ubiquitous in aerobic organisms.It can efficiently remove H₂O₂ which is one of reactive oxygen species(ROS),so that it's crucial for maintaining normal ROS levels in organisms.Aeromonas media WS,a high-yielding melanin strain,was isolated from Wuhan East Lake by our laboratory.Previously experiments proved that the strain could form pyomelanin via homogentisate(HGA)pathway.After the key enzyme encoding gene hpp D of the HGA pathway was knocked out,the mutant strain was completely non-pyomelanogenic,which means the pyomelanin is the major pigment of A.media WS.However,the presence of DOPA and DOPA melanin biomarker were also detected in the culture medium,indicating that a small amount of DOPA melanin were exist in A.media WS.It was previously considered that catalase(Kat E)might be related to the formation of DOPA melanin in WS strain.However,kat E knockout mutant could still form DOPA melanin.Interestingly,the total production of melanin was reduced by about half,and the synthesis process advanced,suggesting that kat E could somehow affect the synthesis of pyomelanin.This study focuses on exploring the influence of catalase on pyomelanin synthesis.First the growth curves of WS?kat E and wild-type strain WS exhibited no significant difference.Then the transcription of HGA pathway related genes were detected via Real-time PCR,the results suggested that the transcription of genes in WS?kat E were similar to genes in wild-type strain,meanwhile the total RNA of WS?kat E cultivated for 24 h couldn't be extracted.Thus,we suspected the mutant's growth was affected.By detecting the Live bacteria of WS?kat E and non-pyomelanogenic strain WS?kat E?hpp D,the data showed WS?kat E died suddenly during stationary phase(around 24 h),but WS?kat E?hpp D still grew.During this period,WS?kat E was synthesizing melanin.In conclusion,it's proved the the reduction of melanin production was caused by the sudden death.Also,the process of melanin synthesis or Hpp D might be the key reason for the death.Subsequently genes of HGA pathway were knocked out the in the genome of WS?kat E respectively,and found WS?kat E?phh A,WS?kat E?tyr B which reduced the pyomelanin production were all dead early.But non-pyomelanogenic strains WS?kat E?tyr B?asp C and WS?kat E?hpp D were growing normally.Therefore,we identified that the pyomelanin biosynthesis process caused the premature death of WS?kat E,once strains didn't synthesize pyomelanin,the premature death wouldn't happen anymore.On this basis,the growth condition of WS?kat E in different culture ways were testified,and the results indicated that the oxygen was important for the premature death.According to the function of catalase,the intracellular ROS levels of WS?kat E,WS?kat E?hpp D and wild-type strain during 19-24 h were measured,then we found ROS level of WS?kat E had shown a dramatic increase,and far more than the wild-type strain WS and WS?kat E?hpp D.At this moment WS?kat E had been synthesizing pyomelanin.Therefore,it's confirmed that the deletion of catalase resulted in the decrease of ROS scavenging ability,while the synthesis of pyomelanin accumulated toxic ROS in a short time,eventually led to the premature death of WS?kat E.In addition,we found it also happened in melanogenic A.salmonicida subsp.salmonicida AB98041?kat E,suggesting that this phenomenon is universal to some degree.We also investigated the advanced pyomelanogenesis of WS?kat E.Extracellular HGA of strains were tested via HPLC.The results showed the HGA of WS?kat E disappeared earlier than wild-type strain,so that HGA autoxidation of WS?kat E happened in advance.This might be related to the changes in the extracellular oxygen environment.Subsequently,we found the dead cells of WS?kat E remained intact by electron microscope.It's possible that WS?kat E could be a bioreactor used in industrial production.Then two industrial enzymes were expressed in WS?kat E.The results showed the crude still kept enzyme activities,which proved the feasibility of WS?kat E as bioreactor.In conclusion,this study revealed the importance of catalase in the biosynthesis of pyomelanin,enriched the understanding of the biosynthesis process of pyomelanin,and laid a foundation for the application and development of pyomelanin.Also,this finding deepened our understanding of the evolutionary adaptation strategies of Aeromonas in aquatic environment,and provided a new idea for the special fermentation strains of industrialization.