Study On HMW-PAHs Metabolic Pathway And Transcriptional Regulation In Novosphingobium Pentaromativorans US6-1

Polycyclic aromatic hydrocarbons （PAHs） are one class of organic pollutants which are widely distributed in the environment. They are carcinogenic, teratogenic, and mutagenic to living organisms through food chain. It is an efficient way to recover PAHs contamination by microbial remediation. Currently, there is only limited information regarding to the bacterial biodegradation pathway and physiological regulation mechanism of HMW-PAHs such as BaP, because they are structurally stable and nonbiodegradable. In order to gain insight into the mechanism of HMW-PAHs, Novosphingobium pentaromativorans US6-1, a marine bacterium isolated from muddy sediment of Ulsan Bay, Republic of Korea, was used to study the PAHs degradative characteristics, identification of the functional genes involved in the biodegradation base on transcroptome and Real-time PCR analysis of N. pentaromativorans US6-1exposed to phenanthrene, pyrene and benzo[a]pyrene, proposing PAHs biodegradation pathway, elucidating the role of megaplamid pLAl and physiological response mechanism involved in benzo[a]pyrene bio remediation.The main results were as follows:1. N. pentaromativorans US6-1could degrade PAHs effectively, the biodegradation rate of10ppm phenanthrene, pyrene and benzo[a]pyrene decreased by99.02%,52.88%and14.54%respectively. Combined with the culture changing to yellow, US6-1started to degrade phenanthrene, pyrene and benzo[a]pyrene at12h,48h,6d respectively.2. To analyze the genome of strain US6-1, we found two central metabolic pathways, protocatechuate metabolic pathway and catechol metabolic pathway. In addition, protocatechuate metabolic pathway existed in chromosome. Otherwise, catechol metabolic pathway indwelled in megaplasmid pLAl. We applied SDS-temperature method to obtain a mutant N. pentaromativorans CPUS6-1（curing megaplasmid pLAl）. To confirm the presence of catechol metabolic pathway in pLAl, we compared different aromatic degradation by strain US6-1and strain CPUS6-1, meanwhile found that pLAl plays a key role in aromatic degradation;3. A total of17ring-hydroxylating oxgenase genes （RHOs） had conserved domains in the genome of strain US6-1. After ClassRHO software running, seven of them had got accurate types. Real-Time PCR was used to compare RHOs and the other genes in pLAl gene expression induced by phenanthrene, pyrene and benzo[a]pyrene. The results indicated that the genes were same unregulated after exposure to PAHs; Gene expression in the Phe and BaP degradation conditions were significantly higher than Pyr, otherwise the NSU₃626and NSU_PLA1141are inverse. Because there were similarities in the structures of Phe and BaP which may lead to express the same genes, but Pyr had a different structure.4strain US6-1were tested OD600, ETSA and CFU in the culture with BaP as the sole source of carbon and energy （hunger environment）. Its OD600values changed little, while the CFU had the large magnitude of change, which means that strain US6-1can be cultured to non-culturable that has be adapted the discomfort environment. We used real-time quantitative PCR to determin relative gene expression levels involed in biodegradation genes and emergency response regulator genes. The results show that the BaP group and Control group regulate gene expression are similar. strain US6-1can regulate gene expression to adapt adverse environmental changes effectively.