The Transcriptomic Induced By Fluoranthene And Genomic Analysis Of Pseudomonas Aeruginosa DN1

Polycyclic aromatic hydrocarbons(PAHs)are widely distributed and persistent organic pollutants in the environment,which are well promising due to their hydrophobic,persistent and toxic.Fluoranthene with four rings was regarded as a model compound for the study on high molecular-weight polycyclic aromatic hydrocarbons(HMW PAHs)because of the structurally similar to carbazole,dioxin,etc.In this thesis,the degradative molecular mechanisms and metabolic pathways of Pseudomonas aeruginosa DN1 induced by fluoranthene were conducted by the way of genomic,transcriptomic,metabolic analysis.The complete genome of P.aeruginosa DN1 was performed using a combined strategy of Pac Bio RSII sequencing and Illumina Miseq paired-end sequencing.The results showed that the whole genome of P.aeruginosa DN1 contains 6,959,251 basepairs with a 6,641,902 bp circular chromosome(G+C content of 67.09%)and a 317,349 bp plasmid(G+C content of 57.01%),and the predicted protein coding sequences(CDSs)are 6,450,and that the genome of DN1 strain contains 64 t RNAs and 12 r RNAs by the analysis of r RNAmmer,t RNAscan and Rfam.There are more than a hundred genes involved in the degradation of PAHs including 25 dioxygenase genes,therein the number of ring hydroxylating dioxygenase is 5.The bioinformatics analysis showed that some genes related to PAHs degradation in P.aeruginosa DN1 had lateral gene transfer or rearranged.In addition,genome-based comparative analysis of Otho ANI showed that DN1 strain was closely related to other reported P.aeruginosa from NCBI,and had good collinearity with the model strain P.aeruginosa PAO1.On the basis of whole-genome sequencing,P.aeruginosa DN1 induced by fluoranthene was used for transcriptomic analysis to investigate the genes expression related to the degradation,regulation and transportation of fluoranthene.The resulted of RNA-Seq revealed that 3,522 genes were differentially expressed,containing 1,919 up-regulated genes and 1,603 down-regulated genes,repectively.Furthermore,they were mainly related to carbon metabolism,degradation of aromatic compounds and benzoate and so on through the comprehensive analysis of the genome and transcriptome.Generally the key enzyme genes involved in the degradation of fluoranthene,such as cat A,pca G and pca H,were up-regulated significantly,hence the homologous recombination technique was used to knockout those genes for exploring their function in fluoranthene degradation.The results showed that the growth of mutant strains were slow due to the gene deletion under the condition of protocatechuic acid and catechins as the sole carbon source,while there was no significant difference in the culture of glucose as the sole carbon source.In particular,the growth of Δpca G and Δcat A exhibited considerable delayed variation in the presence of catechol and protocatechuate as the sole carbon source.Additionally,the degradative rate of fluoranthrene was significantly affected by the gene deletion,such as Δpca G,Δpca H and Δcat A.For example,the degradative rate of fluoranthrene was 84.47% by the wild DN1 strain suppletment with MSM medium shake flask culture with 50μg/m L fluoranthrene as the sole carbon source for nine days.The degradative rate of Δpca G and Δpca H,however,were 64.3% and 71.67% in the same conditions,repectively.It is well known that the conding genes pca H and pca G as protocatechuate 3 4-dioxygenas play crucial roles in the degradation of fluoranthrene of P.aeruginosa.However,the key genes deletion,such as Δpca G,Δpca H and Δcat A,could not completely inhibit the degradation of fluoranthrene,even though they had obvious effects on fluoranthene degradation.Moreover,fluoranthene,9-hydroxyfluorene,1-acenaphthenone,1,8-naphthalic anhydride α/β-methynaphthalene and 2,3-dimethynaphthalene were detected using gas chromatography-mass spectrometry(GC-MS),indicating that the degradation of fluoranthene by P.aeruginosa DN1 might be initiate dioxygenation at the site of C-7,8,although it has been monitor that the degradation of fluoranthene could initiate dioxygenation at C-1,2 and/or C-2,3 in P.aeruginosa DN1.Thus,the detailed degradative pathways need to be further studied in the next step.