Mechanisms Of Fluoranthene And Phenanthrene Degradation In The Deep Sea Pah-degrader Celeribacter Indicus P73~T

Polycyclic aromatic hydrocarbons（PAHs）are among the most persistent organic pollutants in the environment,which are harmful to human and environment due to their carcinogenic,teratogenic and mutagenic properties.Phenanthrene,containing a “bay region” and a “K region” which are common in some PAHs,is usually selected as a model compound in the studies of low molecular weight PAHs degradation.Fluoranthene is a model compound for the studies of high molecular weight PAHs biodegradation,as it is structurally similar to other compounds of environmental concern,such as dibenzofuran,carbazole and dibenzodioxin.Celeribacter indicus P73^T,previously isolated from a deep-sea sediment from the Southwest Indian Ocean,was found to be able to degrade a wide range of PAHs.In this study,genomic,transcriptomic,metabolic and genetic studies of strain P73^T were carried out to investigate the degradative molecular mechanisms and pathways,aimed to provied theoretical proof for the bioremediation of fluoranthene and phenanthrene pollutions.The complete genome was sequenced using Solexa paired-end sequencing technology.The complete genome of strain P73^T contained 4,969,388 basepairs with a G+C content of 65.74 mol%,comprising a circular chromosome of 4,529,105 bp and five plasmids ranging from 7053 to 155,183 bp.The genome contained 4827 predicted protein coding sequences（CDSs）,two sets of r RNA genes（two 5S r RNA,two 16 S r RNA,and two 23 S r RNA）and 48 t RNA genes,with an average length of 909 bp and a coding density of 88.30%.Among the 4827 CDSs,3908（80.96%）were able to be assigned to 22 clusters of orthologous groups（COGs）.One hundred and thirty-eight genes,including 6 ring-hydroxylating dioxygenase subunit alpha genes and 9 ring-cleaving dioxygenase genes,were found to be involved in the degradation of PAHs,and most of these genes were clustered in four regions.P73₀346 was proved to be the first fluoranthene 7,8-dioxygenase to be found and the first fluoranthene dioxygenase identified within the toluene/biphenyl family.Strain P73^T were also found to have used lateral gene transfer to enhance its PAH degradation ability.Analysis of the transcriptome of C.indicus P73^T revealed that 1184 genes（24.5%）were differently expressed and 975（20.2%）were up-regulated when induced by fluoranthene.Joint analyses of the genome and transcriptome showed that at least 110 genes were involved in fluoranthene degradation,including the genes involved in the processes of chemotaxis,signal transduction,regulation,transport and degradation.Four major intermediate metabolites were detected during fluoranthene degradation by strain P73^T using gas chromatography-mass spectrometry（GC-MS）: acenaphthylene-1（2H）-one,acenaphthenequinone,1,2-dihydroxyacenaphthylene and 1,8-naphthalic anhydride,indicating that the metabolism of fluoranthene in strain P73^T was initiated by dioxygenation at the C-7,8 positions.Based on the genomic and transcriptomic analyses,the metabolic results and the pathways reported previously,metabolic pathway was proposed for fluoranthene degradation and the most probable genes were assigned to each enzymatic reaction in the pathway.Strain P73^T degraded fluoranthene through the proposed C-7,8 dioxygenation pathway involving extradiol cleavage of 7,8-dihydroxyfluoranthene.Finally,the metabolites phthalate and protocatechuate were metabolized through the β-ketoadipate pathway.Strain P73^T may be a useful strain for the study of the C-7,8 dioxygenation pathway involving extradiol cleavage of 7,8-dihydroxyfluoranthene,as the C-7,8 dioxygenation pathway was the only pathway detected in this strain.Two major metabolites were detected during phenanthrene degradation by strain P73^T using GC-MS: 1-hydroxy-2-naphthoate and 1-naphthol,indicating that the metabolism of phenanthrene in strain P73^T was initiated by dioxygenation at the C-3,4 positions.Based on the metabolic and genomic analyses,a pathway involved C-3,4 dioxygenation was proposed for phenanthrene degradation by strain P73^T,and the degradation genes involved in the pathway were assigned.The prososed C-3,4 dioxygenation pathway produced the metabolites 1-hydroxy-2-naphthoate and 1-naphthol,which were toxic and led to the weak growth of strain P73^T in phenanthrene.Strain P73^T,a novel PAH-degrader capable of degrading a wide rang of PAHs,can be very useful in the bioremediation applications of marine oil spill and other PAHs pollutions.This study states the degradative molecular mechanisms and pathways in strain P73^T,and may provide theoretical basis for the bioremediation of fluoranthene and phenanthrene pollutions.