Diversity Analysis Of C₁₆H₃₃Cl-degrading Bacteria In Surface Seawater Of The Arctic Ocean And The Genome And PAHs-Degrading Mechanism Analysis Of Alteromonas Sp.P127

This study consist of two parts, diversity analysis of C₁₆H₃₃Cl-degrading bacteriain surface seawater of the Arctic Ocean and the genome and PAHs-degradingmechanism analysis of Alteromonas sp. P127.In order to detect the diversity of the degradation bacterium ofhalogenated-alkane form the surface seawater of the Arctic Ocean. Twelvesurface-water samples from the Arctic Ocean were collected and enriched usingC₁₆H₃₃Cl as the sole carbon and energy source. Bacteria from the enriched cultureswere isolated on marine agar, and followed by16S rRNA gene identification andphylogenetic analysis. Further, their degradation ability was tested with C₁₆H₃₃Cl. Thebacterial community structures were further examined by denaturing gradient gelelectrophoresis （DGGE）.112isolates were obtained from the12samples, amongwhich19isolates can degrade C₁₆H₃₃Cl, bacteria of Alcanivorax and Rhodococcusexerted good emulsification and degradation, while bacteria of Marinobacter alsohave the degradation capacity, but less. DGGE analysis revealed that Alcanivorax,Parvibaculum and Thioclavawere dominated in the enriched consortia. The C₁₆H₃₃Cldegradation bacteria in the Arctic marine environment mainly belonged toα-proteobacteria, γ-proteobacteria, actinobacteria and bacteroidetes. This is the firstreport on the diversity of degradation bacteria of halogenated-alkane in the ArcticOcean. Our result contributed to the knowledge about the arctic environment and thebiodiversity of degrading bacteria.Alteromonas sp. P127was a gram-negative bacteria isolated from the Atlanticdeep-sea water with naphthalene, phenanthrene, anthracene and pyrene four kinds ofpolycyclic aromatic hydrocarbons （PAH） as the carbon source. The bacteria can usebenzoate and naphthalene, phenanthrene, pyrene,2-methyl naphthalene,dibenzofuran,4-methyl dibenzothiophene as the sole carbon and energy source to growth, P127was the first deep-sea Alteromonas strain that have PAHs degradation ability. Genomesequencing show that the P127genome was about4.9Mb, predicted to have4538ORF,68tRNA,5rRNA operons, and the GC content is44.8%. Genome analysisindicated that the P127genome also has some deep-sea genome features, such as alarge number of IS sequence, transposons and phage related genes. P127genome was500Kb large than A. macleodii ’Deep ecotype’（AltDE） genome. Compared genomesanalysis reveal that P127has four major gene cluster that AltDE didn‘t have, thePAHs degradation gene cluster （nag）, the Integrating and conjugative elements genecluster（ice）, the other hydrogenase gene cluster （hy2）, the benzoate degradation genecluster （Ben）. These gene clusters exists in chromosome as genomic island, probablederive from horizontal gene transfer. Heavy metal resistance test show that P127hasthe same heavy metal resistance as the ’Deep ecotype’ Alteromonas,they are betterthan surface Alteromonas.Reverse transcription PCR analysis show that PAHs degradation gene clustermainly consists of two operons and two regulatory genes, the operon contain PAHdioxygenase gene was induced by naphthalene. The two operons located in differentDNA chains, they transcripted in opposite directions. the last ORF in the operoncontain PAH dioxygenase gene encoded a membrane protein that contains signalpeptide and pridicted to be a PAHs outer membrane transporter. It has a structuresimilar to E.coli long chain fatty acid transporters（FadL）. The significant PAHdegradation genes: the large subunit dioxygenase gene orf0227, the two regulatedgenes orf0218and orf0219, the outer membrane protein gene orf0231were deletedmutation. orf0227mutant lost of the PAHs degradation ability, orf0218and orf0231mutant appear not effect on PAHs degradation, but the degradation rate test show thatorf0218mutant had a little dibenzofuran degradation rate increased thanwildtype,show that it is a negative regulator，orf0231mutant almost no influence indegradation rate. But orf0219no successed mutation. Genetic organization ofbenzoate degradation gene cluster were economized and specialed. The gene cluster including all the genes involved in benzoate degradation to catechol and adjacent tocatechol ortho-cleavage through β-ketoadipate pathway into TCA cycle.This part of research through the genome analysis revealed the strain‘s deep-seaenironmental adaptation genetic basis, including some transfer elements beneficial togenome evolution, heavy metal resistance genes, PAHs, benzoate and H₂utilizationgenetic basis. And through the reverse transcription PCR and deleted mutation toverify the function of PAHs degradation genes, but the PAHs association and thegene regulation mechanism was still not clear. This part research deepened ourknowledge about the environment adaptability and PAHs degradation characteristicsof Alteromonas sp. P127, the results have some reference value in the revelation ofdeep-sea PAHs degradation mechanisms and the PAHs-degrading bacteria‘s role toenvironment bioremediation. Additionally, it is benefit for the development andutilization of deep-sea pollutant-degradation bacteria resource, and it is helpful forbiological monitoring, bioremediation and the development and application ofbiological catalyst for PAHs contamination.