Diversity Of Oil-degradation Bacteria And Their Alkane Hydroxylases CYP153A Genes Enriched From Surface Water Of Atlantic Ocean

Nowadays,natural oil seepage,marine oil transport accidents lead to persistent and serious pollution on marine environments and ecosystems.Bioremediation by virtue of microbial biodegradation is now widely recognized as an effective tool to remove marine oil pollution.It has long been recognized that many microorganisms which are abundant and widespread in environments have evolved to use this highly reduced alkanes as a diet. Thus,the isolation and identification of oil-degrading bacteria and study of their mechanisms are very important and necessary on bioremediation of oil spills in both basic research and applied research.20 oil-degrading consortia enriched by a mixture of diesel oil and crude oil(in a ratio of 1:1) from surface seawater accross the Atlantic Ocean were analyzed by cultured and uncultured methods.Consequently,a total of 197 bacterial isolates consisting of 48 genera were obtained,which were characterized by 16S rRNA analysis.They belonged to Proteobacteria,Actinobacteria,the CFB group and Firmicutes,among which theα-andγ-Proteobacteria consisted of the majority.Besides,at least 33 potential novel species were isolated.PCR-DGGE analysis revealed that,the cultivable bacteria belonging to the following genera of Alcanivorax,Thalasspospira,Marinobacter,Novosphingobium, Salinisphaera,Erythrobacter and Parvibaculum dominated these 20 oil-degrading consortia.The major unculturable members included Alcanivorax,Thalasspospira, Salinisphaera,Bortnella and Zymomonas.Further,most of bacterial isolates were confirmed of capability on hydrocarbon utilization.Among them,Salinisphaera spp.can be characterized as potential novel alkane degrader.Besides,bacteria closely affiliated to Halomonas,Tistrella and Martelella were also frequently occurred in these communities. However,they were not dominated members,and showed inert hydrocarbon-biodegradability.According to DGGE results,though genera of Bortnella and Zymomonas were also frequently detected as dominated members,none of them were isolated.Besides,a Gordonia strain S14-10 were found to degrade C₁₀-C₃₆ efficiently. Analysis based on the 16S rRNA sequence,housekeeping gene secAl sequence,as well as physiological and chemical characteristics indicated that it may belong to a new species of Gordonia genus.Then the cloning and analysis of CYP153 P450 and alkB were also carried out.Using newly designed degenerate PCR primers,CYP153A P450 genes were surveyed in these 197 culturable marine bacteria.Together,87 P450 gene fragments were obtained from 68 bacterial strains,which belonged to 19 genera,such as Alcanivorax,Bacillus, Erythrobacter,Martelella,Parvibaculum,Salinisphaera.Among them,the following genera were reported for the first time to harbor P450 genes:Bacillus,Brachybacterium, Halomonas,ldiomarina,Leifsonia,Martelella,Mesorhizobium,Ochrobactrum, Salinisphaera,Sphingobium,Tetrathiobacter,Tistrell and Solimonas.Phylogenetic analysis showed that this kind of genes were quite diverse and formed several clusters, most of which were generated from various Alcanivorax bacteria.Interestingly,some sequences were grouped into a far related novel cluster,such as those from Salinisphaera genus.On the other hand,far related isolates owned similar or even identical sequences, indicating horizontal gene transfer.Moreover,one genotype can exhibit wide geographic distribution.In addition,coexistence of different genes in one organism occurred in ten isolates of Parvibaculum,Erythrobacter,Sphingobium,Bacillus and Tistrella genus.In more details,multiple P450 genes co-occurred in all isolates of Parvibaculum.Summarily, quite diverse P450 genes were detected during our survey with oil-degrading bacteria mainly from pelagic environments.Results of these can help to depict the diversity of P450 in prokaryotes,and the ecological role of their host bacteria in oceanic environments.11 Parvibaculum bacteria isolated from different sea areas were compared based on their 16S rRNA,rpoD and BOX-PCR.The results showed that some strains harboring similar or identical 16S rRNA and rpoD gene sequences had significant different fingerprint patterns in BOX-PCR.These confirmed the reliability and sensitivity of the BOX-PCR technology,also reflected the phylogenetic difference due to different geographic origin.Additionly,by constructing P450 gene libraries,we found that each of the 11 Parvibaculum strains contained 4-6 P450 genes.Parvibaculum isolate S18-4 harboring six genes was chozen for further study by quantitative real time PCR.As a result, only P450-2 and P450-4 genes could be significantly induced by alkanes(C₁₀ and C₁₄, while the others were not induced.Whether they were pseudogenes,or capable of degrading other substrates need further study.In this report,the bacterial community structures of 20 oil-degrading consortia enriched from the surface water across the Atlantic Ocean were investigated.Quite diverse P450 genes and oil-degrading bacteria were observed.These bacteria along with their alkane hydroxylase genes as a large pool everywhere in marine,are ready to respond to oil emergence.These findings facilitate us to understand the fate of oil in marine even oceanic environments.Moreover,these oil-degrading bacteria as well as their alkane hydroxylase genes provide useful materials for the exploitation of bioremediation reagents.