| The halophilic Martelella sp.AD-3(CCTCC M 2011218)can efficiently degrade naphthalene,phenanthrene,anthraquinone and other low molecular weight polycyclic aromatic hydrocarbons(LMW PAHs)in the range of 3-5%salinity and pH 8-9.5.It has the potential to bio-reinforcement of PAHs contaminated high salinity soils.In this study,the whole genome of AD-3 was extracted,and sequenced by Illumina Miseq System and PacBio RS Ⅱ System.And the annotation analysis was combined with a variety of gene identification and protein function prediction software and database for the AD-3 whole genome.The key genes that have been speculated to play an important role in degradation are verified by cloning,expression,and resting cell responses.Through the purification of the encoded protein,the characteristics of the protein and the conditions affecting the enzyme activity were studied from the basic properties and key sites of the enzyme.The main results are as follows:(1)The whole genome of the Martelella sp.AD-3 contains 1 chromosome and 2 plasmids.The coding sequence coverage rate is 88.82%,and the predicted protein is 4,537.The strain AD-3 contains many genes associated with degrading LMW PAHs,most of which are in a gene cluster.The RHOs 2 encoded on the gene cluster is indispensable part of the PAHs metabolic pathway,and it is the first step in the degradation of LMW PAHs.In combination with the reported salt-tolerance mechanism,there are some gene in strain AD-3 which are related to K+accumulation mechanism,Na+efflux mechanism,the synthesis of compatible solute and the transport of compatible solute.In addition,the Type Ⅰ CRISPR/Cas system is contained in strain AD-3.(2)The function of the gene annotated as salicylate 1-hydroxylase in Martelella sp.AD-3 was studied.The gene is 1,212 bp in length and its encoded protein contains 403 amino acids.The optimal expression conditions of the gene in E.coli BL21(DE3)was 0.2 mM IPTG at 30℃.The induced E.coli BL21(DE3)did not catalyze salicylic acid but could catalyze 3hydroxybenzoic acid and generate gentisic acid Thus,it was proved that the enzyme was 3hydroxybenzoate-6-hydroxylase(3HB6H)instead of the salicyllate 1-hydroxylase.(3)The purified 3HB6H which was approximately 46 kDa was homodimeric in active state.3HB6H was yellow and binded FAD as its cofactor.Under the conditions of phosphate buffer(pH 8.0),37℃,salt free(NaCl),and non metal ions,it has a stable activity for a long time,and the enzyme activity is 25.2 U/mg.In addition,Q305,Y306 and A308 in the 3HB6H of strain AD-3 are the loci associated with binding FAD.And Y221 and Q305 are associated with substrate binding.Through the above studies,we can reasonably explain the degradation mechanism of LMW PAHs by strain AD-3 and its salt tolerance mechanism.It provides powerful theoretical guidance for the practical application of strain AD-3 in the high-salt environment to effectively degrade LMW PAHs.In addition,the error annotation of 3HB6H from strain AD-3 was revised by functional verification,it explained the key steps in the degradation of 3-hydroxybenzoic acid and provides a theoretical basis for strain AD-3 to degrade other aromatic compounds.This paper laid the foundation for further research. |
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