| PAHs have two or more fused benzene rings and are broadly existing environmental pollutants that have carcinogenicity,teratogenicity and mutagenicity,with anthropogenic sources.Novosphingobium pentaromativorans US6-1 is a Gram-negative strain obtained in the sediments of Ulsan Bay.It can efficiently degrade PAHs with two to five rings and can use PAHs as the sole carbon source.Strand-specific RNA sequencing showed the expression of many TonB-related genes change significantly under the stress of BaP.To explore the roles of the TBDTs in the degradation of PAHs,we constructed tbdt deletion,replenishant and overexpression strains.Subsequently,the traits of different strains were analyzed by means of real-time PCR and detection of hydrophobicity of cell surface.The main research results are shown below.1.The genomic data showed that US6-1 contains 74 TonB-related genes,including 6 tonB genes,3 exbD genes,3 exbB genes and 62 tbdt genes.Particularly many PAHs degradation-related genes are located around tbdt-06,tbdt-11 and tbdt-23.2.We obtained the gene deletion strains Δtbdt-06,Δtbdt-11 and Atbdt-23 by the homologous recombination of the suicide plasmid pAK405.We got the complemented strains cAtbdt-06,cΔtbdt-11,cΔtbdt-23 and the gene overexpressing strains oex-tbdt-06,oex-tbdt-11,oex-tbdt-23 by using the expression plasmid pBBRl.So far,we established a complete genetic manipulation system for US6-1.And the knock out of the tbdt-11gene caused that US6-1 lost the ability to degrade PAHs.3.The degrading enzyme-related gene’s expression in Δtbdt-11 was lower than US6-1 in the culture with BaP.The laser confocal image with a weaker fluorescence intensity showed the concentration of intracellular PAHs in Δtbdt-11 was low.Moreover,the CSH of the Atbdt-11 no longer increased with the appearance of PAHs.According to those results,we concluded that tbdt-11 may be involved in the recognition and transport of PAHs. |
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