Mechanisms Of Dibutyl Phthalate(DBP)degradation And Cadmium Resistance Of Enterobacter Sp.DNB-S2

Phthalate esters?PAEs?are mainly used as plasticizers in plastics manufacturing.Because PAEs are not chemically combined with these products,they often diffuse into the environment in the process of manufacturing,utilization and disposal of these products.Studies have shown that the carcinogenicity,endocrine and reproductive and developmental interference of PAEs to humans and animals have attracted global attention.Dibutyl phthalate?DBP?is one of the most common and widely used PAEs.Because DBP is difficult to degrade naturally and has strong biological toxicity,it is an urgent environmental problem to remove DBP from the environment.At present,microbial degradation of organic pollutants in the environment is one of the most promising remediation methods.In this study,a DBP efficient degradation bacterium,Enterobacter sp.DNB-S2,was used as the experimental strain,which was isolated from the black soil in Northeast China by our laboratory in the early stage.The whole genome sequencing of the strain DNB-S2 was finished and some key degradation genes of DBP were explored in this study.The mechanism of DBP degradation was systematically and comprehensively revealed at the genetic level.In addition,the resistance of the strain to heavy metals was also investigated,and the resistance genes were discovered,and the mechanism of cadmium resistance was elucidated at the cellular and molecular levels.Finally,based on the cytological characteristics of DNB-S2 and the changes of functional gene expression,the effect of cadmium stress on DBP degradation and the effect of DBP on cadmium resistance were studied,providing theoretical basis for the efficient bioremediation of DBP.The main results of this study are as follows:1.Firstly,the effects of culture conditions on the DBP degradation efficiencies were investigated.When the initial DBP concentration is 500 mg/L and the initial p H value of the medium is set to 5,6,7,8,9,10,the degradation rates of DBP were 4.4%,10.40%,96.39%,99.95%,99.47%,98.67%,respectively after 7-day cultivation.The optimum temperature for DBP degradation is 35?,and the degradation rate could exceed 96%.Surprisingly,at 5?and 50?,the degradation rates of DBP by strain DNB-S2 were 44.10%and 91.08%,respectively.The metabolites of DBP were detected by GC-MS,and three key compounds,n-butyl benzoate?BP?,diethyl phthalate?DEP?and mono butyl phthalate?MBP?,were identified.2.The whole genome of strain DNB-S2 was obtained successfully.The results showed that the whole genome of strain DNB-S2 was 5155736 bp long,which was composed of a circular chromosome with a length of 4725935 bp,a circular plasmid with a length of 304224 bp and six linear plasmids with different lengths.A total of 4975 protein coding sequences?CDSs?were predicted.According to the KEGG classification,overall 254 genes were related to the biodegradation and metabolism of exogenous substances,including several key genes that may be involved in the degradation of DBP,such as multiple esterase coding genes,one 3,4-dihydroxybenzoate decarboxylase?also known as protocatechuic acid decarboxylase?and nine catechol 2,3-dioxygenase coding genes.The amino acid identities of almost all esterases in DNB-S2 to that of the four previously reported esterases were all lower than 40%,indicating that the conserved amino acid sequence of esterase was low.However,the amino acid sequence identity of3,4-dihydroxybenzoic acid decarboxylase and that of cp017087.1?from Enterobacter sp.hk169?was 100%,and the identities were higher than 99%comparing with many other strains,indicating that the amino acid sequence of 3,4-dihydroxybenzoic acid decarboxylase is highly conservative.The results of subcellular localization showed that all the key enzymes were located in the cytoplasm,indicating that the degradation of DBP and its intermediate metabolites belonged to intracellular degradation.3.Strain DNB-S2 can utilize mono butyl phthalate,phthalic acid,benzoic acid,protocatechuic acid and pyrocatechol?also called catechol?as the sole carbon source.The results of real-time fluorescence quantitative PCR?RT-q PCR?showed that the m RNA expression level of 3,4-dihydroxybenzoate decarboxylase when the cells grew in DBP was 69 times?relative multiple?as much as that of citric acid as the sole carbon source.This suggested that DBP or DBP metabolites might induce the efficient expression of 3,4-dihydroxybenzoate decarboxylase.We speculated the possible metabolic pathway?catechol pathway?of DBP degradation by strain DNB-S2.In addition,a large number of other aromatic compounds such as nitrotoluene,dihydroxybiphenyl and phenol degradation genes were also annotated on the DNB-S2 genome.4.The minimum inhibitory concentrations?MICs?of copper,zinc,mercury,chromium and arsenic to DNB-S2 were 3.13,1.56,0.20,3.13 and 0.78 m M,respectively.A large number of heavy metal resistant genes were found in the genome of DNB-S2.Among them,copper resistant genes were clustered on the chromosome?junni GL000397-413?,including copper binding protein,copper resistant protein,copper oxidase,copper efflux protein,copper homeostasis protein,copper chaperone protein and other coding genes.Genes related to zinc resistance,including Znt R?belonging to Mer R family transcription regulators?,Znt B?zinc transporter?and Znt A?zinc transporter ATPase?coding genes,were basically located on chromosomes.Genes related to mercury resistance include genes encoding mercury resistance protein,mercury reductase and mercury transporter.These genes are located on circular plasmids,forming a complete mercury resistance gene cluster?junni GL004601-4607?.Chromium resistant genes include chromium transporter Chr A and chromium reductase genes,both of which are located on chromosomes.Arsenic resistant genes include Ars C,Ars H,Ars B,and Ars R family transcriptional regulators,most of which were scattered on the chromosomes and a few were scattered on the plasmid.5.Enterobacter sp.DNB-S2 was proved to be highly resistant to cadmium?MIC was 12.5 m M?when it was grown in the LB medium and the involved mechanisms were verified in this study.The results showed the maximum values of intracellular accumulated Cd²⁺contents accounted for less than 5%of the initial Cd²⁺content in the medium.The extreme low intracellular Cd accumulation reduced the risk of cadmium interfering with normal cell metabolism.TEM exhibited cell structure of DNB-S2 kept intact even at high Cd concentrations.Some precipitation particles were formed inside cells,which were further identified to be Cd-O-P,Cd?OH?₂,Cd CO₃,and Cd S by XPS.6.Furthermore,Cd resistant genes were identified on DNB-S2 genome,and the presence of 11alkaline phosphatase encoding genes provided genetic basis for the formation of cadmium phosphate precipitation,which illustrated intracellular precipitation is one of the important intracellular Cd detoxification mechanisms in strain DNB-S2.Noteworthily,8 efflux proteins were annotated possibly responsible for cadmium efflux and they were all predicted located on cytoplasmic membrane,suggesting that partial cadmium ions were discharged into the periplasm.Most alkaline phosphatases were predicted to be located in cytoplasm,only a few of them were predicted to be located in cell membrane,and only one was predicted to be located in periplasm,suggesting that cadmium phosphate mainly existsted in the cytoplasm,which is consistent with the results observed by TEM.7.When DBP and cadmium were added to the MSM medium,the hydrophobicity of the cell surface of the strain was obviously reduced,which was unfavorable to the absorption and degradation of DBP.DBP-Cd treatment increased the permeability of cell membrane,which would increase the chance of cadmium entering cells,thus reducing the resistance to cadmium.The coexistence of the two pollutants changed the composition of cell surface material,which indicated that they had a great influence on the metabolism of the strain.The activity of intracellular esterase in DBP-Cd treatment decreased obviously,which indicated that the metabolism of the strain was affected obviously.Although DBP had no significant effect on the expression of Cd efflux gene?czc D?,cadmium significantly reduced the expression of protocatechuic acid decarboxylase gene in the degradation pathway of DBP.