Classification And Identification Of Four Strains Of Rhodococcus And Their Mechanism Of Polycyclic Aromatic Hydrocarbons Degradation

Polycyclic aromatic hydrocarbons（PAHs）are organic chemical pollutants containing more than two benzene rings that are widely distributed in the environment.Because of the potential toxicity,carcinogenicity and teratogenic mutagenesis,PAHs have great potential harm to human health and ecological environment.In the previous study,four PAHs-degrading strains were screened from farmland soil in Shandong province and named NJ-11,NJ-18,NM-2 and LJ-10,respectively.In this paper,four PAHs-degrading strains isolated from shandong farmland soil were classified and identified,and the pathway of PAHs-degrading naphthalene and biphenyls was analyzed by whole-genome annotation method,and the genome analysis of four Rhodococcus was carried out,and the following results were obtained:1.The sequence analysis of 16S r RNA gene of strain NJ-11,NJ-18 and Rhodococcus pyridinvorans PDB9^Tshowed that the similarity of 16S r RNA with R.pyridinvorans PDB9^Twas 100.0%and 99.6%respectively;The ANI values of R.pyridinvorans PDB9^Twith NJ-11 and NJ-18 were 99.1%and 98.4%respectively,and the d DDH values were 94.3%and 89.6%respectively.The two strains were identified as R.pyridinvorans PDB9^T.Strain LJ-10 is also clustered with R.pyridinvorans PDB9^T,and the similarity of 16S r RNA with R.pyridinvorans PDB9^Tis also the highest,with 99.7%,but the ANI and d DDH between the two strains are94.5%and 61.3%respectively.In addition,there are also differences in physiology and biochemistry between the two.The similarity of 16S r RNA between strain NM-2and R.jostii IFO 16295^Twas 99.5%;The ANI and d DDH values of R.jostii IFO16295^Tand R.opacus DSM 43205^Twere 41.8%and 88.6%,62.5%and 93.7%respectively.Strains LJ-10 and NM-2 are quite different from all existing standard strains,and meet the standard of new species.LJ-10 and NM-2 are identified as two new species of Rhodococcus respectively.2.Strain LJ-10 can grow with biphenyl as the only carbon source,and can grow within 72h at 2%inoculation amount.The degradation rate of 200 mg/L biphenyl was92.8%,and the maximum value of OD₆₀₀reached about 0.3966;strain NJ-11,NJ-18and NM-2 can grow with naphthalene as the only carbon source,in which strain NJ-11 can degrade 200 mg/l naphthalene within 48 hours,with a degradation rate of94.1%,and the maximum value of strain OD₆₀₀is 0.1970;strain NJ-18 can degrade200 mg/l naphthalene within 48 hours,the maximum degradation rate is 99.6%,and the maximum value of OD₆₀₀is 0.1245;strain NM-2 can degrade 200 mg/l naphthalene within 48 hours,and the maximum degradation rate is 96.4%.The maximum OD₆₀₀of strain growth OD₆₀₀is 0.1789.3.Genome analysis showed that strain LJ-10 catalyzed biphenyl to produce2,3-dihydro-2,3-dihydroxybiphenyl by biphenyl 2,3-dioxygenase,and then transformedinto2,3-dihydroxybiphenylunder the actionof2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase;Under the action of2,3-dihydroxybiphenyl 1,2-dioxygenase,2,3-dihydroxybiphenyl was further converted to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid;Under the action of hydrolase,2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid generates benzoic acid and enoate.Under the catalysis of benzoic acid 1,2-dioxygenase,benzoic acid is converted to 1,6-dihydroxy-2,4-cyclohexene carboxylic acid,and then converted to catechol under the action of dehydrogenase.Catechol is decomposed into myxofulvic acid under the action of diol ring cleavage dioxygenase;Mucofuranic acid is converted into mucofuranic lactone by isomerase;Mybranic acid lactone is againδ-Under the action of isomerase,it is transformed into 2-adipate ketene lactone,and then enters the tricarboxylic acid cycle,finally realizing the degradation of biphenyl.4.Strains NJ-11,NJ-18 and NM-2 catalyze naphthalene to produce cis-naphthalene dihydrodiol by naphthalene dioxygenase small subunit,and then convert it to L,2-dihydroxynaphthalene under the action of cis-naphthalene dihydrodiol dehydrogenase;Under the action of L,2-dihydroxynaphthalene 1,2-dioxygenase,it is converted to 2-hydroxy-4-（6-oxo-2,4-cyclohexadienyl）-3-butenoic acid.The hydrolase converts 2-hydroxy-4-（6-oxo-2,4-cyclohexadienyl）-3-butenoic acid to catechol and 2-hydroxy-3-butenoic acid.Catechol is converted to myxofulvic acid under the catalysis of catechol dioxygenase,and myxofulactone under the action of myxofulvic acid isomerase;Mucofuranic acid lactone in muconic acid lactoneδ-Under the action of isomerase,it is converted into 2-adipate ketene lactone,and then enters the tricarboxylic acid cycle.Finally,naphthalene can be degraded.