| Dicamba is a kind of synthetic auxin herbicide.Because of its high efficiency,broad weed control spectrum,and low mammalian toxicity,dicamba is considered to be a suitable target herbicide for construction of herbicide-resistant transgenic crops.Thus,new dicamba-degrading strain,as well as gene resources have very important application in herbicide-resistant transgenic engineering.In this study,a bacterial strain 3-3 capable of degrading dicamba with high efficiency was isolated from soil sample from Nanjing.The strain was preliminary identified as Ochrobactrum sp.according to its physiological&biochemical characters and 16S rDNA gene sequence analysis.Strain 3-3 could completely degrade 100 mg/L dicamba within 48 h incubation.The optimal temperature and pH for dicamba degradation were 30? and 7.0,respectively.The high-performance liquid chromatography(HPLC)and tandem mass spectrometry analysis indicated that the first step of dicamba degradation in strain 3-3 was demethylation to generate the herbicidally inactive metabolite 3,6-dichiorosalicylic acid(DCSA).The crude enzyme of strain 3-3 displayed dicamba demethylase activity only when NADH was present.PCR amplification and bioinformation analysis results of the genome of the strain indicated that the reported dicamba demethylases genes DMO,Mtv,and Dmt or their homologous sequence were not found in the genome of the strain.These data suggested that there was a new NADH-dependent dicamba demethylase gene in strain 3-3.Ndbnu20T is a dicamba-degrading strain preserved by our lab.Research shows that cells of the strain Ndbn-20T were gram-negative,non-motile,rod shaped,lacking flagella and 0.4-0.6×0.8-1.2?m in size.Colonies were pale yellow,circular,opaque and convex with entire margins after 7 days incubation on R2A agar.The strain Ndbn-20T was negative for the test of catalase and oxidase.The strain Ndbn-20T were found to be positive for nitrate reduction to generate N2 and hydrolysis of aesculin and urea,but negative for indole production,glucose fermentation,hydrolysis of gelatin and assimilation of arabinose,mannose,mannitol,N-acetylglucosamine,maltose,potassium gluconate,adipic acid.The strain Ndbn-20T tested positive for lipase(C14),cystinearylamidase,chymotrypsin,?-galactosidase,?-galactosidase,?-glucuronidase,?-glucosidase,?-glucosidase,?-mannosidase and ?-fucosidase,but negative for alkaline phosphatase,esterase(C4),esteraselipase(C8),leucinearylamidase,valinearylamidase,trypsin,acid phosphatase and naphthol-AS-BI-phosphohydrolase.The strain Ndbn-20T produced acid from ribose,D-xylose,fructose,starch and 5-ketone-glucose.The strain Ndbn-20T resist Streptomycin,clindamycin.The predominant respiratory quinone was Q-10.The major fatty acids(>5%of total fatty acids)were C18:1?7c/C18:1?6c 46.17%,C161?7c/C16:1?6c 11.68%,C17:1 ?6c 10.96%,C16:0,10.36%? C14:0 2-OH 7.56%.The polar lipid profile consisted of diphosphatidylglycerol,phosphatidylethanolamine,phosphatidylglycerol,glycolipid,phosphatidyldimethylethanolamine,phosphatidylcholine and sphingoglycolipid.The DNA G+C mol%was 65.4 mol%.Analysis of the 16S rDNA gene sequence indicated that strain Ndbn-20T shared the highest identity with Rhizorhabdus argentea SP1T(98.8%),followed by Sphingomonas wittichii RW1T(97.9%),Sphingomonas starnbergensis 382T(97.7%)and Sphingomonashistidinilyica UM2T(97.7%).The DNA-DNA hybrid homology is 45.6±1.9%,33.5 ± 2.3%,39.4 ±3.6%,42.1±4.1%between the strain Ndbn-20T and the type strain R.argentea SP1T,S.wittichii Rw1T?S.histidinilytica UM2T?S.starnbergensis 382T,which is less than the threshold of 70%identified as a novel bacterial species.Therefore,we can conclude that strain Ndbn-20T is a novel species of the genus Rhizorhabdusy for which the name Rhizorhabdus dicambivorans sp.nov.is proposed. |
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