| Tetracyclines antibiotics are broad-spectrum antibiotics with low price which was produced by actinomycetes.It is one of the largest types of antibiotic and widely used in the world.The tetracycline antibiotics residues in the environment may lead to ecological risk and potential toxic effects due to its large dosage and most of them are discharged in the form of the parent compounds.The continuous accumulation of tetracycline residues in soils poses a great threat to ecosystems and thus attracts extensive attention recently.Therefore,isolate a microbial which can degradation tetracycline and get clear the mechanism of degradation,can lead to better estimation of the fate and transport of antibiotics in the environment and has the potential to be utilized in designing engineering processes to remove tetracycline from water and soil.This work aims to isolate tetracycline degrading bacterial,characterize the biotransformation of tetracycline by degrading strain,analysis the key proteins in the process of biotransformation tetracycline and clear the role of them in metabolism,gene and protein levels.Reveal the mechanism in biotransformation of tetracycline and provide scientific support for the bioremediation of tetracycline in soil and water.The main results were as follows:One tetracycline degrading bacterial strain DT1 was isolated from long-term contaminated soil by tetracycline.DT1 was identified as a Stenotrophomonas maltophilia according to morphological characteristics,physiological characteristics and16 S rRNA homology analysis.The single factor experiment on the growth conditions of DT1 showed that the DT1 was a mesophilic bacteria.It can grow well in 30 oC,pH 7-10,liquid volume 25-50mL/250 m L.The effects of inoculation size,liquid volume,culture temperature,initial pH,and degradation time on the degradation of tetracycline by DT1 were studied.The results showed that inoculation size and liquid volume had no significant effect on the degradation of tetracycline.The biotransformation rate was the highest when the initial pH was 9 and the reaction temperature was at 30oC.Both the overall degradation process and the hydrolysis process of tetracycline can be described using first-order kinetics.The change of tetracycline concentration due to biotransformation equals the observed overall degradation（experiments containing the bacterial strain DT1）minus the change of tetracycline concentration due to hydrolysis（control experiments containing no bacterial cells）.The progress under different initial tetracyclineconcentrations can be described using the Michaelis-Menten model.The vmax and were62.14 mg L-1d-1 and 80.52 mg L-1 respectively.The biotransformation products exhibited lower antimicrobial potency than the parent compound by the antibacterial activities test of tetracycline transformation products.Two hydrolysis product（ETC or ISO-TC）and six possible biotransformation products（TP 431,TP 415,TP 387,TP 370,ISO-TP 415,and ISO-TP 387）were also identified and a potential biotransformation pathway was proposed that included sequential removal of N-methyl,carbonyl,and amine function groups.The whole genome of Stenotrophomonas maltophilia DT1 was sequence by Illumina HiSeq X Ten system.The draft genome sequence was obtained by filtering and joining the original data.It consists of 41 scaffolds included 4,532,597 bp.There were71 RNA,10 rRNA and other 43 non coding RNA.The GC content was determined to be 66.48%.There were 4052 predicted open reading frames（ORFs）.Out of all ORFs,2,787 could be functionally annotated.A total of 1153 hypothetical proteins were predicted for S.maltophilia DT1.Fifty seven ORFs were predicted to encode for proteins related to resistance to antibiotics,such as aminoglycoside,fluoroquinolone,and beta-lactam.Among them,forty ORFs encode multidrug efflux pumps and twelve encode beta-lactamase.Twelve other ORFs encode parts of cation/metal efflux pumps for heavy metals such as cobalt,zinc,cadmium,copper and arsenic.Twenty one ORFs encode oxygenases,including eight ORFs encoding monooxygenases,eleven encoding dioxygenases and two encoding oxygenases which may be involved in the metabolism of enobiotic.Eight peroxidase genes were also identified in the draft genome.In order to investigate the molecular mechanisms of tetracycline resistance and biotransformation by Stenotrophomonas maltophilia DT1.Quantitative proteomic analyses suggest that seven proteins involved in repair and detoxificationfour of cell.Four proteins in DT1 might have been involved in tetracycline resistance: nodulation protein transported tetracycline outside of cells;HXGPT facilitated the activation of the ribosomal protection proteins to prevent the binding of tetracycline to the target site on the ribosome;and superoxide dismutase and peroxidase transformationed tetracycline.By comparing the fold changes under different nutrition conditions,it is showed that the biotransformation rates of tetracycline matched the expression levels of superoxide dismutase under the background nutrient conditions tested.The superoxide dismutase maybe a rate limiting protein for tetracycline degradation.Under starvationconditions,the expression of protein related to tetracycline resistance and detoxification related decreased.The cells can maintain the survival and integrity of the outer membrane through the consumption of endogenous nutrients,in order to resist external pressure.Tetracycline degradation cannot be carried out at this condition.This study proposed a kinetic model for microbial degradation of tetracycline and also suggested a biotrasformation pathway.Combine the previous reports and the conclusions of this research,proposed the molecular mechanism of microbial degradation of tetracycline. |
