The Degradation Pathway Of Chloramphenicol By Sphingobium Sp. CAP-1 And The Catalytic Properties Of Key Degradative Enzyme

Chloramphenicol（CAP）,a halogenated nitroaromatic antibiotic,can cause aplastic anemia.In addition,CAP can inhibit the diverse metabolic function of activated sludge microbial communities in wastewater treatment system.At present,CAP was still widely detected in the natural environment and organisms（0.07-47400 ng/L）.ARGs related to CAP also appeared in a variety of environmental samples.Therefore,the deep removal of CAP,the identification and minimization of ARGs transmission or amplification is urgent.Microorganisms are the main drivers of degradation and transformation of emerging risk pollutants such as antibiotics/antimicrobial agents.However,few bacteria with the capable of efficiently degradaing/mineralizing CAP has been isolated,a complete CAP catabolism pathway has yet to be proved and the related molecular mechanism has not been clear.Meanwhile,it is in press for exploring the molecular markers of metabolic activity to identify and evaluate the catabolism/resistance of CAP.The purpose of this study is to deeply explore novel high-efficiency CAP-degradaing/mineralizing bacteria from the activated sludge of sewage treatment plant and systematically study the biodegradation process by these bacteria to propose a relatively complete CAP catabolism pathway by the interdisciplinary methods.Moreover,the key enzyme genes involved in the detoxification and degradation of CAP will be cloned and characterized in order to obtain a clear corresponding molecular catalytic mechanism.Due to the common detection of CAP and ARGs in water environment,an efficient CAP-mineralizing consortium was obtained and named DCAP,which is capable of completely degrading 50 mg/L CAP within 24 h and subsisting on CAP as the sole carbon,nitrogen and energy sources under aerobic conditions with the activated sludge from sewage treatment plant as the initial inoculum.During the degradation of CAP,TOC and Cl^-were removed and released respectively.With the repression of addtional carbon source glucose,CAP degradation efficiency was significally decreased at the initial stage.P-nitrobenzoic acid（PNBA）during CAP degradation was capable of providing carbon and nitrogen sources for DCAP,and 25 mg/L PNBA was completely degraded by DCAP within 24 h.Accoding to high throughput sequencing analysis,Sphingobium was the most dominant genus in DCAP microbial community with the relative abundance ranged from 63.4%to69.2%.Most of the bacteria in the enriched microbial communities can degrade aromatic compounds including PNBA,suggesting that these dominant bacteria are potentially involved in the catabolism of CAP.In the case of 152 times-passaged,DCAP can still maintain efficient CAP degradation efficiency and microbial community stability.In the case of the lack of CAP mineralizing/degrading bacteria resources,Sphingobium sp.CAP-1 was isolated from DCAP and shown the outstanding ablility of completely degrading 50 mg/L CAP within 30 h under aerobic conditions.Compared with DCAP,strain CAP-1 can also subsist on CAP as the sole carbon,nitrogen,and energy sources and CAP degradation efficiency was repressed by glucose within 24 h as well.In addition to PNBA,p-nitrobenzaldehyde（PNBD）,protocatechuate（PCA）,and a novel side chain C₃-hydroxy-oxygenated product of CAP（named O-CAP）were identified by nuclear magnetic resonance（NMR）and high-resolution mass spectrometry during CAP degradation.Strain CAP-1 can convert the oxidative product O-CAP to PNBA.PNBA was further converted to PCA which could be ring-cleavaged to formed oxaloacetic acid and alanine into tricarboxylic acid cycle（TCA）with CO₂ and H₂O as the final products.According to genome sequencing and comparative proteomic analysis,all functional gene clusters involved in the catabolism of PNBA towards TCA,the initial CAP oxidase gene（cmo）catalyzing CAP to O-CAP,and the complete gene cluster of floc formation and regulation were located and identified.Based on the high-throughput omics information,physiological and biochemical data,and analytical chemistry data of CAP degradation products,a novel CAP catabolism pathway mediated by the initial oxidation reaction was proposed.Considering the unclear molecular mechanism of CAP degradation and the lack of metabolic molecular markers,an oxidase Cmo which is responsible for catalyzing CAP to O-CAP was firstly cloned and biochemically characterized.Cmo shared low amino acid similarity（41.05%）with the glucose-methanol-choline oxidoreductase family.The optimum temperature and p H of catalytic reaction by Cmo were 30℃and 7.4,respectively.In the range of 10-30℃or under the neutral condition,the stability of Cmo remained the highest.1 m M Co²⁺,Cr³⁺,Cd²⁺,Hg²⁺and Ag⁺can obviously inhibit Cmo activity.While Zn²⁺and Mg²⁺had a weak promoting effect on Cmo activity.All the chemical inhibitors and detergents had different inhibitory effects on Cmo.Among them,1 m M iodoacetamide,nitrilotriacetic acid（NTA）,phenylmethanesulfonyl fluoride（PMSF）,diethyl pyrocarbonate（DEPC）and 0.1%cetyltrimethylammonium bromide（CTAB）inhibited Cmo activity seriously,with the inhibition efficiency of 85.23%-98.30%.Cmo kinetic parameter,K_m and k_cat was 183.94±11.20μM and125.48±7.63 s^-1,respectively.Based on homology modeling and molecular docking,the active amino acid sites of oxidase Cmo binding to CAP were located:Ala344,Tyr380,Met474,Gly99,and Asn518.This study provides the efficient bacterial degradative/mineralized resource and theoretical guidance for the microbial enhanced treatment of CAP-polluted environment,and provides an indicator gene for the microbial mediated fate and transformation activity of CAP in the actual environment.