| Nowadays,antibiotics and resistance genes are one of the most concerned emerging pollutants in the 21st century,and among the common antibiotics,sulfonamides(SAs)have attracted much attention because of their tremendous usage and high detection rates in natural water.Bacterium has been widely used in the removal of organic pollutions due to its rapid propagation,less influenced from the environment,and no secondary pollution.The main contents of this paper include the screening of SAs bio-transforming and biodegrading bacteria strains,SAs bio-transforming and biodegrading pathways metabolized by different bacteria,the molecular mechanism of SAs biodegradation and bio-transformation,and the degradation mechanism of mixed bacteria.The details are as follows:1.Screening and identification of SAs transforming/degrading strainsWith sulfamethoxazole(SMX),the most commonly detected SAS antibiotics,as the only carbon source,and estuarine water and sediments with rich microbial resources as the source of strains,three strains with SMX transformation/degradation ability were screened,which could adapt to freshwater river seawater environment.They were Vibrio diabolicus strain L2-2,Aeromonas caviae strain GLB-10 and Catellibacterium terrae strain MMR-2-1,and the degradation rates of SMX were27.79%,15.85%and 9.96%,respectively.2.Researches of bio-transformation pathway and bio-transforamtion efficiency of Vibrio diabolicus strain L2-2 to sulfonamidesA marine bacterium Vibrio diabolicus strain L2-2 was isolated which could metabolize 9 selected SAs to different extents.What's more,compared with SAs containing phenylamino group or p-aminobenzene sulfonamide and its analogs,SAs with N-oxides of heterocyclic structure were more easily to be transformed to their N~4-acetylated metabolites or their isoxazole ring rearrangement isomers by strain L2-2.Multiple SAs antibiotics could be metabolized simultaneously by strain L2-2,and higher bio-transformation rates were observed in five SAs including sulfathiazole(STZ),sulfamonomethoxine(SMT),sulfadiazine(SDZ),sulfamethoxazole(SMX)and sulfisoxazole(SIX),reaching 29.39±5.63,24.97±4.45,79.41±4.05,64.64±1.71,32.82±4.46%in 6 days,respectively.Besides,influential factors on SAs bio-transformation were investigated and the results showed that irrespective of the slight difference among the five SAs in response to influential factors,the overall optimal conditions for SAs bio-transformation were determined to be less than 100mg/L for total SAs in neutral or weakly alkaline medium with the salinity of 10-20‰and additional nutrients like glucose,sucrose or glycerine.Furthermore,the toxicity was demonstrated to be significantly reduced after bio-transformation,suggesting that the N~4-acetylated metabolites showed less toxicity to Escherichia coli,so the bio-transformation of strain L2-2 to SAs could effectively reduce their toxicities.3.The molecular mechanism of Vibrio diabolicus strain L2-2 bio-transforming SMXThe potential resistance and bio-transformation mechanism of strain L2-2 to SMX of antimicrobial concentration(10 mg/L)were studied by comparative transcriptome analysis and related genes responses to SMX of environmental concentrations(0.1-10?g/L)were researched by RT-qPCR.According to the significantly enriched KEGG pathways analysis,pathways of L-Leucine,L-Isoleucine,and fatty acid metabolism were significantly affected,and they all would decrease acetyl-Co A.The resistance mechanism could be summarized as follow:the enhancement of membrane transport,antioxidation,ribosome protection and over-expression of response regulator and repair proteins.The key genes of SMX bio-transformation might be gene nat,cyc-355,and acs.When strain L2-2 exposed to SMX of environmental concentrations,SMX hydroxylation related gene cyc-553significantly up-regulated under SMX stress of 1?g/L,however,SMX acetylation related gene nat might not be activated.Moreover,the membrance tranportion and antioxidant activity of strain L2-2 were also activated under SMX stress of 10?g/L.There also found some different responses comparing to the antimicrobial concentration.The drug-efflux pump might be inhibited under SMX stress of environmental concentrations,which were significantly enhanced under antimicrobial concentration.Furthermore,SMX of the environmental concentration would cause strain L2-2 to produce more energy to keep normal physiological activity,while SMX of antimicrobial concentration would suppress some metabolites to survival.These results provided a better understanding of resistance and bio-transformation of bacteria to SMX and would support related researches about the impacts of environmental antibiotics.4.Cloning and expression of arylamine N-acetyltransferase(VDNAT),the key enzyme of Vibrio alginolysis strain L2-2 bio-transforming SMXAccording to the genome analysis and RT-qPCR verification,gene sequence of VdNAT was identified which was the key enzyme of strain L2-2 bio-transforming SMX,and VdNAT was cloned and expressed in Escherichia coli.The VdNAT-positive clones could transform SMX,and with addational acetyl-Co A,almost 100%transformation rate achieved,which confirmed that acetyl-Co A might be a key factor limiting the transformation of SMX by VdNAT.VdNAT had the highest homology with NAT of Salmonella typhimurium,and also had higher homology with NAT1 and NAT2 of human according to amino acid sequence alignment.Meanwhile,the predication of 3D structure showed that VdNAT formed a deep and wide active site pocket,which was conducive to the binding of acetyl-Co A.This specialized protein structure might be the reason for the higher SMX transforming ability of VdNAT.5.The biodegradation pathway and biodegradation ability of Aeromonas caviae strain GLB-10 to SMXAeromonas caviae strain GLB-10 was isolated which could degrade SMX,but the biodegradation rate of strain GLB-10 to SMX was relatively low,which was 17.46±0.57%in 3 days.The main products of SMX degraded by strain GLB-10 were aniline(AN)and 3-amino-5-methylisoxazole(3A5MI),p-aminobenzene sulfonamide(SA)with low yield was also detected.The possible degradation pathway was that4-amino-N-hydroxy-benzene sulfonamide(4ANHs)was generated by the broken of N-C bond due to the hydrolyzation of the linear terminal of SMX,and 4ANHs was dehydrated to SA,then SA or 4ANHs was desulfurized to AN by sulfur reduction 4S pathway(the broken of C-S bond).At the same time,the amino side chain of SMX was fragmented to 3A5MI.6.The biodegradation pathway,biodegradation effect,and key proteins prediction of mixed bacteria to SAsThe mixed bacteria could degrade SAs with high-efficiency,and strain L2-2 and GLB-10 were both isolated from the mixed bacteria.The degradation pathway of SMX by mixed bacteria combined L2-2 and GLB-10 pathways,and the main products were AN and 3A5MI,N4-acetylated SMX and hydroxylated SMX were also detected.And meanwhile,Acetanilide and hydroquinone were also detected which were not detected in the single bacteria degradation process.The mixed bacteria had a higher degradation rate to SAs with nitrogen heterocycles,and a lower degradation rate to sulfanilamide and sulfacetamide which did not have nitrogen heterocycles,and the mixed bacteria could not degrade AN.The mixed bacteria could completely degrade 250 mg/L SMX in 3 days,and the degradation rate was not affected by pH,but the high salinity would reduce the degradation ability of SMX.Through the identification of differentially expressed proteins,it was found that the expression of outer membrane protein and peroxidase were significantly up-regulated,which meant mixed bacteria could resist SMX stress by regulating membrane proteins and enhancing the antioxidant system.Besides,6-hydroxyniacin-3-monooxygenase and ammonooxygenase were up-regulated,in which 6-hydroxyniacin-3-monooxygenase might be involved in the degradation of SMX,while ammonooxygenase maybe related to the co-metabolism of mixed bacteria.The mixed bacteria also could degrade SMX in actual water samples and could adapt to a variety of water environments,such as rivers,lakes,estuarine and seawater,so it has a high application prospect in environmental SAs pollution treatment.The research expanded the bacteria strains capable of transforming or degrading sulfonamides,and provided the theoretical basis and excellent strains for the mechanism exploration and practical application of environmental sulfonamides antibiotics pollution removal,through the screening of sulfonamides metabolic(transformation/degradation)strains,the study of single and mixed strains on sulfonamides metabolic pathway,transformation/degradation performance and metabolic molecular mechanism. |
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