Screening And Analysis Of Tetracycline Resistance Genes From Soil Microbiomes

As its important role in therapy and control in bacterial infectious diseases,antibiotics are now recognised as one of the most important discoveries in 21st century.In recent years,antibiotics are widely unreasonable used and overused in clinic and animal husbandry,which result in the wide spread of antibiotic resistant bacteria and antibiotic resistance genes in global world.These antibiotics resistance genes(ARGs)can transmit among human,animal and environment via horizontal gene transfer,which caused environmental and food safety problems.Today,most researches are mainly focused on clinical bacteria isolates to discover ARGS,but it showed that ARGs which identified from clinical bacteria isolates are greatly originated from soil microbiomes.Thus,vast majority of unknown ARGs in soil microbes are unexplored by the traditional method.It is known that microbes in soil are enormous and varied,but only less than 1%of species could be cultured by standard laboratory techniques,and a large majority of bacteria cannot be cultured.With the emergence of metagenomics,this problem can be sloved.Metagenomics,by using the culture-independent method,can study the whole DNA derived from environmental bacteria,including cultured and uncultured bacteria,which offered an effective approach to reveal novel resistance genes.The tetracyclines is one of the most widely used classes of broad spectrum antibiotics in clinic and agriculture in the past years,as its low cost,oral administration and few side effects.After several decades of extensive use of tetracyclines,the prevelance and dissemination of tetracycline resistances have alarmingly increased.To date,about several tens tetracycline resistance genes have been identified in clinic.In this thesis,novel tetracycline resistance genes were identified from three soil metagenomic DNA libraries by functional metagenomics.It is important in controlling the transmitting of ARGs in clinic.In this research,we identify novel tetracycline resistance genes by screening three metagenomic DNA libraries,including Yunnan,Tibet,and Sichuan libraries,via functional metagenomics.Sequences were analyzed by bioinformatics.The results demonstrate that the deduced eDNA derived amino acid of efflux pump transporters show the amino acid 80%-90%identity to those found MFS transporter.However,the tetracycline-inactivating enzymes,which show low identity(20%,21%)to Tet(X),the only one confirmed tetracycline inactivation enzyme from clinical bacterium,could be considered as novel tetracycline-inactivating enzymes.Then we verify the function of tetracycline enzymatic inactivation genes in vitro by doing biochemical reactions.The results are as follows:1.We obtained 24 positive clones by screening three soil libraries.By analyzing the sequences,we found that the 22 clones of tetracycline resistance genes showed homology to previously reported tetracycline transporters,including 21 major facilitator superfamily(MFS)transporters,1 multidrug and toxic compound extrusion(MATE)family efflux and 2 novel tetracycline-inactivating enzymes,demonstrating that a wide distribution of tetracycline resistance genes in the area and diverse soils.The 21 MFS transporter show 40%-60%identity to each other and 80%-90%identity to those found MFS transporter genes,and among them,YN1469 from Yunnan library showed identity 98%to the MFS transporter from Burkholderia sp.BT03.The MFS transporters SC6 and SC2451 from Sichuan library shared the same amino acid sequences with YN2306 and YN1469 from Yunnan library,respectively,implying a horizontal transfer among antibiotic resistance genes.A putative MATE efflux protein showed 59%identity to a MATE efflux from cidobacteria bacterium OLB17,and it conferred resistance only to tetracycline.The two tetracycline-inactivating enzymes MQ776 and MQ1435 shared 73%amino acid identity with each other and the same MIC(64?g/mL)to tetracycline.They showed low identities of 20%and 21%,to Tet(X),respectively,and could be considered as novel tetracycline destructases2.The two tetracycline-inactivating enzymes were heterologously expressed in E.coli BL21,and the proteins were purified for analysis of the activities of enzymes in vitro.The progressions were monitored by reverse-phase HPLC.We found that the substrate was degraded and the new product was observed,suggesting the destruct activities in vitro.The substrate was degraded and new stable product was observed,but the amount was not accumulated in case of MQ776 with time going.In case of MQ1435,the substrate was degraded and new unstable product was observed.Then the new products of this reaction were monitored by LC-MS,the m/z showed 461.20.We determined the optimal pH value of inactive reaction.The results showed that the reaction product of MQ776 was stable under the condition of weak acid or alkalinity,but the amount was not accumulated and the reaction product of MQ1435 was unstable under the condition of alkalinity,but it was stable under the condition of acid and the amount was not accumulated.So we conclude that the mechnism of two tetracycline-inactivating enzymes MQ776 and MQ1435 was different.We identified novel tetracycline resistance genes from soil microbes by functional metagenomics,which contributes to understand the mechanisms of tetracycline resistance.