Research On The Mechanism Of Resistant Gene GmrA And Accessory Genes In The Biosynthesis Of Gentamicin

Gentamicin has proved an invaluable aminoglycoside antibiotic since it was discovered and isolated from Micromonospora echinospora ATCC15835 in 1963, and then first introduced into clinical use as a subtenon injection agent in 1971. Gentamicin in particular is used worldwide as a mainstay of the treatment of life-threatening sepsis cased by Gram negative bacterial infections. It also shows new pharmacodynamic activity for treatment of inherited diseases associated with premature stop codons. So far, it still plays an irreplaceable role in the clinical treatment.This study aims to verify gmr A is an essential gene in gentamicin biosynthesis at molecular level so as to set the resistance testing screening system, with which other potential resistance genes can be screen out more quickly and effectively. Then it will lay the foundation for exploring collaborative mechanisms of multi-drug resistant on aminoglycoside antibiotics.Firstly, based on the sequence of gentamicin biosynthetic gene cluster and with the help of bioinformatics analysis, it can be seen that gmr A may be a resistance gene which is involved in the biosynthetic pathway of gentamicin. To test the function of gmr A, we tried to knock out the gmr A by in-frame deletion, however, the mutant was not obtained. This result implies that gmr A is probably an essential gene in getamicin biosynthetic gene cluster. We thus doubled the gmr A in the wild type and knocked out the original gmr A again by in-frame deletion, and then the mutant can be got easily. This result shows that the gmr A is an essential gene.Secondly, in order to screen out other potential resistance genes, a rapid screening system of gentamicin resistance gene testing, which is based on E.coli is successfully established. By testing resistance candidate genes gmr B, gen P, gen H, gen I, gen V, it reveals that: 1) 16 s r RNA methyltransferase Gmr B with Gmr A both Changes in the affinity of gentamicin and ribosomal RNA of the target site; 2) Phosphorylation enzymes Gen P passivates gentamicin activity by Phosphorylating Specific sites of drugs; 3) Extracellular proteins Gen H and Gen V are responsible for extracellular transport, so as to reduce the intracellular concentration of gentamicin.The study reveales a variety of resistance genes and their functions, so as to lay the foundation of solving the problem of drug resistance of aminoglycoside antibiotics in the future.