Degradation Of Kanamycin At High Concentration By Two Gram-negative Bacterial Strains

Kanamycin is one of the most commonly used aminoglycoside antibiotics.Aminoglycoside antibiotics,which also include streptomycin,gentamicin,neomycin,paromomycin,spectinomycin,tobramycin,ribostamycin,netilmicin,amikacin,isepamicin,micronomicin and etimicin,are glycoside antibiotics having an aminocyclitol nucleus(streptamine,2-deoxystreptamine,or streptidine)linked to amino sugars through glycosidic bonds.Because of their low price,broad spectrum activity against gram-negative bacteria and synergistic effect with other antibiotics,aminoglycosides are widely used in the treatment of infections caused by gram-negative aerobic bacilli;in addition,they also commonly used in veterinary drugs or as husbandry growth promoters.However,these antibiotics are very stable and difficult to decompose,resulting in a raising concern about the wide occurrence of antibiotic resistance bacteria(ARG)and a potential healththreat risk for their ototoxicity and nephrotoxicity.At present,there is a lack of safe and effective treatment method for different environmental media containing aminoglycoside antibiotic residues,such as wastewater and bacterial residue produced during the production of kanamycin and waste containing kanamycin residue produced by animal husbandry.In our previous work,we fortunately obtained two gram-negative bacteria exhibiting high kanamycin-degrading capability,designated Paracoccus kondratievae A2 and Aquamicrobium sp.A3.In this paper,the degradation mechanism of kanamycin in these two strains was studied,in order to provide theoretical basis for the development of environmental and feasible kanamycin-degrading methods.In addition,given that bacterial degradation of antibiotics is closely related to their drug resistance,the study in this paper can also reveal the mechanism of kanamycin resistance in Paracoccus kondratievae A2 and Aquamicrobium sp.A3,which has certain medical value.The results obtained were listed as follows:(1)Both strain A2 and A3 can tolerate kanamycin in high concentration(>100 g/L);in addition,strain A2 can use kanamycin as the sole carbon and nitrogen source for growth.The two strains could degrade kanamycin,the aminocyclitol-4,6-amino sugar,into aminocyclitol-6-amino sugar.The result suggested a novel mechanism other than enzymatic modification at-OH or-NH₂ groups of the2-deoxystreptamine nucleus or the sugar moieties by aminoglycoside modifying enzymes(AMEs).(2)Kanamycin lost its bacteriostatic activity after degradation by strain A2 or A3;in addition,the cytotoxicity of the degradation products was significantly reduced compared with the same concentration of maternal kanamycin.The result signified that kanamycin removal with the degradation enzymes from strain A2 or A3 is feasible and safe.(3)Strain A3 differentiates into a phenotype that does not degrade kanamycin,and the strain capable of degrading kanamycin was renamed as A3-1 while the one uncapable of degrading was renamed as A3-6.From the genomes of strain A3-1 and strain A3-6,we found that A3-1 plasmid contains complete genetic information,and A3-6 plasmid 5 is the product of A3-1 plasmid missing four fragments.One or both ends of these missing segments contain transposase.The results indicated that gene loss is probably responsible for the non-degradation of kanamycin phenotype in strain A3-1.In addition,the genes that encode enzymes for catalyzing the breaking of 4-site glycoside bond to form kana-1 may be located in these missing fragments.(4)Kanamycin degrading enzymes were isolated and purified from the bacterial body breaking liquid of strain A2 and A3-1 by ammonium sulfate precipitation,ion exchange and native-page.The size of A2-protein 1 isolated from strain A2 and A3-protein 1 isolated from strain A3-1 was about 60 KDa,while the size of A2-protein 2 isolated from strain A2 and A3-protein 2 isolated from strain A3-1 was about 20 KDa.From the results of N-terminal sequencing,we found that the gene coding for A2-protein 1 is pka2 2598,while the gene coding for A3-protein1 is Aqu A3.1 0009;in addition,pka2 2598 and Aqu A3.1 0009 are the same gene encoding a glucose-methanol-choline(GMC)oxidoreductase.Similarly,the gene coding for A2-protein 2 is pka2 2599,while the gene coding for A3-protein 2 is Aqu A3.1 0010;in addition,pka2 2599 and Aqu A3.1 0010 are the same gene encoding twin-arginine translocation pathway signal.The Aqu A3.1 0009 and Aqu A3.1 0010 genes are located in the A3-1 plasmid 1 and are missing in the corresponding A3-6 plasmid 5.This result further indicated that pka22598(Aqu A3.1 0009)and pka2 2599(Aqu A3.1 0010)genes are key enzymes in the degradation of kanamycin into kana-1.Then we cloned genes pka2 2598 and pka2 2599 into E.coli BL21(DE3)for heterologous expression,and the results showed that coexpression genes pka2 2598 and pka2 2599 in an E.coli BL21(DE3)cell can degrade kanamycin into kana-1;however,the enzyme expressed by pka2 2598 or pka2 2599 alone or the enzyme mixture of PKA2 2598 and PKA2 2599 failed to degrade kanamycin into kana-1.In addition,no additional cofactors are required for the reaction,but artificial electron receptors such as phenazine methyl sulfate(PMS),2,6-dichloroindophenol sodium(DICIP)or potassium ferricyanide are required.(5)The pka2 2599 gene encodes twin-arginine translocation pathway signal, suggesting that PKA2 2598 and PKA2 2599 protein complexes may be secreted into the periplasmic space or medium through the Tat transport system.By analyzing the genomes of strain A2 and A3-1,it was found that both strain A2 and A3-1 have complete Tat transport system,which implied that both strain A2 and A3 can transport PKA2 2598/PKA2 2599 complex to the periplasmic space or medium.Given the inactivity of the culture supernatant fluid of strain A2 and A3-1,the PKA2 2598/PKA2 2599 complex was most likely be secreted into the periplasmic space for degradation.Based on these results,the proposed model for the strong kanamycin degradation in strain A2 can be described as follows:kanamycin enter into periplasmic space and be decomposed into kana-1 and cryptic compound(s)by PKA2 2598/PKA2 2599 complex,the cryptic compound(s)is(are)used as nutrients for growth of strain A2 and kana-1 was excreted from periplasmic space into medium.