Research of Possibility the Transformation and Resistance Na-Tellurite Originated Plasmide at the Soil Bacteria Isolated From Natural Environment

Abstract

Bu çalısmada Na-tellurit bilesiginin toprak bakterilerinden alkalifilik Bacillus spp. suslarında inhibitör etkisi arastırıldı. Bakteriler Na-tellurit bilesigine 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 175, 178 ve 180 ?g/ml artan konsantrasyonlara denendi. zolatların 1, 5, 10, 20, 30, 40, 50 ve 60 ?g/ml konsantrasyonlardaki Na-tellurit denemesinde çogunlukla dirençlilik gösterirken 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 178 ve 180 ?g/ml olarak denenen konsantrasyonlarda ise hassasiyet profili gösterdigi saptandı. Aynı sekilde 60, 70, 80, 90, 100, 120, 130, 150 ve 160 ?g/ml konsantrasyonlarda da plasmid kodlu dirençlilik gözlendi. 80 ?g/ml konsantrasyon denemesinde en fazla plasmid kodlu dirençlilik gösteren 5 ayrı sus belirlendi. zolatlar için MICs degeri ve 0.8 ?g/ml olarak belirlendi. Diger parametrelerde ise plasmid kodlu dirençlilige rastlanmadı. 180 ?g/ml konsantrasyon denemesinde de denenen hiç bir izolatın üremedigi ve %100 hassasiyet gösterdigi bulundu. zolatların Na-tellurit bilesigine ve Chloramphenikol antibiyotigine plasmid kodlu dirençliliginin saptanması için Acridine orange (20 ?g/ml)ve Ethidium Bromid (10-5 M) ile plasmid eliminasyonu yapıldı. zolatlarda Bacillus spp.'ye ait %60.86 Na-tellurit bilesigine hassasiyet, %39.13 dirençlilik fenomeni ve %21.10 oranında plasmid kodlu Na-tellurit dirençliligi bulundu. Na-tellurit bilesigine ve antibiyotige karsı dirençlilik genini tasıyan plasmidler (B. cereus T112-Na-TeR), Na-tellurit bilesigine ve antibiyotige hassasiyet gösteren susa (B. megaterium DSM 32-Na-TeS)transformasyonu yoluyla aktarıldı. zolatları arasında protoplast transformasyon yoluyla 108 transformant elde edildi. Agaroz jel elektroforezde seperasyonu yapıldı. Na-tellurit bilesigine ve antibiyotige dirençli suslardan izole edilen plasmidin 22.514 bp moleküler agırlıga sahip oldugu saptandı. Anahtar Kelimeler: Na-tellurite, Alkalifilik Bacillus spp. Plasmid, Protoplast Transformasyonu iIn this study, researched the inhibitor effect at the Bacillus spp.strains from the soil bacteria of Na-tellurit compound. Bacteria treatmented at the remaining concentrations 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 175, 178 ve 180 ?g/ml the Na-tellurite compound. Isolates which was at the experiment Na-tellurite in the 1 5, 10, 20, 30, 40, 50 ve 60 ?g/ml concentrations mostly showed resistant but determined the sensitiveness profile at the its experiment concentration as 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 175, 178 ve 180 ?g/ml Na-tellurite compound. In the same way we observed resistance originated plasmid at the 60, 70, 80, 90, 100, 120, 130, 150 ve 160 ?g/ml concentrations. At the experiment of 80 ?g/ml concentration determined five different satrains showing most resistance originated plasmid. At the other parameter wasn?t happen at the same and at the experiment of 180 ?g/ml concentration weren?t growth of any izolate too. However they determined to showing %100 sensitiveness. For Bacillus spp. determined MICs of 0.8 ?g Na-tellurit. Of present izolate were maked to plasmid eliminate with for determine of the resistance originated plasmid with acridine orange (20 ?g/ml) and ethidium bromid (10-5 M) with plasmid eliminated Na-tellurite compound and Chloramphenicol antibiotic. At the isolates were determined the Na-tellurite resistance originated plasmid at the %21.10 ratio and the %39.13 resistance phenomenon, sensitive the % 60.86 Na-tellurite compound belong to Bacillus spp.. Plasmids B. cereus T112-Na-TeR carriering the resistance gene against to antibiotic and Na-tellurite compound were transfered by way of protoplast transformation to B. megaterium DSM 32-Na-TeS strain showing sensitiveness to antibiotic and Na-tellurit compound. From the soil izolates, 108 transformants via protoplast transformation were obtained. The seperation of plasmid was made in the agarous gel electrophoresis after the isolation. It was determined that the plasmids isolated from the strains resistant to Na-tellurit and antibiotic had 22.514 bp molecular weight. Key Words: Na-tellurite, Alkalophilic Bacillus spp. Plasmide, Protoplast Transformation