A molecular study of the copper resistant genes in the microbial population of industrial bioreactors

[摘要] Copper ions play an essential role in many biological systems but above optimumconcentrations it exerts toxic effects. Some micro-organisms mediate the toxic effects ofcopper through resistance mechanisms the organisms possess. These vary with theresistance systems.Microbial diversity studies for 37°C and 70°C bioreactors were done to characterize theorganisms present in the industrial copper bioreactors used for bioleaching of copperfrom its ore. After 16S rDNA amplification both reactors communities were defined. TheBLASTn results obtained from the selected clones of the 37°C bioreactor revealed thepresence of Sulfobacillus thermosulfidooxidans, Leptospirillum ferriphilum (these wereassociated) and Acidithiobacillus caldus (which was 100% similar), and Acidianus sp.,Solfobales archaeon and Metallospaera sp. were present in the 70°C bioreactor. Thisconforms to the suggested organisms present in the bioreactor by MINTEK.Prior to determination of MIC of copper exhibited by the organisms, suitable mediumwas selected for the 37°C bioreactor organisms and the organisms present in thisselected medium was assessed through amplification and sequencing analysis of theDGGE products. The result obtained showed the presence of the three organismspresent in the 37°C reactor. Each species was isolated through selective media and theminimum inhibitory copper concentrations were determined thereafter.The single species were not able to tolerate the high copper concentrations theconsortium was able to withstand. The MIC of copper of the consortium of bacteria(organisms from 37ºC bioreactor sample) were determined to be 400 mM, while isolationof each of the organisms present in the 37ºC bioreactor sample led to a drastic drop incopper MIC; Sulfobacillus isolate exhibited MIC of copper at 6 mM, Leptospirillum isolatea minimum inhibitory copper concentration at 3 mM and Acidithiobacillus sp. showed aminimum inhibitory copper concentration at 10 mM.Standard curves for copper(I) and copper(II) were set up at 390 nm to distinguishbetween the two valence states. Whole cell interaction with copper showed the ability ofProteus mirabilis and the consortium of bacteria to take up copper(II) and release coppercopper(I) at a certain period of time. The individual isolates were subjected to copper(II)environment to assess the ability to interact with copper. The results obtained showedless uptake of the divalent state of copper in Sulfobacillus sp. and Leptospirillum sp. andno copper(I) was detected. In contrast, Acidithiobacillus sp. was able to take upcopper(II) and actively reduce it to copper(I). Acidithiobacillus sp. may be dominant in theability to reduce copper(II) and in the consortium. Efflux of copper(I) has beendemonstrated by Rensing and co-workers (2000).The copper resistance mechanism of these organisms was further characterized and thewhole cell reduction showed that Acidithiobacillus sp. identified as Acidithiobacilluscaldus was the organism responsible for active efflux of copper(I) and was confirmed byamplification and sequencing analysis of the copA fragment.There was no PCR amplification of a copper resistance fragment in Sulfobacillus sp. andLeptospirillum sp. but amplification thereof in Acidithiobacillus sp. was obtained, whilethere was also amplification of the fragment in the consortium of bacteria. The BLASTnresults obtained after sequencing analysis showed similarity to Acidithiobacillusferrooxidans copA gene. As a result, full length gene specific primers were designedusing the Acidithiobacillus ferrooxidans copA sequence. The efforts to amplify a fulllength copA fragment from Acidithiobacillus caldus were fruitless. As a result, for qualitycontrol, the previous set of designed primers was used for amplification of a copA inAcidithiobacillus ferrooxidans and there was amplification. The results confirm thatAcidithiobacillus caldus possesses a copper translocating P-type ATPase which it usesto protect itself from copper toxicity and the remaining organisms in the bioreactor.

[发布机构] University of the Free State