A versatile and efficient markerless gene disruption system for Acidithiobacillus thiooxidans: application for characterizing a copper tolerance related multicopper oxidase gene. (28th May 2014)

• Record Type: Journal Article
• Authors: Wen, Qing; Liu, Xiangmei; Wang, Huiyan; Lin, Jianqun
• Is Part Of: Environmental microbiology. Volume 16:Number 11(2014:Nov.)
• Page Start: 3499
• Page End: 3514
• Publication Date: 2014-05-28
• Subjects: Microbial ecology -- Periodicals; Environmental Microbiology -- Periodicals; 579.17
• DOI: 10.1111/1462-2920.12494
• Languages: English
• ISSNs: 1462-2912

Abstract

<abstract abstract-type="main"> <title>Summary</title> <p>The acidophilic bioleaching bacteria can usually survive in high concentrations of copper ions because of their special living environment. However, little is known about the copper homeostatic mechanisms of <italic>A</italic><italic>cidithiobacillus thiooxidans</italic>, an important member of bioleaching bacteria. Here, a putative multicopper oxidase gene (<italic>cueO</italic>) was detected from the draft genome of <italic>A</italic><italic>. thiooxidans</italic> ATCC 19377. The transcriptional level of <italic>cueO</italic> in response to 10 mM CuSO<sub>4</sub> was upregulated 25.01 ± 2.59 folds. The response of <italic>P<sub>cueO</sub></italic> to copper was also detected and might be stimulated by a putative CueR protein. Then, by using the counter‐selectable marker <italic>lacZ</italic> and enhancing the expression of endonuclease I‐SceI with <italic>tac</italic> promoter, a modified markerless gene disruption system was developed and the <italic>cueO</italic> gene disruption mutant (Δ<italic>cueO</italic>) of <italic>A</italic><italic>. thiooxidans</italic> was successfully constructed with a markedly improved second homologous recombination frequency of 0.28 ± 0.048. The Δ<italic>cueO</italic> mutant was more sensitive to external copper and nearly completely lost the phenoloxidase activity; however, the activity could be restored after complementing the <italic>cueO</italic> gene. All results suggest the close relation of <italic>cueO</italic> gene to copper tolerance in <italic>A</italic><italic>. thiooxidans</italic>. In addition, the developed efficient markerless gene knockout method can also be introduced into other <italic>A</italic><italic>cidithiobacillus</italic> strains.</p> </abstract>