Construction Of Markerless Deletion In Acidithiobacillus Caldus Genome And The Characterization Of SoxYZ Mutant

Acidithiobacillus caldus is a gram-negative, obligated autotrophic sulfur-oxidizing bacteria, and the optimal pH and temperature are1.5to2.5and40to50℃, respectively. It obtains energy and reducing power by oxidizing inorganic reduced sulfur compounds, and fixes CO2from atmosphere through Calvin cycle as carbon source. It plays a great role in biohydrometallurgy, desulfurization of coal and the treatment of waste water. However, its long generation time and low cell concentration limit its application in industry. Therefore, a deep research of its sulfur metabolism and genetic modification of A. caldus is necessary.For the reasons of slow growth and difficulties in genetic operation of A. caldus, there is little progress in the research of genetic operation of this strain. Even if the gene transfer method has been established, the method is still sophisticated and there is no report of markerless gene replacement method of this strain.The sequencing of A. caldus genome has been finished. Bioinformatic analysis of it showed that the sulfur-oxidizing genes may contain two incomplete sox gene clusters soxX-soxY-soxZ-soxA-hyp-soxB (sox clusterl) and soxY-soxZ-soxB-hyp-resB-soxX-soxA-resC (sox clusterⅡ), as well as sqr, doxD, tth, hdr, sor and terminal oxidase, NADH-quinone oxidoreductase. What the function of each gene is, and how they work together to oxidize the reduced sulfur compounds are still in the exploration.We firstly established the "in-out" markerless gene replacement method in A. caldus based upon its own Rec homologous recombination system and the extensive endonuclease I-Scel. At the same time, we constructed the universal suicide plasmid vector pSDUDI and the plamid pSDU1-I-SceⅠ expressing I-Scel enzyme. We knocked out the gene soxYZ (ORF2520-2521) of sox clusterⅢ successfully by using this method, and the efficiency is7%. It is the first report on markerless gene knock out in A.caldus internationally, which contributes to the research of gene functions and regulations of A.caldus.We then investigated the growths of A. caldus wild strain and soxYZ (ORF2520-2521) mutant strain using sulfur powder and tetrathionate, respectively, as energy source. The results showed that there was no obvious difference between them when sulfur powder was used as the energy source. However, the mutant strain entered into the exponential phase much late and growed more weakly than the wild strain when tetrathionate was used as the energy source. The results were well agreed with the report of Mangold et al (Mangold et al.,2011). Therefore, we considered that the Sox sulfur oxidizing system encoded by sox clusterll played important role in oxidizing tetrathionate.