Differentially Expressed Gene Analysis Of The Heat Shock Response Of Acidithiobacillus Ferrooxidans ATCC 23270

As one of the most important bioleaching bacterial species, Acidithiobacillus ferrooxidans(A.ferrooxidans)was applied in bioleaching of low grade copper,uranium mine initially,and then expanded to gold,zinc and cobalt.A.ferrooxidans is subjected to different kinds of environmental stress such as temperature changes,presence of some toxic heavy metals or pH changes which normally take place on bioleaching operations and that may affect the activity of the bacteria.So obtaining further knowledge of the protective mechanisms employ A.ferrooxidans in response to elevated growth temperature is meaningful to the national bioleaching industry.To define this organism's molecular response to elevated growth temperatures,temporal gene expression profiles were examined in cells subjected to heat stress by using whole-genome DNA microarrays for A. ferrooxidans,and then validated by Real-time PCR method.Lastly,a putative regulatory site was predicted upstream of a number of heat-inducible genes.cDNA derived from the heat shock A.ferrooxidans ATCC 23270 sample(42℃for 25 min)and untreated sample(30℃)were dual-color fluorescence(Cy^TM3-dUTP and Cy^TM5-dUTP)hybridized,as the result showed,approximately 14.70%(n=473,248 induced and 225 repressed)of the total predicted A.ferrooxidans ATCC 23270 genes(n=3217)represented on the microarrays were significantly up- or downregulated(P＜0.05)over 25-min period after shift to the heat shock temperature(42℃).These differentially expressed genes involved in stress response,signal transduction,substance transport,protein fate,lipid metabolism and carbohydrate metabolism etc.As expected,a large number of the genes homologous to known chaperones and heat shock proteins in other organisms were highly induced.Gene encoding membrane proteins were differentially expressed,suggesting that cells possibly alter their membrane composition or structure in response to variations in growth temperature.A substantial number of the genes encoding ribosomal proteins and flagellar proteins displayed downregulated coexpression patterns in response to heat stress.Real-time PCR were used to verify the hybridization result.Among the 14 genes we have examined,13 genes exhibited the similar results to the microarrays.The majority of predicted genes,including those encoding chaperones and heat shock proteins were significantly up-regulated(P＜0.05 and the fold change≥2.0)over a 25-min period after upshift to the heat shock temperature and then decrease to readjust their transcript levels to a new steady state at 42℃.Finally,based on the gene expression data and computational analysis,a putative regulatory site having T-T-T-T-T-T-n-n in the -35 region and n-T-A-T-n-A-T-C in the -10 region with average 19 base pairs separating the two elements was predicted upstream of these heat-inducible genes.