Global Transcriptome Analysis Of The Alkaline Shock Response Of Acidithiobacillus Ferrooxidans ATCC 23270

As one of the acidophilic bioleaching bacterial species, Acidithiobacillus ferrooxidans(A.ferrooxidans) is prone to grow in environment of pH at about 2.0.In practical industry,it is proved that the highly acidic environment is hardly maintainable due to the dissolution of alkaline matrix and further to impede the bioleaching efficiency of A. ferrooxidans.Therefore,it is very valuable to explore how to domesticate the bioleaching bacteria of this kind,and it is theoretically meaningful to investigate how they express and regulate at whole-genome level in response to pH upshock.In this study,we constructed the whole-genome oligonucleotide array based on the whole 3,217 ORFs of A.ferrooxidans ATCC 23270 genome. The transcriptome analysis of this bacterial species in response to pH upshock was conducted by the whole-genome array,and illuminate its expression dynamics,further to explore the response mechanism to surrounding stress.In this study,a whole-genome 50-mer-based oligonucleotide microarray was effectively developed covering～97%of the total predicted protein-encoding open reading frames(ORFs) in A.ferrooxidans ATCC 23270.Based on artificial oligonucleotide probes,our results showed that the optimal hybridization temperature was at 45℃.Specificity tests with the purified PCR amplifications of 5 genes(Sulfide-quinone reductase, Cytochrome C,Iron oxidase,Mercuric resistance protein,Nitrogenase iron protein) of A.ferrooxidans ATCC 23270 indicated that the probes on the arrays appeared to be specific to their corresponding target genes.Based on the WGA hybridization to global transcriptional difference between A. ferrooxidans ATCC 23270 strains cultured with Fe(Ⅱ) and S(0),the results indicated the developed 50-mer WGA could be used for global transcriptome analysis of A.ferrooxidans ATCC 23270.The detection limit was estimated to be approximately 5 ng with the genomic DNA,and at 100 ng of the DNA concentration,all of the signals reached to saturation.In addition,strong linear relationships were observed between hybridization signal intensity and the target DNA concentrations(r2=0.977 and 0.992). The results indicated that this technology had potential as a specific, sensitive and quantitative tool for detection and identification of the strain A. ferrooxidans ATCC 23270 at the whole-genome level.In this study,cDNA derived from the alkaline shock A.ferrooxidans ATCC 23270 sample(pH up-shift from 1.58 to 3.00 by 2 mol/L KOH,shock time at 10,20,40,80,160 min) and corresponding untreated sample were dual-color fluorescence hybridized by the whole-genome array,as the result showed,the number of the genes differentially expressed for 160 min reached to 600,approximately 19.16%,which revealed the expression system of A.ferrooxidans ATCC 23270 could quickly reponse to the stress when shocked by alkali.There is 297(9.48%) genes induced for 160 min, while 174(5.56%) genes repressed for 160 min,and 407 genes induced and 614 genes repressed only a certain time.Statistical analysis of the genes differentially expressed in 10 min revealed,there is 1217(38.8%) genes obviously differentially expressed in alkaline shock for 10 min(969 genes induced and 248 genes repressed).And 74.82%induced genes differentiated at 2～3 times,while 78.23%repressed genes differentiated at more than 10 times.Majority of genes could response quickly to alkaline shock in 10 min. By statistical analysis,the result showed,these differentially expressed genes involved in various metabolic pathway,such as membrane activities, transport and binding proteins,cellular processes,regulatory functions, protein fate and synthesis etc.Also,the genes encoding membrane activities, transport and binding proteins,cellular processes,regulatory functions obviously induced,this is similar to heat shock,cold shock and salt shock. However,the genes encoding energy metabolism also obviously induced, this is similar to heat shock and salt shock,but contradictory to cold shock. Besides,it was almost same number between induced and repressed genes for the genes encoding hypothetical proteins or unknown function proteins, this is similar to other shocks.To validate the microarray results,six open reading frames(ORFs) showing obvious induction after alkaline shock in microarray analysis were subjected to real-time PCR with the same RNA samples used in the array hybridizations.The real-time PCR results showd the same trend with the whole-genome array,proved the credibility of the microarry results.Finally,based on the gene expression data and computational analysis,a putative regulatory site having T-n-T-T-n-n in the -35 region and n-T-A-T-n-A-T-C in the -10 region with average 21 base pairs separating the two elements was predicted upstream of these alkali-inducible genes.