Comparative Genome Analysis Of Acidithiobacillus Ferrooxidans Using Whole Genome DNA Microarrays

Acidthiobacillus ferrooxidans is an important microorganism used in biomining operations for metal recovery. To effectively study A. ferrooxidans, a comprehensive 50-mer-based oligonucleotide microarray was developed based on A. ferrooxidans ATCC 23270 complete genome published in TIGR. This array contained 3100 ORFs with <85% similarity and non-redundant sequence.Based on artificial probes, our results showed that under hybridization conditions of 50℃, the 50-mer microarray hybridization had best hybridized results. To evaluate the sensitivity and quantification of 50-mer microarray hybridization, 1ng, 2.5ng, 5ng, 10ng, 15ng, 50ng, 100ng A. ferrooxidans DNA were hybridized to the array. The results showed that when DNA was 50ng, the array could detect almost all ORF spotted in the array. And strong linear relationships between the signal intensity and the target DNA were observed (r~2 = 0.98). Five functional genes (sulfide-quinone reductase, cytochrome C, iron oxidase, mercuric resistance protein, nitrogenase iron protein) were amplified from A. ferrooxidans to test specificity of 50-mer microarray, the results indicated that the designed probes on the arrays appeared to be specific to the five corresponding target genes.Whole genomic diversity analysis based on oligonucleotide microarray was used to analyze the gene content of 12 strains of A. ferrooxidans purified from various mining areas in china. Among the 3100 ORFs on the slides, 1235 ORFs were absent in at least one strain of bacteria and 1385 ORFs were conserved to all strains. The hybridization results showed that these strains were highly diversified. The hybridization results of five major functional gene categories: electron transport, carbon metabolism, extracellular polysaccharides, detoxification, and regulatory were analyzed. Based on the hybridization signals obtained, phylogenetic tree was built to analyze the evolution of 12 tested strains, which indicated that the spatial factor was the main factor influencing the diversity of these strains. Based on the hibridizaiton signals of genes associated with bioleaching, another phylogenetic tree coupling the activation of bioleaching and geochemistry was built. The result revealed that the main factor was geochemistry, among which the following seven factors: pH, Ca, Mg, Cu, S, Fe, Al were most important.