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Reverse-transcription qualitative PCR (RT-qPCR) was used to analyze the changes in transcription levels of the sulfur metabolism-related periplasmic protein genes of Acidithiobacillus ferrooxidans ATCC 23270 grown on sulfur or ferrous. Seven periplasmic proteins with apparently higher abundance grown on elemental sulfur than on ferrous sulfate were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Expression analysis of the corresponding genes by RT-qPCR shows that the constitutive expression of all those genes are more up-regulated grown on sulfur than those grown on ferrous (>10 folder). Study on the corresponding genes of the identified periplasmic proteins by RT-qPCR confirmed the results of two-dimensioned gel electrophoresis, indicating they may be related with sulfur metabolism in A. ferrooxidans.