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In order to contribute to our understanding of Acidithiobacillus taxonomy, we determined 16S rDNA sequences and the 16S-23S internally transcribed spacer (ITS) sequence of 35 Chinese Acidithiobacillus isolates and three reference strains representing three validly described species and used them to construct phylogenetic trees. The two phylogenetic trees were roughly similar topologically, and Acidithiobacillus strains were assigned to eight phylogenetic groups. In addition, the results of phylogenetic analysis were consistent with those obtained by randomly amplified polymorphic DNA (RAPD) cluster analysis. Compared with a phylogenetic tree based on the 16S rRNA sequences, the ITS tree showed more clearly the inter- and intraspecific genealogical relationships of the genus Acidithiobacillus. Similarity values of the ITSs varied from 60.5 % to 84.7 % between representative strains of different species, and the maximum level of ITS divergence between strains belonging to the same species was 13 %. Coupling phylogenetic analysis and phenotypic characteristics, we concluded that at least each of the three Acidithiobacillus ferrooxidans phylogenetic groups should be considered a separate subspecies, and that five sulfur-oxidizing Chinese Acidithiobacillus-like isolates represent one or two new species of the genus Acidithiobacillus. The ITS may be a potential target for the development of fluorescent in situ hybridization probes for more accurately detecting distinct ecotypes of Acidithiobacillus strains and other closely related sulfur-oxidizing bacteria.

16S rDNA and 16S–23S internal transcribed spacer sequence analyses reveal inter- and intraspecific Acidithiobacillus phylogeny