Molecular Systematcs Of Acidophilic Bioleaching-Associated Bacteria From Diverse Habitats In China

The genetic heterogeneity among thirty-five strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans , including 32 field strains isolated from coal, copper, molybdenum mine wastes or sediments from 15 different geoclimatic sampling sites located in ten provinces of China, reference strain ATCC19859 and the type strains of the two species, was characterized by using a combination of molecular systematic methods, namely restriction fragment length polymorphisms (RFLPs) of PCR-amplified 16S rRNA genes and 16S-23S rDNA intergenic spacers (ITS), repetitive element PCR (rep-PCR) , arbitrarily primed PCR (AP-PCR) fingerprinting and comparative 16S rDNA sequence analyses.The complete sequences of 16r RNA genes and intergenic spacer regions (1SR) from Acidithiobacillus strains were determined. These data, together with other sequences available in the GenBank database, were used to carry out the first detailed assessment of the inter- and intraspecific genomic variability of the ISR sequences and to infer phylogenetic relationships within the genus. All of Acidithiobacillus strains were separated into three phylogenetic major clusters and eight phylogenetic groups.In most case only small variation in ISR size (less than 3%) were observed with strains belonging to the same species. The size of the ISRs of the 8 A.thiooxidans isolates studied varied from 451 bp to 464 bp, whereas the size of the ISRs of the 22 A. ferrooxidans isolates was comparatively shorter: only 434 to 456 bp. Acidithiobacillus strains DBS-2, TSK-3, originally assigned to A.thiooxidans, presented the smallest spacer, with 414 bp and 416 bp, respectively. The observed size heterogeneity was mainly due to length variations in the ISRc region. Most of the nucleotide changes also occurred in the ISRc region.The degree of intrageneric ISR sequence similarity was higher than the degree of intergeneric similarity, and the overall similarity values of the ISRs varied from 60.49 to 84.71% between representatives of different species of the genus Acidithiobacillus. Sequences from the spacer of the A.thiooxidans and A.ferrooxidans strains ranged from 86.71 to 99.56% and 92.36 to 100% similarity, respectively. ISR may be a potential target for the development of in situ hybridization probe aimed' at accurately detecting acidithiobacilli in the various acidic environments.All of the A.ferrooxidans strains fell into three phylogenetic groups. Most of Chinese A.ferrooxidans mine strains were assigned to phylogenetic Group 1 and 2, respectively. Unfortunately, A.ferrooxidans field strains assigned to the phylogenetic Group3 (representative strain A.ferrooxidans ATCC 33020) were not discovered in the present study. An interesting finding was that members belonging to each of the three phylogenetic groups possess the distinct ISR, ranging in size from 440 to 441bp (Groupl), 448 to 456 bp (Group2), 452 to 454bp (Group3), respectively. Strains ATCC23270~T, SSP and ATCC33020 were used as representative strain of each of the three A.ferrooxidans phylogenetic groups, respectively.It can be, therefore, suggested that each of the three phylogenetic groups may represent separate species or subspecies.22 A.ferrooxidans isolates can be divided into five distinct physiological groups based on minimum pH values, maximum temperature for growth and the capacity for element sulfur oxidation. Strains belonging to Group A and B failed to grow or were very poor growth at 40℃or pH 1.3, whereas 8 strains of Group C were capable of growth at 40℃or pH 1.3. Furthermore, 12 strains from Group A and B grew slowly in Erlenmeyer flasks with 50ml of iron-free 9k medium (pH 3.5) supplied with 1% sulfur powder and pH values of media declined only to 2.0-2.5 after 1 week. In contrast, 8 strains in Group C were able to oxidize rapidly elemental sulfur and pH values of media were able to decline to 1.3-1.6 after 1 week. Obviously, strains belonging to Group C were more acid-tolerant. A.ferrooxidans isolates BY-3 and DX-2 were very much like strains of Group C. One significant distinguishing feature of both strains and strains of Group C was difference in the minimum pH for growth. Isolate DX-2 was unable to grow at pH 1.3, whereas Strain BY-3 was able to slowly oxidize ferrous iron at pH 1.2. Therefore, A.ferrooxidans isolates BY-3 and DX-2 was assigned to physiological Group D and E, respectively. At the same time, we have found the correlation between some physiological traits and molecular information in the grouping of A.ferrooxidans strains. Physiological Group A and B were combined into genotypic Group Ia, whereas genotypic Group Ib, Ic, and Id corresponded to physiological Group C, D and E, respectively. An exception was observed for A.ferrooxidans strains HNQ and TGS as they were assigned to genotypic Group Ia, but were recovered in physiological Group C. The sequence analyses of 16S rRNA genes and ISRs showed the strains from physiological Group C, D, E were almost assigned to phylogenetic Group 1, while the strains in physiological Group A and B were affiliated with phylogenetic Group 2.In addition, the phylogenetic groups 7 (DBS-4,FY-2,YP-5) and 8 (DBS-2, TSK-3) may represent one or two new species of the genus Acidithiobacillus or do not belong to the genus Acidithiobacillus at all, although five unidentified sulfur-oxidizing isolates are very much like to A.thiooxidans type strain ATCC19377~T.Additional data need to be acquired before these taxons can described as new species.