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1 School of Food Sciences, Shihezi University, Shihezi, PR China
2 School of Life Sciences, Lanzhou University, Lanzhou, PR China
Correspondence
Kaiyu He
niyqlzu{at}sina.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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These authors contributed equally to this work.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA nucleotide sequence and 16S–23S rDNA intergenic spacers data reported in this study are DQ676505–DQ676511, EF059755–EF059762 and EU084695–EU084714.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). These bacteria appear to be present in a wide range of environments, especially in sites where pH is low, such as mining areas, hot sulfur springs and bioleaching operations. Many attempts have been made to describe a large number of Acidithiobacillus strains (Harrison, 1982; Karavaiko et al., 2003; Bergamo et al., 2004). More recently, bacteria of the genus Acidithiobacillus have been widely studied for bioleaching, biodegradation and desulfurization of coal and natural gas (Acharya et al., 2004; Rawlings & Johnson, 2007). However, since their natural habitats are ecologically extremely diverse, different Acidithiobacillus strains of the same species developing in various ecological niches are characterized by differences in growth rate, tolerance to heavy metal ions and activities of ferrous iron and/or sulfide mineral oxidation (Leduc & Ferroni, 1994; Kondratyeva et al., 1999; Ageeva et al., 2001). Moreover, strains belonging to the same Acidithiobacillus species exhibit a high degree of interstrain genetic variability with respect to the DNA G+C content, level of DNA–DNA homology of total genomes, the number and sizes of plasmids and chromosomal DNA structure (Harrison, 1982; Amils et al., 1998; Karavaiko et al., 2003; Rawlings, 2005; Valenzuela et al., 2006).
To date, the revised genus Acidithiobacillus consists of four validly described species: Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans, Acidithiobacillus albertensis and Acidithiobacillus caldus (Kelly & Wood, 2000). However, it has been found that some Acidithiobacillus-like isolates could not be assigned to any of the known Acidithiobacillus species (Harrison, 1982; Lane et al., 1992; Karavaiko et al., 2003), and some other sulfur-oxidizing strains were proposed to represent a novel species of Acidithiobacillus (Norris, 2007). Therefore, large-scale investigations of the genotypic features of indigenous Acidithiobacillus strains in relation to their physiological properties might give a better understanding of the inter- and intraspecific genealogical relationships of this genus, and could be used for clarifying the taxonomic status of controversial strains and the rapid screening of strains important in industrial environments, such as bioleaching operations.
While the 16S rDNA sequence is a good tool for inferring inter- and intrageneric relationships, sequencing of the 16S–23S rDNA internal transcribed spacer (ITS) has been suggested to be well suited for typing and identification of bacteria at both the species and the strain level (Barry et al., 1991; Gurtler & Stanisich, 1996), because of marked variation of the ITSs in both sequence and size between strains and species. Based on diversities between ITS sequences, it will be possible to construct species- and strain-specific oligonucleotides that can be used to detect bacteria in their natural environments.
In this work, in order to contribute to our understanding of Acidithiobacillus taxonomy, 35 Chinese Acidithiobacillus isolates and four reference strains were examined by randomly amplified polymorphic DNA (RAPD) and sequencing of the 16S rDNA and the 16S–23S rDNA ITS. Phylogenetic data obtained from the ITS sequences were compared with corresponding data derived from the 16S rDNA analysis, considering both the resolution and branching of the phylogenetic trees. To clarify the taxonomic status of some controversial strains, we analysed levels of ITS sequence divergence between the Chinese Acidithiobacillus isolates and representative strains from various parts of the world. In addition, molecular data on the grouping of the Acidithiobacillus isolates are discussed relating to phenotypic characteristics.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Samples were serially diluted and spread onto a variety of solid media specifically designed for isolating acidophilic sulfur-oxidizing and iron-oxidizing bacteria, or subjected to enrichment in liquid medium and then streaked onto solid medium (Hallberg & Lindstrom, 1994; Johnson, 1995). Each isolate was purified by repeated single-colony isolation, and preliminary identification of isolates was carried out on the basis of colony morphologies and cell characteristics (Johnson, 1995). The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g. oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined (Kelly & Wood, 2000, 2005). Of the 35 isolates, 20 were identified as representing A. ferrooxidans, seven as representing A. thiooxidans and three as representing A. caldus; the remaining five were not identified to species level and were termed Acidithiobacillus-like strains. The strains used in this study and their sources are listed in Table 1.
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and Bergamo et al. (2004), with modifications. After treatment with 10 µl RNase (20 mg ml–1), DNA concentrations and purity were determined by spectrophotometric readings. The DNA was stored at –70 °C until needed.
RAPD-PCR.
Ten 10-mer primers (OPA-02, OPA-07, OPA-13, OPC-02, OPC-08, OPC-09, OPC-11, OPC-15, OPC-19, OPC-20) from Operon Technologies and 20 10-mer primers (S-21 to S-40, Sangon Biological Engineering Technology) were chosen randomly for the PCR. In addition, five longer primers (17–22-mer) were tested for the ability to differentiate Acidithiobacillus isolates: PJ108, PJ118 (Lado & Yousef, 2003); M13 (Jonas et al., 2000); ERIC2 (Versalovic et al., 1991); and BG2 (van Belkum et al., 1993).
PCR was carried out in 25 µl containing 20 ng genomic DNA, 2.5 mM MgCl2, 0.4 µM primer, 2 U Taq DNA polymerase (TaKaRa Biotechnology), 200 µM of each dNTP in 10 mM Tris/HCl, pH 8.3, 50 mM KCI and 0.001 % gelatin, 0.1 % Triton X-100, under a drop of mineral oil. A Bio-Rad iCycler Thermal Cycler was used for amplification. The cycling programme when using 10 nt primers was essentially as described by Welsh & McClelland (1990). The cycling programme when using longer (>17 nt) primers was as described by Williams et al. (1990), with minor modifications. Each amplification was done in triplicate and results were reproducible when DNA obtained by different extraction procedures of the same strain was used (Novo et al., 1996; Bergamo et al., 2004). The amplification products (10 µl) were separated by electrophoresis on 1.5 % agarose 0.5x TBE gels. The gels were stained with ethidium bromide and photographed under UV. The 200 bp DNA ladder (TaKaRa Biotechnology) was used as a size marker in all gels.
Data from the RAPD-PCR were scored in binary form (presence/absence) and similarity matrices were generated using the Jaccard coefficient (Sneath & Sokal, 1973). A dendrogram was constructed by using the UPGMA (unweighted pair group method with arithmetic means) with the software package NTSYS-pc 2.01 (Rohlf, 1997). Cluster analyses were performed on the combined datasets generated by RAPD-PCR fingerprinting.
Sequencing and phylogenetic analysis of 16S rDNA and 16S–23S rDNA intergenic spacers.
The 16S rRNA genes were amplified using the primers 27F and 1525R (Lane 1991). The primers 5'-GACTGGGGTGAAGTCGTAAC-3' and 5'-TGGCTGGGTTGCCCCATTCGG-3' were used to amplify the 16S–23S rDNA intergenic spacers (Sagredo et al., 1992). PCRs (50 µl) contained 30–50 ng DNA, 0.4 µM each primer, 200 µM each dNTP, 2 mM MgCl2 and 2 U Taq DNA polymerase (TaKaRa Biotechnology), in the buffer supplied by the manufacturer. Amplifications were performed using a Bio-Rad iCycler Thermal Cycler and included an initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min and final extension at 72 °C for 7 min. Direct sequencing of the PCR products was performed with an ABI PRISM BigDye terminator v3.1 sequencing Ready Reaction kit (PE Applied Biosystems) and an ABI PRISM 3730 genetic analyser (PE Applied Biosystems), using both forward and reverse PCR amplification primers. All sequencing procedures were repeated at least twice for each strain.
The 16S rRNA gene sequences and 16S–23S rDNA intergenic spacers were compared to sequences in the GenBank database by using BLAST (Altschul et al., 1990). The sequences were aligned by using the CLUSTAL_X program (Thompson et al., 1997). Phylogenetic trees were constructed using the MEGA v. 4.0 (Tamura et al., 2007) packages with p-distances and the neighbour-joining method (Saitou & Nei, 1987). Bootstrap values were calculated from 1000 replications using the MEGA program.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Amplified DNA fragments that were reproducible were scored as 0 (absence) and 1 (presence) in a matrix dataset and the relationships between the strains were estimated using Jaccard's coefficient of similarity (data not shown). The cluster analysis, performed by distance data matrix from RAPD-PCR profiles, divided the 35 Acidithiobacillus strains into two major clusters (Fig. 1). Cluster I comprised all the A. ferrooxidans strains, whereas cluster II was composed exclusively of sulfur-oxidizing strains, and contained all these strains. Within cluster I, the 22 strains of A. ferrooxidans could be divided into four distinct groups at the 34 % similarity level. Group Ia included most of the A. ferrooxidans isolates from different regions in China and isolates TKY-2 and JDC were the most divergent strains (35 % average similarity, Fig. 1); group Ib comprised isolates DBS, YP-3, DX-1, TSK-1, BY-3, type strain ATCC 23270T, and reference strain ATCC 19859; group Ic consisted of isolates FY-3 and TGS. Isolate DX-2 was the most divergent organism in cluster I and was assigned to single-member group Id. In comparison with cluster I, cluster II, formed by the 13 sulfur-oxidizing strains, included two groups: group IIa consisted of isolates BY-s, JY, YP-2, LYS, DX-3, TKY-t and the A. thiooxidans type strain ATCC 19377T; group IIb comprised five sulfur-oxidizing Acidithiobacillus-like isolates YP-5, FY-2, DBS-2, TSK-3, DBS-4 and A. thiooxidans strain SZS. Similarity between strains from the two clusters ranged from 5.7 % to 19 %. Among strains from cluster I (strains of A. ferrooxidans), the similarities ranged from 16.2 % (ATCC 23270T and JDC) to 71.6 % (ATCC 23270T and ATCC 19859), and among strains of cluster II (only sulfur-oxidizing strains) was 18.3 % (LYS and TSK-3) to 66 % (LYS and YP-2). Among the strains within A. ferrooxidans group Ib, the similarities ranged from 33.3 % to 71.6 %. The similarity between group Ic strains TGS and FY-3 was 45 %.
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, who concluded that various A. ferrooxidans strains have a very wide similarity coefficient range (almost 0 % to over 98 %). There is a good correlation between the relationships of the Chinese Acidithiobacillus strains determined by RAPD analysis and corresponding data derived from repetitive element (rep)-PCR, with the exception of A. ferrooxidans isolates TGS and FY-3 belonging to the same group with most of the Chinese A. ferrooxidans isolates in the rep-PCR analysis (Ni et al., 2008). In addition, as shown in Table 1
and in Fig. 1, no clear correlation was found between the genotypic groups of the Acidithiobacillus sp. strains and either the geographical location or type of habitat/sample from which the strains were isolated; this is consistent with results obtained by other workers (Novo et al., 1996; Karavaiko et al., 2003).
Comparative analysis of 16S rDNA sequence and 16S–23S rDNA intergenic spacers
Sequences of the 16S rDNA and the 16S–23S rDNA ITS were determined for 35 Acidithiobacillus isolates recovered from wastes or sediments in China. Although it is known that the genome of A. ferrooxidans has two rrn operons (Salazar et al., 1989), in each case only one ITS-PCR product was obtained, suggesting that the two ITS copies in the genome of each of the Acidithiobacillus isolates did not differ in size. According to our sequencing results, the structure and organization of the 16S–23S intergenic spacers of the Chinese Acidithiobacillus isolates were consistent with the data obtained by other researchers (Venegas et al., 1988; Sagredo et al., 1992). The 16S–23S intergenic spacers of all Chinese Acidithiobacillus isolates contain two highly conserved genes for tRNAIle and tRNAAla, which split the ITS into three regions (ITS1, ITS2 and ITS3). The tRNAIle and tRNAAla genes of Acidithiobacillus spp. are 87.3 % and 86.8 % homologous, respectively, to the corresponding tRNA genes of E. coli. The other feature of the ITS of all the Acidithiobacillus strains is the presence of a 9 bp antitermination consensus sequence (TGTTCTTTTG) resembling box A, which is found in all the 16S–23S intergenic spacers of E. coli, as well as in a number of other bacterial species (Berg et al., 1989). A detailed comparison of the sequence revealed variations in the length of the ITS among the Acidithiobacillus strains examined (Table 2). The divergence between the ITS sequences arises from insertions and deletions of short sequences located in the ITS3 region (see Fig. 4). These sequences could be interesting candidates for the development of species- and strain-specific probes. The size of the ITS of the A. ferrooxidans strains varied from 434 to 456 bp. The A. thiooxidans strains possessed different ITS in sizes ranging from 451 to 490 bp. The Acidithiobacillus-like strains YP-5, FY-2 and DBS-4 possessed an identical ITS of 421 bp, whereas the ITS of the other two sulfur-oxidizing strains DBS-2 and TSK-3 was shorter: only 413 and 415 bp. A. caldus stains presented the smallest ITSs, of 379–382 bp. A cfoI site was found in the ITS of A. ferrooxidans and A. thiooxidans, while no cfoI site was present in the ITS of A. caldus and five Acidithiobacillus-like strains.
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and 3). The phylogenetic tree constructed on the basis of the ITS sequences was topologically roughly similar to that generated by 16S rDNA sequences. The Acidithiobacillus strains were separated into eight phylogenetic groups. The strains assigned to a species showed higher phylogenetic coherence on the topology tree, suggesting the identities of all tested strains to be correct. An analysis of the 16S rRNA gene sequence indicated that all the A. thiooxidans and A. ferrooxidans strains constituted a monophyletic cluster with an 89 % bootstrap value and that the species A. caldus was phylogenetically close to A. thiooxidans and A. ferrooxidans. As shown in Fig. 2, the A. caldus strains and A. thiooxidans DSM 612 took an intermediate position between A. ferrooxidans, A. thiooxidans and the five sulfur-oxidizing Acidithiobacillus-like isolates. In contrast, the 16S–23S rDNA spacer sequences revealed that A. caldus strains and five sulfur-oxidizing Acidithiobacillus-like isolates showed a higher degree of relatedness, and constituted a monophyletic cluster (C) with a bootstrap value of 100 %, whereas A. thiooxidans strains also constituted another monophyletic cluster (B) with a bootstrap value of 99 %. The ITS nucleotide sequence divergence between representative strains belonging to different phylogenetic groups is shown in Fig. 4. Moreover, results of phylogeny analysis, especially the phylogenetic tree constructed on 16S–23S rDNA spacer sequence, were in line with the dendrogram obtained by RAPD cluster analysis based on the whole genome. In the RAPD clustering analysis, the most divergent A. ferrooxidans strain DX-2 fell into none of the phylogenetic major groups; the Chinese A. thiooxidans isolate SZS most distantly related to A. thiooxidans ATCC 19377T was assigned to the independent phylogenetic group 5 (Fig. 3). There was an exception, in that the representative strains of RAPD groups Ib and Ic were combined into the phylogenetic group 1.
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; Paulino et al., 2001) have demonstrated that the 16S rRNA gene sequences of strains belonging to different Acidithiobacillus species are less than 5 % divergent, except for presumptively identified Acidithiobacillus strains, and that most strains from the same Acidithiobacillus species show greater than 98 % identity in their 16S rDNA sequences. The 16S rRNA gene sequence divergence between A. thiooxidans and A. ferrooxidans strains is less than 3 %, whereas the 16S rRNA gene sequence of A. thiooxidans has more than 99 % identity to that of the currently presumed A. albertensis strains. Consequently, the variations in the 16S rRNA gene sequences are not sufficient to provide useful information to allow clear differentiation of closely related Acidithiobacillus strains (Fox et al., 1992). Compared with the 16S rRNA gene sequence, the 16S–23S rDNA ITS exhibited considerable variation in structure and is more appropriate for studies at the intraspecies level. The ITS-based phylogeny markedly improved the resolution and complemented the phylogeny of the genus Acidithiobacillus based on 16S rRNA gene sequences. Our analysis results showed that the levels of ITS sequence similarity within members of the same species were markedly greater than the levels of similarity between members of different species. Overall similarity values of the ITSs varied from 60.5 to 84.7 % between representative strains of different species of the genus Acidithiobacillus (data not shown). Sequences from the spacer of the A. thiooxidans and A. ferrooxidans strains ranged from 86.7 to 99.6 % and 92.4 to 100 % similarity, respectively. The Acidithiobacillus strains DBS-2, TSK-3 and the erroneously identified A. thiooxidans KCTC8928P, which were assigned to phylogenetic group 7, showed about 80 % and 69–74 % ITS identity with A. caldus DSM 8584T and A. thiooxidans ATCC 19377T, respectively. Phylogenetic group 8 consisted of Acidithiobacillus strains YP-5, FY-2 and DBS-4, which showed about 80 % and 72.5 % ITS identity with A. caldus DSM 8584T and A. thiooxidans ATCC 19377T, respectively. The ITS and the 16S rRNA gene sequence similarity between the two groups was 85.6–87.7 % and 96–97 %, respectively. In addition, although A. albertensis was tentatively assigned to the new genus Acidithiobacillus, there have up to now been no research reports about the ITS of A. albertensis. As its 16S rDNA sequence and phenotypic traits are highly similar to most A. thiooxidans strains (more than 99 % 16S rDNA sequence identity) (Kelly & Wood, 2005), the information on ITS is expected to contribute to improvement of the taxonomy and phylogeny of A. albertensis.
From the alignment, we could also verify the existence of intraspecific polymorphism in the ITSs of Acidithiobacillus spp. Most of the A. ferrooxidans strains, including the type strain ATCC 23270T, fell into three phylogenetic groups. Each of the three A. ferrooxidans phylogenetic groups possessed a distinct ITS, ranging in size from 440 to 441 bp (group 1), 448 to 456 bp (group 2) and 452 to 454 bp (group 3), respectively. A. thiooxidans strains were assigned to phylogenetic groups 4 and 5 (Fig. 3). The majority of A. thiooxidans strains, including the type strain ATCC 19377T, were recovered in a very tight group (group 4) with more than 97 % ITS sequence similarity. The Chinese A. thiooxidans isolate SZS was assigned to a separate group 5, which contained a unique A. thiooxidans strain STAM61 with the largest ITS of 490 bp. While ITS sequence similarity levels between members of the two A. thiooxidans groups were only 88–90 %, the two groups showed more than 98 % identity in their 16S rRNA gene sequence. A. thiooxidans strain CASQ22, described by Bergamo et al. (2004), which possessed an ITS with a smallest size of 409 bp, and fell into none of groups, showed only ITS sequence similarity on average of less than 87 % to other A. thiooxidans strains, suggesting that this strain might correspond to a different genospecies or genomovar from the remaining A. thiooxidans strains. Consequently, the ITS could also be used as a tool to clarify controversial strains.
Determination of cutoff points of genomic data
As seen in Fig. 3, the tree constructed by using 16S–23S ITS sequences showed clearly the inter- and intraspecific genealogical relationships of the Acidithiobacillus species. Thus, levels of ITS sequence divergence may also be considered as a genomic criterion for distinguishing closely related Acidithiobacillus strains and species definitions. In this study, Acidithiobacillus strains that share less than 87 % similarity in the 16S–23S rDNA spacer sequence are nearly always members of different species, which is consistent with the data obtained by Leblond-Bourget et al. (1996) with the genus Bifidobacterium. The maximum levels of the 16S–23S rDNA spacer sequence divergence for the species A. thiooxidans were about 13 %. For A. ferrooxidans and A. caldus, the maximum levels of the 16S–23S rDNA spacer sequence divergence were about 8 %.
Although levels of ITS sequence divergence between strains belonging to the same species may help determine relationships of closely related strains, there is up to now no precise boundary limit to clarify species definitions and controversial strains. In this study, the levels of the 16S–23S rDNA spacer sequence divergence between five sulfur-oxidizing Acidithiobacillus-like isolates and two closely related species A. caldus and A. thiooxidans were more than 13 %. This result was also consistent with those derived from 16S rDNA sequences analysis (level of similarity <95 %). Preliminary phenotypic experiments revealed that Acidithiobacillus-like isolates YP-5, FY-2 and DBS-4 were distinguished from A. thiooxidans ATCC 19377T not only in that they were moderate acidophiles (optimum growth pH between 4.0 and 4.5) when growing between 30 and 35 °C, but also in that they were also able to oxidize elemental sulfur at temperatures ranging from 40 °C to 45 °C. In addition, isolates YP-5, FY-2 and DBS-4 grew faster in the media supplied with 0.01 % yeast extract than without yeast extract on the basis of their specific growth rate (Ni et al., 2008), indicating that the three isolates are physiologically similar to A. caldus DSMT 8584, and should be mixotrophic. In contrast, Acidithiobacillus strains DBS-2 and TSK-3 are unable to grow above 40 °C (Ni et al., 2008). Following the recommendation of Stackebrandt et al. (2002) that phenotype continues to play a salient role in the decision about cutoff points of genomic data, these novel isolates should represent one or two new species of the genus Acidithiobacillus or even indicate a new genus. Further studies on these sulfur-oxidizing isolates are in progress in our laboratory.
Previous work has shown that A. ferrooxidans strains could be divided into several genomic groups (genomovars) on the basis of DNA–DNA hybridization similarity, and that strains from the different A. ferrooxidans genomovars had genome similarity of only 10–50 % (Harrison, 1982; Karavaiko et al., 2003). However, genomovars are commonly distinguished at a genome similarity level of 70 %. Even if the genomic similarity value for circumscribing the species could be lowered to 50 % according to the suggestion of Rossello-Mora & Amann (2001), the current situation, that strains from the different genomovars were assigned to a single species A. ferrooxidans, should be controversial. On the other hand, in most cases only small variations in 16S rRNA gene sequence were observed with strains belonging to A. ferrooxidans (less than 2 % sequence divergence).
In this study, the majority of A. ferrooxidans strains isolated from various acid habitats were assigned to three phylogenetic groups by 16S rDNA sequences and the 16S–23S rDNA spacer sequence analysis. ITS sequence similarity levels within and between the phylogenetic groups of A. ferrooxidans were about 97.5–100 % and 92–97 %, respectively (data not shown). Strains ATCC 23270T, SSP and ATCC 33020 were used as representative strains of the three A. ferrooxidans phylogenetic groups. The Chinese A. ferrooxidans isolates were assigned to phylogenetic groups 1 and 2, as shown in Figs 1 and 2. A. ferrooxidans isolates related to strain ATCC 33020 were not recovered in this study. Previous studies have demonstrated that A. ferrooxidans ATCC 33020 (representing the phylogenetic group 3) possesses distinctive phenotypic characteristics, including more sensitivity to uranium and an ability to accumulate a large amount of uranium in comparison with other ecotypes of A. ferrooxidans strains (Merroun & Selenska-Pobell, 2001). Our phenotypic analysis (Ni et al., 2008) also revealed that A. ferrooxidans strains from phylogenetic group 1 were able to grow at 40 °C or pH 1.3, and able to oxidize elemental sulfur rapidly in iron-free 9k medium (pH 3.5) (pH values of media decreased to 1.6–1.8 after 1 week). In contrast, A. ferrooxidans strains belonging to phylogenetic group 2 failed to grow or grew very poorly under the corresponding conditions and grew slowly in iron-free 9k medium (pH 3.5) supplied with 1 % sulfur powder (pH values of media decreased only to 2.0–2.5 after 1 week). Thus each of the three A. ferrooxidans phylogenetic groups corresponds to a different physiological group. As was the case with A. ferrooxidans ATCC 33020, each of the three phylogenetic groups has been proposed to be considered as a distinct species or a separate subspecies (Selenska-Pobell et al., 1998; Johnson et al., 2005). At least, each phylogenetic group should be represented by a subspecies, a concept currently being defined (Brenner et al., 2004). This problem needs further elucidation by other phenotypic characteristics or genomic information.
In a wide range of sulfur-containing environments, diverse members of the genus Acidithiobacillus and other closely related sulfur-oxidizing bacteria are abundant and their metabolism versatile (Friedrich, 1998). However, pure cultures of these sulfur-oxidizing bacteria are difficult to isolate because of their slow growth and synergistic interactions with heterotrophic organisms (Baker & Banfield, 2003). It is generally believed that there is a selection for certain sulfur-oxidizing bacteria during the process of purification and that purified cultures reflect the conditions used for isolation, rather than the actual dominance of the particular strains in the environment (Schrenk et al., 1998). Therefore, pure cultures of acidithiobacilli available today are considered unrepresentative of the true diversity of the genus Acidithiobacillus in the environment. In fact, a bacterial clone from acidic volcano samples was proposed to represent a novel species of Acidithiobacillus (Simmons & Norris, 2002). Recently, there have been several attempts to construct specific oligonucleotides based on 16S rRNA gene sequences (Peccia et al., 2000; Bond & Banfield, 2001; Bouchez et al., 2006; Okabe et al., 2007), with the aim of detecting and identifying a variety of the sulfur-oxidizing bacteria at the species level or above in situ. As described in this study, however, Acidithiobacillus strains from various econiches possess only small variations in 16S rRNA gene sequences. Acidithiobacillus strains sharing identical 16 rRNA gene sequences show only much less than 50 % genome DNA similarity (Goebel & Stackebrandt, 1994). In contrast, Acidithiobacillus strains show higher ITS polymorphisms, including either species-, genospecies- and/or even strain-specific nucleotide changes. Thus, as was the case with the iron-oxidizing species Leptospirillum ferrodiazotrophum (Tyson et al., 2005), ITS may be a potential target for the development of fluorescent in situ hybridization probes for more accurately detecting distinct ecotypes of strains within the Acidithiobacillus species and other closely related sulfur-oxidizing bacteria, pure cultures of which are difficult to obtain from environmental samples.
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ACKNOWLEDGEMENTS |
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Edited by: G. Muyzer
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Received 24 December 2007;
revised 29 April 2008;
accepted 5 May 2008.
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