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1 Institute of Microbiology, Russian Academy of Sciences, pr. 60-letiya Oktyabrya 7, k. 2, Moscow 117811, Russia
2 Centre for Bioengineering, Russian Academy of Sciences, pr. 60-letiya Oktyabrya 7, k. 1, Moscow 117312, Russia
Correspondence
Grigorii I. Karavaiko
gregor{at}inmi.host.ru
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). On the basis of the results of 16S rDNA sequence analysis, Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Thiobacillus caldus were combined into a new genus, Acidithiobacillus, within the 
-subclass of the Proteobacteria. Arbitrarily, the species Thiobacillus albertensis was also assigned to this genus, though the 16S rRNA genes of this species have yet to be sequenced.
The distinguishing feature of the species Acidithiobacillus ferrooxidans is its ability to derive energy from the oxidation of ferrous ions. Various strains of this species have been isolated from natural (rocks, ores and mine waters) and technological (ore concentrates and pulps of the gold and non-ferrous industries) sources. The habitats of A. ferrooxidans strains are geographically extremely diverse and vary in their physico-chemical conditions (presence of particular sulfide minerals and their ratio, pH, temperature and the content of toxic compounds in the liquid phase). This might explain the polymorphism of A. ferrooxidans strains, in terms of both their physiological properties (Tuovinen et al., 1971; Harrison, 1982; Valenti et al., 1989; Leduc & Ferroni, 1994; Chisholm et al., 1998; Frattini et al., 2000; Ageeva et al., 2001) and genotypic characteristics (Martin et al., 1981; Harrison, 1982; Shiratori et al., 1991; Kondratyeva et al., 1993, 1995; Rawlings & Kusano, 1994; Rawlings, 1999). The study of the natural genotypic and phenotypic variability of bacterial strains is of great importance in discerning the microevolutionary mechanisms of species formation and for their monitoring in biohydrometallurgical processes and in nature.
This study involved a polyphasic genotypic analysis of a large group of A. ferrooxidans strains isolated from different habitats and the evaluation of the phylogenetic heterogeneity of these strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). All of the novel isolates were Gram-negative, motile, acidophilic, mesophilic, chemolithoautotrophic bacteria that obtained energy by the oxidation of Fe2+, S0 or sulfide minerals (Karavaiko et al., 1997; Kondratyeva et al., 1999; Ageeva et al., 2001). This allowed us to assign all of the novel isolates to the species A. ferrooxidans. This was supported by the genotypic analysis performed in this work. Nineteen strains, two of which (strains VKM B-458 and VKM B-1160) have been deposited in the All-Russia Collection of Microorganisms, were isolated in the Laboratory of Chemolithotrophic Micro-organisms, the Institute of Microbiology and the Russian Academy of Sciences. Two strains (ATCC 23270T and ATCC 19859) were obtained from the American Type Culture Collection. Four identified strains (TFY, TF-16, TFP and TFR-2) were kindly donated by M. Vrvi
, A. Torma, J. Hurtado and E. S. Kalyaeva.
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. The flasks were shaken at 160 or 240 r.p.m. The initial pH of the medium was 1·8-2·0. The inoculum size comprised 10 % (v/v) of the growth medium.
DNA isolation.
A. ferrooxidans cells for the isolation of DNA were grown in unshaken 5 l bottles containing 3 l growth medium aerated at a rate of 3 l air min-1. Native DNA was isolated from cells as described by Kondratyeva et al. (1995). In DNA-DNA hybridization experiments, DNA was isolated by the method of Marmur (1961).
DNA analysis.
Chromosomal DNA was analysed by PFGE (Kondratyeva et al., 1995). The G+C content of the DNA was determined by the thermal denaturation method. The genome size and the level of DNA-DNA hybridization were evaluated from the DNA renaturation rates (De Ley et al., 1970).
16S rRNA gene amplification and sequencing.
16S rRNA genes were amplified and sequenced using universal prokaryotic primers (Edwards et al., 1989). PCR amplifications were carried out using thermostable BioTaq polymerase (Dialat), according to the manufacturer's instructions. The volume of the reaction mixture was 20 µl. The PCR program consisted of one cycle of DNA denaturation at 94 °C for 3 min, primer annealing at 50 °C for 3 min and DNA synthesis at 72 °C for 3 min, five cycles of 94 °C for 30 s, 50 °C for 2 min and 72 °C for 30 s, 30 cycles of 94 °C for 30 s, 40 °C for 30 s and 72 °C for 30 s and a final incubation at 72 °C for 7 min. PCR products were analysed by electrophoresis in 1 % agarose gel. Bands were visualized by illuminating ethidium bromide-stained gels on a BioKom UV transilluminator. 16S rDNA fragments were extracted from agarose gels using a Wizard PCR Preps kit (Promega) and sequenced by the method of Sanger et al. (1977) using a Silver Sequencing kit from the same manufacturer. These procedures were performed according to the manufacturer's instructions, with minor modifications. Electrophoresis was conducted using a Macrophor device (Pharmacia) and an SQ3 Sequencer (Hoefer) at a gel thickness of 0·19 mm.
Phylogenetic analysis.
The XbaI restriction profiles of native DNA of the strains under study were formalized according to the presence or absence of DNA fragments of a certain length and analysed using the maximum-parsimony algorithm realized in the PHYLIP software package (Felsenstein, 1989) and the neighbour-joining algorithm of the FREETREE program (Pavlicek et al., 1999). A dendrogram based on DNA-DNA hybridization data was constructed using an approximate similarity matrix, whose unknown terms were calculated based on mean values for the cluster, with the aid of the UPGMA algorithm of the PHYLIP software. The 16S rRNA gene sequences of analysed strains were aligned with those of the closest micro-organisms using CLUSTAL W (Thompson et al., 1994). An unrooted phylogenetic tree was generated with the aid of the neighbour-joining algorithm realized in the TREECON package (Van de Peer & De Wachter, 1994).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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): (i) gold–arsenic ores and their concentrates; (ii) copper and copper–zinc ores and their concentrates; and (iii) other sources. The major sulfide minerals of gold–arsenic ores are pyrite (FeS2), arsenopyrite (FeAsS) and, in ores from the Olimpiadinskoe and Maiskoe deposits, pyrrhotite (FeS). Copper and copper–zinc ores from different deposits were mainly polymetallic, except that the copper ore from the Volkovskoe deposit (from which strain TFV-1 was isolated) contained primarily copper sulfide minerals. The major source of A. ferrooxidans strains isolated from coal deposits is pyrite. The pH of ore pulps depends on the composition of sulfide minerals and the activity of bacteriochemical processes in bioreactors. Correspondingly, the amounts of metal ions leached from ores into the liquid phase of ore pulps are different.
Analysis of chromosomal DNA relatedness by PFGE
PFGE of XbaI-digested DNA of various A. ferrooxidans strains showed that their genomes differ somewhat (Kondratyeva et al., 1993, 1995). Analysis of the DNA restriction profiles of 20 A. ferrooxidans strains by the maximum-parsimony algorithm showed that they fell into three clusters (Fig. 1). However, the confidence levels of most branching points of the dendrogram evaluated by bootstrap analysis were less than 70 %. This dendrogram correlated with that generated by the neighbour-joining algorithm (data not presented) with respect to only three branching points, whose bootstrap values exceeded 70 %.
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). The most frequent genome size was 2·3–2·4x106 kDa and the most frequent G+C content was about 57 mol%.
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). The strains were divided into four groups characterized by a high degree of genome similarity. Such genome groups are presently called genomovars (Ursing et al., 1995; Rossello-Mora & Amann, 2001).
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). However, some authors believe that this value for genome variability within a species should be raised to 50 % (Ursing et al., 1995; Vandamme et al., 1996). According to this criterion, the A. ferrooxidans strains under study were divided into four genomovars: (1) strains ATCC 23270T, ATCC 19859, TF-1292 and TFBk, with genome similarity levels of 59–96 %; (2) strains VKM B-1160, TFM, TFR-2, TFO, TFN-d and TFT, with genome similarity levels of 86–97 %; (3) strains TFY and TFL-2, with a genome similarity level of 92 %; and (4) strains TF-16, TFN, TFD, TFI and TFT-92, with genome similarity levels of 50–99 %. The remaining six A. ferrooxidans strains could not be assigned to any of the genomovars, since the similarity of their genomes to one another and to those of the strains comprising the genomovars was only 13-43 %.
Phylogenetic analysis of 16S rRNA genes
We determined nearly complete nucleotide sequences (about 1450 nt) of the 16S rRNA genes of five A. ferrooxidans strains: the type strain, ATCC 23270T (genomovar 1), and strains TFN-d (genomovar 2), TFY (genomovar 3), TFI and TFD (genomovar 4). A comparative phylogenetic analysis of the 16S rRNA genes was performed with respect to a large group of A. ferrooxidans strains, two validly described species of the genus Acidithiobacillus (Acidithiobacillus thiooxidans and Acidithiobacillus caldus), a species of this genus that has not yet been validly described (‘Acidithiobacillus cuprithermicus’) and several strains of this genus whose 16S rDNA nucleotide sequences are available from the GenBank database.
According to the results of this analysis, most of the A. ferrooxidans strains, including the type strain, ATCC 23270T, constituted a monophyletic cluster with a 100 % bootstrap value (Fig. 3). Within this cluster, A. ferrooxidans strains fell into three phylogenetic groups. The only exception was A. ferrooxidans strain IFO 14245, which showed sequence similarity of 97·8-98·7 % to the 16S rRNA genes of the other members of this cluster and fell into none of the groups. The strain Acidithiobacillus sp. SSP, which was not identified to the species level, was assigned to phylogenetic group III, while phylogenetic group IV included the strains Acidithiobacillus spp. NO-8, NO-25, NO-37 and KSC-1, which were also not identified to the species level. The major phylogenetic cluster included not only A. ferrooxidans strains but also several strains of A. thiooxidans (including the type strain of this species) and the unidentified strain Acidithiobacillus sp. THA, all of which constituted phylogenetic group V. The high bootstrap values of the branching points of the phylogenetic tree (not less than 87 %) confirm the statistical significance of the above phylogenetic grouping. DNA similarity levels within and between the phylogenetic groups were respectively 97·8-100 and 96·7-99·2 %. The highest level of intergroup divergence of 16S rRNA gene sequences (1·8-3·3 %) was observed for phylogenetic group V of A. thiooxidans strains.
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Some A. ferrooxidans and A. thiooxidans strains could not be affiliated with the major phylogenetic cluster. Strains [A. thiooxidans] DSM 612 and [A. thiooxidans] KCTC 8929P took an intermediate position between A. ferrooxidans, A. thiooxidans and A. caldus, showing 88·7-96·9 % 16S rRNA gene sequence similarity (square brackets indicate that the current identification of a given strain is not confirmed by 16S rRNA gene sequence data). Strain [A. ferrooxidans] LM2 was found to be close to the A. caldus type strain, DSM 8584T (99·2 % similarity). On the basis of 16S rRNA gene sequence similarity, three Acidithiobacillus strains, [A. ferrooxidans] DSM 2392 (79·9-88·4 % similarity), [A. thiooxidans] KCTC 8928P (76·1-82·2 %) and [A. thiooxidans] KCTC 8930P (68·1-76·6 %), could not be assigned to this genus.
All newly studied A. ferrooxidans strains belonged to the major phylogenetic cluster, which also included A. thiooxidans strains (Fig. 3). The type strain of A. ferrooxidans fell within phylogenetic group I (99·3-100 % 16S rRNA gene sequence similarity), strain TFN-d fell within group II (99·7-99·9 %) and strains TFI, TFD and TFY fell within phylogenetic group III (99·0-100 %).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The genotypic heterogeneity of the species A. ferrooxidans is evident even from the analysis of 16S rRNA gene sequences, which are generally much more conservative than the total bacterial genome sequence. This heterogeneity shows up in the grouping of strains belonging to this species into four phylogenetic groups. The data on DNA-DNA hybridization also indicate high genotypic divergence of A. ferrooxidans strains, which, with the exception of six independent considerably divergent strains, constitute four groups characterized by a high level of genome similarity (genomovars). These data agree well with the earlier findings of Harrison (1982), who also revealed five groups of A. ferrooxidans strains corresponding to genomovars and several strains that could not be affiliated with any of these groups. The high level of DNA–DNA relatedness (96 %) of the genomes of strain ATCC 19859 and the type strain ATCC 23270T suggests that all the allied strains studied by Harrison and us constitute one genomovar and therefore, that the members of this genomovar must be widespread in nature. It can also be suggested that analysis of a larger group of A. ferrooxidans strains than those studied by Harrison and us may disclose several additional genomovars among these strains.
Analysis of 16S rRNA gene sequences of the strains studied by DNA–DNA hybridization by Harrison (1982) allowed us, in addition, to compare the results yielded by the two methods, and showed that A. ferrooxidans strains ATCC 19859 and F221 from phylogenetic group I belong to one genomovar, while strains IFO 14262, from phylogenetic group II, and IFO 14245, from a separate phylogenetic branch, represent two other genomovars.
Therefore, in general, the results of the DNA-DNA hybridization analysis of A. ferrooxidans strains do not contradict the results of analysis of 16S rRNA gene sequences, except that the latter analysis combines the representatives of two different genomovars (strain TFY from genomovar 3 and strains TFI and TFD from genomovar 4) into one phylogenetic group. The data presented provide evidence that there are at least two levels of genotypic divergence for A. ferrooxidans. Phylogenetic grouping based on analysis of 16S rRNA gene sequences represents a higher level of the genotypic divergence of A. ferrooxidans strains, whereas the division of these strains into genomovars in accordance with the species criterion of Wayne et al. (1987) reflects the lower level of their divergence.
The DNA-DNA hybridization analysis of A. ferrooxidans and A. thiooxidans strains carried out by Harrison (1982) showed low genome similarity (not more than 23 %). Moreover, most of the A. thiooxidans strains studied, including the type strain but excluding strain DSM 612, were found to belong to one genomovar. Analysis of the 16S rRNA gene sequences of A. thiooxidans strains, including the type strain, showed that they belong to the same phylogenetic cluster as the majority of A. ferrooxidans strains, constituting a separate phylogenetic group equidistant from the other phylogenetic groups of this cluster. These data indicate a close relatedness of the species A. ferrooxidans and A. thiooxidans and allow the suggestion to be made that the latter species has diverged from the former species because of a gradual accumulation of phenotypic differences. According to the findings of Harrison (1982), A. thiooxidans strain DSM 612 does not belong to the same genomovar as other A. thiooxidans strains. Analysis of 16S rRNA gene sequences confirmed the incorrect taxonomic identification of this strain, as well as of some other strains such as KCTC 8929P and LM2 (the latter strain probably belongs to the species A. caldus). As for strains DSM 2392, KCTC 8928P and KCTC 8930P, they do not belong to the genus Acidithiobacillus at all.
Analysis of 16S rRNA gene sequences showed that the degree of relatedness of the species A. caldus to A. ferrooxidans and A. thiooxidans is lower than the degree of relatedness between the latter two species. The species ‘A. cuprithermicus’ was affiliated with the phylogenetic cluster of the species A. caldus (with a bootstrap value of 98 %), which casts doubt on the existence of ‘A. cuprithermicus’ as a separate species.
The genotypic heterogeneity of A. ferrooxidans and other species brings up the question of whether they appear as a result of microevolutionary processes within one species or arise through the convergence of several species. The data presented here, in particular those indicating the monophylicity of strains, suggest that their genotypic differences are due to microevolutionary processes. As is evident from the results of analysis of chromosomal DNA by PFGE, these processes may change the location of some genes, while their expression will be retained.
Another problem is the degree of intraspecies variability and the validity of using various physiological, biochemical and molecular genetic methods in the taxonomy of A. ferrooxidans. If we insist that the isolates studied in this work do belong to A. ferrooxidans, then this heterogeneous species is a combination of several groups of strains that have similar phenotypes but differ in their 16S rRNA gene sequences and in the composition of their total genomes. This difficulty is a specific example of the general taxonomic problem concerning agreement between the results of phenotypic and genotypic analyses. The taxonomic division of a phylogenetically heterogeneous species into genomovars and then into separate species occurs as the number of distinguishing phenotypic characteristics of these genomovars increases. It can be suggested that some genomovars of A. ferrooxidans may turn out to be separate species, as was the case with the species A. thiooxidans.
The DNA restriction profiles, obtained by PFGE, for A. ferrooxidans strains cannot adequately characterize the degree of their genome similarity, as is evident from the differences in the dendrograms (of these strains) that were generated using different algorithms. Even the most statistically significant clusters of these dendrograms do not coincide with genomovars determined by DNA–DNA hybridization. This can be accounted for by the fact that PFGE is able to detect point mutations at restriction sites, changes in the locations of IST elements (insertion sequences; Yates & Holmes, 1987) and plasmid–chromosome recombinations, i.e. events that do not alter significantly the nucleotide sequence of the total genome. PFGE is the most sensitive method of DNA analysis applied to the evaluation of intraspecies genome divergence. Unlike chromosomal DNA restriction patterns, the less-sensitive 16S rDNA fingerprinting technique fails to differentiate A. ferrooxidans strains from each other, but can differentiate them from A. caldus strains (Kamimura et al., 2001). In other words, study of the structure of the chromosomal DNA of A. ferrooxidans strains by PFGE cannot evaluate the degree of similarity of the genomes of these strains (and, therefore, the degree of their relatedness) but can detect genetic variability. This method is also of particular value in the individual characterization (‘passportization’) of microbial strains, especially those used in biohydrometallurgy, and for monitoring natural microbial communities.
It is interesting to ask whether there is a geographical correlation between the diversity of A. ferrooxidans strains and their origin. Like Harrison (1982), we failed to reveal such a correlation, although Harrison interpreted his data as an indication of selection of particular genotypes in microeconiches of particular geographical zones. Our data may be interpreted as indicating the existence of a certain degree of correlation between the similarity of the genomes of A. ferrooxidans strains and the mineralogical characteristics of their habitats. For instance, most strains of genomovar 2 (TFO, TFM, TFN-d and TFT) were isolated from gold–arsenic pyrite–arsenopyrite ores and concentrates, although some other strains of this genomovar, such as VKM B-1160 and TFR-2, were isolated from other natural substrates. It is conceivable that large-scale investigations of the genotypic features of indigenous bacterial strains in relation to the properties of their habitats might provide an insight into the role of natural substrates in the microevolution of A. ferrooxidans. This is illustrated by our data on the degree of adaptation of particular strains to oxidation substrates (Fe2+, S0 and sulfide minerals) (Ageeva et al., 2001): each strain attained its own adaptation level determined by the strain's history. At the same time, each indigenous A. ferrooxidans strain isolated from an econiche with a particular mineralogical composition of oxidation substrates was characterized by its own chromosomal DNA restriction pattern. In biohydrometallurgical technologies, indigenous strains are always dominant in the pulps of gold and non-ferrous metal ores and concentrates, gradually displacing other introduced strains. Intense oxidative processes in these pulps activate microevolutionary processes and thus promote the formation of new A. ferrooxidans strains. These strains differ in terms of the structure of chromosomal DNA, plasmid composition, optimal pH and temperature values for growth, the growth and substrate oxidation rates, resistance to heavy-metal ions, adaptability and adaptation thresholds. This may explain the failure of attempts to introduce, into natural environments, artificially constructed microbial strains that are highly productive under laboratory conditions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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