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ENVIRONMENTAL AND EVOLUTIONARY MICROBIOLOGY

Phylogenetic and genetic variation among Fe(II)-oxidizing acidithiobacilli supports the view that these comprise multiple species with different ferrous iron oxidation pathways

Agnès Amouric, Céline Brochier-Armanet, D. Barrie Johnson, Violaine Bonnefoy, Kevin B. Hallberg

  • 1Laboratoire de Chimie Bactérienne, UPR-CNRS 9043, Institut de Microbiologie de la Méditerranée, Aix-Marseille Université, 31 chemin Joseph Aiguier, 13009, Marseille, France
  • 2School of Biological Sciences, College of Natural Sciences, Bangor University, Bangor LL57 2UW, UK
  • Correspondence
    Violaine Bonnefoy
    bonnefoy{at}ifr88.cnrs-mrs.fr

    • Microbiology 2011; 157(1):111–122 · https://doi.org/10.1099/mic.0.044537-0

    View at publisher PubMed

    Abstract

    Autotrophic acidophilic iron- and sulfur-oxidizing bacteria of the genus Acidithiobacillus constitute a heterogeneous taxon encompassing a high degree of diversity at the phylogenetic and genetic levels, though currently only two species are recognized (Acidithiobacillus ferrooxidans and Acidithiobacillus ferrivorans). One of the major functional disparities concerns the biochemical mechanisms of iron and sulfur oxidation, with discrepancies reported in the literature concerning the genes and proteins involved in these processes. These include two types of high-potential iron–sulfur proteins (HiPIPs): (i) Iro, which has been described as the iron oxidase; and (ii) Hip, which has been proposed to be involved in the electron transfer between sulfur compounds and oxygen. In addition, two rusticyanins have been described: (i) rusticyanin A, encoded by the rusA gene and belonging to the well-characterized rus operon, which plays a central role in the iron respiratory chain; and (ii) rusticyanin B, a protein to which no function has yet been ascribed. Data from a multilocus sequence analysis of 21 strains of Fe(II)-oxidizing acidithiobacilli obtained from public and private collections using five phylogenetic markers showed that these strains could be divided into four monophyletic groups. These divisions correlated not only with levels of genomic DNA hybridization and phenotypic differences among the strains, but also with the types of rusticyanin and HiPIPs that they harbour. Taken together, the data indicate that Fe(II)-oxidizing acidithiobacilli comprise at least four distinct taxa, all of which are able to oxidize both ferrous iron and sulfur, and suggest that different iron oxidation pathways have evolved in these closely related bacteria.

    • ↵†These authors contributed equally to this work/paper.

    • The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are provided in Figs 2⇑–4⇑ and Supplementary Table S2.

    • A supplementary figure, showing determination of the presence or absence of rusA and rusB in Fe(II)-oxidizing acidithiobacilli that reportedly contain both genes, and two supplementary tables, listing oligonucleotides used and database accession numbers of the nucleotide sequences analysed, with supplementary references, are available with the online version of this paper.

    Edited by: W. Liesack

    INTRODUCTION

    The majority of obligately chemolithoautotrophic acidophilic bacteria that can oxidize ferrous iron [Fe(II)], sulfur and reduced inorganic sulfur compounds (RISCs) have for many years been considered a priori to be strains of the well-documented species Acidithiobacillus ferrooxidans (At. ferrooxidans). The genus Acidithiobacillus has generally been considered to be a member of the Gammaproteobacteria, though recent phylogenetic analysis suggests it arose after divergence from the Alphaproteobacteria but before the Betaproteobacteria/Gammaproteobacteria split (Williams et al., 2010). There have been numerous reports suggesting that Fe(II)-oxidizing acidithiobacilli are a heterogeneous collection of bacteria with sufficient genetic variability to warrant classification as more than one species (Harrison, 1982, 1984; Novo et al., 1996; Amils et al., 1998; Selenska-Pobell et al., 1998; Paulino et al., 2001; Karavaiko et al., 2003; Mitchell et al., 2003; Bergamo et al., 2004; Akbar et al., 2005; Waltenbury et al., 2005; Peng et al., 2006; Ni et al., 2007, 2008a, b). For instance, a recent study using microarrays of the whole genome of the At. ferrooxidans type strain (ATCC 23270) showed that 12 strains isolated from different locations, and presumptively identified as At. ferrooxidans, were highly diverse at the genomic level (Luo et al., 2009). In addition to genetic variability, several phenotypic differences have been observed among Fe(II)-oxidizing acidithiobacilli, such as their motility and the presence/absence of flagella (Dispirito et al., 1982; Valdés et al., 2008; Hallberg et al., 2009, 2010; Li et al., 2010), as well as their optimal pH and temperature for growth (Harrison, 1982; Kupka et al., 2007; Dave et al., 2008; Ni et al., 2008b; Hallberg et al., 2009, 2010). On the basis of several of these differentiating characteristics, a new species (Acidithiobacillus ferrivorans; At. ferrivorans) has recently been proposed to circumscribe some of the Fe(II)-oxidizing acidithiobacilli (Hallberg et al., 2009, 2010).

    One of the major functional disparities among the Fe(II)-oxidizing acidithiobacilli concerns their propensities and mechanisms for oxidation of Fe(II) and RISCs. Different strains have been reported to display very different specific rates of Fe(II) oxidation (e.g. Suzuki et al. 1990). Harvested biomass of some strains is able to oxidize Fe(II) immediately, irrespective of the substrate on which they have been grown, while others only do so when grown on Fe(II) (Suzuki et al., 1990), suggesting either constitutive or inducible expression of the genes involved. Differences concerning RISC oxidation have also been observed, including (i) low RISC-oxidizing activities of some strains compared with others (Wakao et al., 1991; Ni et al., 2008b; Hallberg et al., 2010) and (ii) the absence in at least one strain of the thiosulfate ubiquinone oxidoreductase that is found in the type strain (Wakai et al., 2004). Furthermore, some strains oxidize Fe(II) and sulfur simultaneously, while others oxidize Fe(II) prior to sulfur (Suzuki et al., 1990; Yarzábal et al., 2004).

    In addition to these observations, inconsistencies have been reported in the literature regarding the genes encoding redox proteins involved in Fe(II) and RISC oxidation pathways. For instance, the unique high-potential iron–sulfur protein (HiPIP) present in At. ferrooxidans has been proposed to be involved in the sulfur oxidation respiratory chain in a number of strains, including the type strain and ATCC 33020 (Bruscella et al., 2005, 2007; Quatrini et al., 2006, 2009; Valdés et al., 2008), while it was described as the first electron acceptor from Fe(II) in some other strains (e.g. JCM 7811) (Fukumori et al., 1988; Kusano et al., 1992; Cavazza et al., 1995) (Fig. 1a⇓). The HiPIP has been referred to as Hip in the former case, and Iro (iron-oxidizing enzyme) in the latter. Also, several strains presumptively identified as At. ferrooxidans have been reported to have two distinct types of the blue copper protein rusticyanin (Sasaki et al., 2003). The first (RusA) was detected in some At. ferrooxidans strains including the type strain and ATCC 33020 (see references in Holmes & Bonnefoy, 2007), whereas the second (RusB) was found in some other strains (e.g. JCM 3865, JCM 7811 and IFO 14246) (Sasaki et al., 2003) and recently in most strains of At. ferrivorans (Hallberg et al., 2009, 2010). While most strains carry only one type of rusticyanin gene, some strains have been reported to possess both (JCM 7811, JCM 3865 and IFO 14246) (Sasaki et al., 2003). The rusA gene belongs to the rus operon, which encodes two cytochromes c and the aa3 cytochrome oxidase (Fig. 1b⇓), all of which have been shown to be involved in Fe(II) oxidation (see references in Holmes & Bonnefoy, 2007; Quatrini et al., 2009). While RusA is considered to play a central role in this process, the function of RusB, which has a lower kinetic rate constant for electron transfer from Fe(II) (Ida et al., 2003), has not been established. Interestingly, neither rus nor rusticyanin has been detected in one strain (CF27) of At. ferrivorans, though this strain is capable of growth with Fe(II) as electron donor (Blake & Johnson, 2000; Hallberg et al., 2009, 2010). In addition, strain AF2, presumptively identified as At. ferrooxidans, contains neither the rusA nor the coxC (aa3-type cytochrome oxidase subunit III) genes from the rus operon (Chen et al., 2009). This suggests that both of these strains use a different Fe(II) respiratory chain from that of At. ferrooxidans ATCC 23270T.

    Figure image not available in archive
    Fig. 1.

    Genetic organization of the rusticyanin- and HiPIP-encoding genes and the pathways in which they are involved. (a) hip in At. ferrooxidans ATCC 23270T and ATCC 33020 involved in the electron transfer chain between RISCs and O2; iro in JCM 7811 proposed to be involved in the electron transfer chain between Fe(II) and O2. (b) rusA in At. ferrooxidans ATCC 23270T and ATCC 33020 involved in the Fe(II) oxidation pathway; rusB in JCM 3865, JCM 7811 and IFO 14246, for which a role is unknown. Solid arrows represent transcriptional units; dotted arrows represent electron transport.

    All of these observations support the hypothesis that the Fe(II)-oxidizing acidithiobacilli encompass distinct species, including At. ferrooxidans and At. ferrivorans, that are metabolically similar but taxonomically distinct. In this paper, we address two questions. (i) Is there a correlation between phylogenetic relationships and genetic heterogeneity among the Fe(II)-oxidizing acidithiobacilli, in particular concerning rusA/B, hip/iro genes? (ii) Could such heterogeneity be useful in delineating different species of these bacteria? To answer these questions we have reconstructed the phylogeny of 21 Fe(II)-oxidizing acidithiobacilli based on a multilocus sequence analysis (MLSA), and investigated the presence of genes encoding the two rusticyanins (rusA/B) and the two HiPIPs (hip/iro).

    METHODS

    Strain selection and cultivation conditions.

    The Fe(II)-oxidizing acidithiobacilli used in this study are listed in Table 1⇓, of which the genome sequences of two (ATCC 23270T and ATCC 53993) are in the public domain. Strains CC1, CB5 and B20 were isolated from arsenic-rich mining sites, while strains R1, A1 and A2 have been described as producing more extracellular polysaccharide (EPS) than At. ferrooxidans ATCC 23270T (Harneit et al., 2006).

    Table 1.

    Strains of Fe(II)-oxidizing acidithiobacilli used in this study

    Purity of cultures was tested by streaking onto both ferrous sulfate and yeast extract overlay solid media (Johnson & Hallberg, 2007) to screen for the presence of single-colony morphologies of Fe(II)-oxidizing bacteria and heterotrophic contaminants, respectively. Following the inference that JCM 7811 comprises more than one strain of Fe(II)-oxidizing acidophile, the freeze-dried biomass obtained was suspended in 200 μl acidified (pH 2.5) basal salts solution and portions were inoculated into a 20 mM ferrous sulfate/basal salts/trace elements medium (Wakeman et al., 2008) that had been adjusted to either pH 1.6 or 2.2. When the cultures had oxidized most of the ferrous iron (as indicated by a colour change from colourless to orange), they were streak-inoculated onto ferrous iron overlay plates, and representative colonies of differing morphologies were picked off and maintained as separate cultures (denoted as JCM 7811-P1 and JCM 7811-P4).

    Most of the strains were grown under aerobic conditions at 30 °C in acidified (pH 1.6) ferrous sulfate/basal salts liquid medium, as described previously (Yarzábal et al., 2003). Strains of At. ferrivorans (which is less acidophilic than At. ferrooxidans) were also grown at 30 °C but at pH 2.2 (Hallberg et al., 2010).

    DNA extraction, amplification and sequencing.

    Genomic DNA from most of the Acidithiobacillus strains was prepared with the NucleoSpin Tissue kit (Macherey Nagel). At. ferrivorans cells and those from the original JCM and IFO strain suspensions (100 μl) or from liquid cultures of the latter (1 ml) were harvested, and crude DNA was extracted as described previously (Okibe et al., 2003).

    Primers used for PCR amplification of each targeted gene and the sizes of the expected PCR products are described in Supplementary Table S1 available with the online version of this paper. The PCR program consisted of an initial denaturation step at 94 °C for 2 min 30 s, followed by 30 (or 40) cycles of 30 s at 94 °C, 30–60 s at primer annealing temperature (see Supplementary Table S1), 30–90 s at 72 °C, with a final extension step at 72 °C for 7 min. PCR products were analysed by electrophoresis in a 1 % agarose gel, then concentrated and purified using Microcon PCR (Millipore) or QIAquick (Qiagen) reagents.

    Nucleotide sequences of the amplified DNA were determined by Cogenics or Eurofins MWG operon, or as described previously (Hallberg et al., 2006).

    Phylogenetic analysis.

    The sequences of the phylogenetic markers used in this study [rrs (16S rRNA gene), 16S–23S ITS1, atpD, nifH, recA, rusA/B and iro/hip] were obtained from the published genomes of At. ferrooxidans ATCC 23270T and strain ATCC 53993, or, for other strains, were determined as described above. For the analysis of the rrs gene, we used sequences from two closely related species (Acidithiobacillus thiooxidans and Acidithiobacillus albertensis) as outgroups. These sequences were retrieved from the nr database at the National Center for Biotechnology Information (NCBI) (accession numbers AJ459803 and AJ459804, respectively). For each marker, sequences were aligned using clustal w2 default parameters (Larkin et al., 2007). The resulting alignments were inspected using the must software (Philippe, 1993). Regions where the alignment was ambiguous were removed prior to phylogenetic analysis using must. The concatenation based on the alignments of atpD, nifH and recA was constructed using in-house software.

    For each marker, and for the concatenated sequences, phylogenetic trees were reconstructed using maximum-likelihood and Bayesian methods implemented, respectively, in treefinder (Jobb et al., 2004) and MrBayes (Ronquist & Huelsenbeck, 2003). For maximum-likelihood analyses, the evolutionary models used were selected according to the ‘propose model’ tool implemented in treefinder. The branch robustness of the resulting trees was estimated by the non-parametric bootstrap procedure implemented in treefinder using the same parameters. For Bayesian analyses we used the GTR model and a gamma distribution to account for evolutionary site rate variations (four categories and an estimated alpha-parameter). MrBayes was run with four chains for 1 million generations and trees were sampled every 100 generations. To construct the consensus tree, the first 1500 trees were discarded as ‘burn-in’.

    DNA–DNA hybridization.

    Cells from 80 l cultures of Fe(II)-grown At. ferrooxidans ATCC 23270T and strain ATCC 33020 were lysed using a French pressure cell (Thermo Spectronic), and genomic DNA from these lysates was purified by chromatography on hydroxyapatite, as described by Cashion et al. (1977). DNA–DNA hybridization was carried out by the Deutsche Sammlung von Mikroorganismen und Zellkulturen, as described by De Ley et al. (1970) with the modifications of Huß et al. (1983), using a Cary 100 Bio UV/Vis spectrophotometer equipped with a Peltier-thermostatted 6×6 multicell changer and a temperature controller with an in situ temperature probe (Varian).

    RESULTS

    MLSA-based phylogeny

    While the rrs gene sequences of the strains examined showed less than 2 % divergence, which is less than the often-cited threshold of 3 % recommended to differentiate species (Stackebrandt & Goebel, 1994), phylogenetic analysis clearly placed them into four distinct clusters (I, II, III and IV, Fig. 2a⇓). Group I is represented by At. ferrooxidans ATCC 23270T and also includes ATCC 53993, ATCC 19859 and strains A1 and A2. Group II is represented by ATCC 33020, and also includes strains CC1, CB5, B20 and BRGM1. Group III is composed exclusively of strains of At. ferrivorans, and Group IV of strains JCM 7812, JCM 3865, IFO 14246 and R1. However, while these four clusters were well supported by high bootstrap values (BVs) (≥96 %) and posterior probabilities (PPs) (≥0.98), the relationships to each other are not fully resolved. Indeed, while the link between Groups III and IV was well supported (BV=96 % and PP=1.0), their relationship with Group II strains was less clear (BV=78 % and PP=0.74, Fig. 2a⇓). This lack of resolution is likely to be linked to the high similarity between rrs gene sequences, which also confounds the classification of the Fe(II)-oxidizing acidithiobacilli strains based on this gene sequence alone.

    Figure image not available in archive
    Fig. 2.

    Bayesian phylogenetic trees of the Fe(II)-oxidizing Acidithiobacillus rrs gene (a) and the 16S–23S rRNA ITS1 (b) (1387 and 577 positions, respectively). The rrs tree is rooted with sequences from Acidithiobacillus thiooxidans (Att) and Acidithiobacillus albertensis (Ata). The ITS1 tree is unrooted. Italicized numbers at nodes represent PPs, whereas numbers in bold type represent BVs. For clarity, only PPs greater than 0.5 and BVs greater than 50 % are shown. The scale bars represent the average number of substitutions per site. At. ferrooxidans ATCC 23270T and At. ferrivorans DSM 22755T are in Group I and Group III, respectively. Accession numbers of the nucleotide sequences are given in parentheses. Type strains are indicated in bold type.

    Four additional markers were analysed to refine the phylogeny of Fe(II)-oxidizing acidithiobacilli. Importantly, these markers are located in different regions of the chromosome, at least in those of At. ferrooxidans ATCC 23270T and strain ATCC 53993, limiting biases due to single horizontal gene transfer (HGT) of adjacent loci. The unrooted phylogenies based on each of these genes were congruent with the tree based on rrs gene sequences. In particular, Groups I to IV were recovered and well supported in all trees (Figs 2b⇑ and 3a–c⇓). The only exceptions were Group I and Group IV, in ITS1 and nifH trees, respectively. In the former case, ITS1 sequences from strains A1 and A2 did not cluster with other Group I sequences (Fig. 2b⇑), while in the latter case, the JCM 3865 nifH sequence emerged as a sister of Group I, albeit with moderate statistical support (BV=82 % and PP=0.69, Fig. 3b⇓). Interestingly, in the ITS1 tree, the grouping of the strains correlated with sequence lengths, as observed by Ni et al. (2007, 2008a). The size of the ITS1 was 441, 454, 442–443 and 452 bp for strains from Groups I–IV, respectively. Exceptions were strains A1 and A2, which harboured an atypical and divergent ITS1 resulting from the insertions of short runs of nucleotides, which also might skew the phylogenetic analysis, and resulted in the separation of these two strains from the rest of the Group I strains with a very long branch (Fig. 2b⇑). The close relationship between Group III and Group IV based on rrs analysis was also observed in the atpD and recA trees (BV=100 and 82 %, and PP=1.0 and 0.89) (Fig. 3a, c⇓).

    Figure image not available in archive
    Fig. 3.

    Bayesian unrooted phylogenetic trees of Fe(II)-oxidizing Acidithiobacillus atpD (a) (1256 positions), nifH (b) (346 positions) and recA (c) (805 positions) genes and of their concatenation (d) (2407 positions). Italicized numbers at nodes represent PPs, whereas numbers in bold type represent BVs. For clarity, only PPs greater than 0.5 and BVs greater than 50 % are shown. The scale bars represent the average number of substitutions per site. At. ferrooxidans ATCC 23270T and At. ferrivorans DSM 22755T are in Group I and Group III, respectively. Accession numbers of the nucleotide sequences are given in parentheses. Type strains are indicated in bold type.

    These results support the hypothesis that Fe(II)-oxidizing acidithiobacilli comprise at least four distinct taxa. However, as with the rrs gene phylogeny, the branching pattern within each group was poorly supported (most BVs <90 %, Figs 2⇑ and 3⇑), indicating that the phylogenetic signal carried by these individual markers is not sufficient to resolve the relationships between the corresponding strains, possibly due in part to their relatively small size but also to the great similarity between sequences.

    To improve the resolution of the relationships within each group, sequences of the recA, atpD and nifH genes were concatenated, leading to a supermatrix of 2407 nt positions. The resulting phylogenetic tree supported the split of the Fe(II)-oxidizing acidithiobacilli into four clades (BV ≥98 % for groups I, II and III and 66 % for group IV, Fig. 3d⇑). Interestingly, whereas most strain relationships within groups remained poorly resolved, some well-supported groupings emerged: A1 and A2 strains within Group I (BV=100 % and PP=0.99); the four remaining strains of Group I (BV=100 % and PP=1.0); BRGM1 and ATCC 33020 strains within Group II (BV=100 % and PP=1.0); OP14 and CF27 strains in Group III (BV=92 % and PP=0.92); Peru6 and SS3-P1 strains, together with At. ferrooxidans ATCC 23270T in Group III (BV=83 % and 98 %, and PP=0.72 and 0.79, respectively); and IFO 14246, JCM 7812 and JCM 7811-P4 (BV=88 %), together with R1 (BV=83 % and PP=0.94), within Group IV.

    Taxonomy of Fe(II)-oxidizing acidithiobacilli

    MLSA showed that At. ferrooxidans ATCC 23270T and strain ATCC 33020 belong to two different phylogenetic groups (I and II), though their rrs genes have 99 % sequence identity. The same gene of At. ferrooxidans ATCC 23270T and Group IV strain JCM 7812 shared 98.5 % sequence identity. All DNA–DNA hybridization values (DDHVs) for strains identified by MLSA to be in Group I were >80 % (W. Sand, personal communication) (Table 1⇑), confirming that these do indeed belong to the same species as At. ferrooxidans ATCC 23270T. By contrast, Group II strain ATCC 33020 and At. ferrooxidans ATCC 23270T were shown to have a DDHV of only 63 % (Table 1⇑), a value similar to that (60 %) previously reported for strain ATCC 33020 and another Group I strain (ATCC 19859) (Harrison, 1982). Elsewhere, it has been found that At. ferrooxidans ATCC 23270T and Group IV strain R1 have a DDHV of 40 % (W. Sand, personal communication), while Harrison (1982) had earlier reported an even smaller DDHV (24 %) for Group I strain ATCC 19859 and Group IV strain IFO 14246 (Table 1⇑). Since DDHVs of <70 % are generally considered to indicate different species (Wayne et al., 1987), it is apparent that neither Group II nor Group IV bacteria should be regarded as strains of At. ferrooxidans, like those in Group III, which are strains of At. ferrivorans.

    Representative isolates from Groups I–IV revealed some phenotypic differences from each other (Table 2⇓). These included: (i) a lesser propensity for growth on sulfur or tetrathionate (as evidenced by protracted lag phases) in strains of Groups III and IV compared with strains of Groups I and II; (ii) similar cell sizes to each other of bacteria of Groups I, II and IV, although notably smaller than that of At. ferrivorans (Group III); (iii) non-motility of cells of Groups I and IV, and motility of Groups II and III; (iv) growth at 4 °C of Group III acidithiobacilli alone; and (v) sensitivity to low pH and absence of growth at pH 1.6 of Group III strains.

    Table 2.

    Differentiation of Fe(II)-oxidizing Acidithiobacillus species and isolates based on selected phenotypic traits

    All strains grew at 30 and 37 °C and in medium of pH 2.2. The main differences are indicated in bold type.

    Differences in the ferrous iron oxidation pathway among acidithiobacilli

    The biochemical mechanism by which At. ferrooxidans mediates Fe(II) oxidation has been the subject of considerable debate and some controversy for a number of years. To ascertain whether some of the disparate results reported correlate with the phylogenetic clustering of strains revealed in the present study, PCR amplification of the hip, iro, rusA and rusB genes in the strains examined was performed. The results revealed a strong correlation between the distribution of rusA/rusB and hip/iro among the strains and the four phylogenetic groups detected by MLSA (Fig. 4⇓). Notably, the presence of a rusA gene and the absence of a rusB gene were detected in strains from Groups I and II, while one, two or no copies of rusB, and no rusA gene were observed in strains from Groups III and IV with the primers used. Furthermore, whereas the hip gene was amplified from all strains in Groups I and II, only the iro gene was detected in strains from Groups III and IV with our primers. Phylogenies based on the rusA/rusB and hip/iro genes were in agreement with those obtained by MLSA (Fig. 4⇓).

    Figure image not available in archive
    Fig. 4.

    Bayesian unrooted phylogenetic trees of Fe(II)-oxidizing Acidithiobacillus iro/hip (a) and rus (b) genes (358 and 392 positions, respectively). Italicized numbers at nodes represent PPs, whereas numbers in bold type represent BVs. Stars highlight the two different copies of rusB amplified from JCM 3865. For clarity, only PPs greater than 0.5 and BVs greater than 50 % are shown. The scale bars represent the average number of substitutions per site. At. ferrooxidans ATCC 23270T and At. ferrivorans DSM 22755T are in Group I and Group III, respectively. Accession numbers of the nucleotide sequences are given in parentheses. Type strains are indicated in bold type.

    Rusticyanin in strains JCM 7811, JCM 3865 and IFO 14246

    As mentioned above, some Fe(II)-oxidizing acidithiobacilli reportedly have both rusA and rusB genes, and JCM 3865 has been reported to have two copies of the latter (Sasaki et al., 2003). The presence of two copies of rusB in JCM 3865 could be explained by a single and specific gene duplication event in this strain. However, since none of the confirmed pure cultures of Fe(II)-oxidizing acidithiobacilli analysed in this study was found to contain both rusA and rusB, we examined the possibility that the cultures previously examined might have comprised more than a single strain.

    DNA extracted from the original lyophilized culture of JCM 7811 gave a strong rusA and a weak rusB PCR product, whereas DNA from subcultures grown at either pH 1.6 or 2.2 gave relatively stronger rusB PCR products (Supplementary Fig. S1). The two colony variants obtained from the original culture, strains JCM 7811-P1 and JCM 7811-P4, matched the description of those of strains Fe1 and Fe2 (Wakao et al., 1991), which were deposited as JCM 7811 and 7812, respectively. MLSA showed that JCM 7811-P1 belongs to Group I, and it was found to contain hip and rusA (but not rusB) genes (Figs 2⇑–4⇑). Like other Group I strains, it was not motile, grew at pH 1.6 but not at 4 °C, and oxidized RISCs efficiently (Table 2⇑). In contrast, the second isolate, JCM 7811-P4 belongs to Group IV, which also includes JCM 7812. It harbours rusB (but not rusA) and iro genes (Figs 2⇑–4⇑). In addition, it shares physiological similarities with other representatives of Group IV; in particular, it exhibited a long lag phase prior to growth on RISCs (Table 2⇑). It was thus apparent that the original JCM 7811 culture was mixed and contained at least two distinct strains of Fe(II)-oxidizing acidithiobacilli.

    In contrast, the JCM 3865 culture was confirmed to be pure. However, while it was initially described as having two copies of rusB and one of rusA, we detected only the two different rusB genes (stars in Fig. 4b⇑) and no rusA (Supplementary Fig. S1). The same result was obtained with a few pure colonies isolated from the original culture supplied. MLSA (Figs 2⇑–4⇑) and physiology analysis (Table 2⇑) placed JCM 3865 in Group IV, and like other strains in this group it also contained the iro gene (Fig. 4⇑).

    We failed to get growth from two lyophilized cultures of IFO 14246 provided independently by the National Institute of Technology and Evaluation (NITE) Biological Resource Center, Japan. However, DNA was extracted directly from one of the lyophilized cultures, and this served as template for PCR and subsequent sequencing. Each of the DNA fragments amplified was highly identical to those of JCM 3865, JCM 7812 and JCM 7811-P4. MLSA placed IFO 14246 unambiguously in Group IV (Figs 2⇑–4⇑). As with all strains of Group IV, rusB and iro genes were amplified from the lyophilized IFO culture, but rusA and hip were not (Supplementary Fig. S1).

    DISCUSSION

    DNA–DNA hybridization and phylogenetic analysis of rrs gene sequences have been the main techniques used in recent decades to determine bacterial taxonomy (Stackebrandt & Goebel, 1994). More recently, MLSA, which has a number of advantages over more traditional approaches (Maiden, 2006), has been used to clarify areas where phylogenetic relationships are unclear, allowing the refinement of the taxonomy of composite bacterial groups such as the Geobacteraceae (Holmes et al., 2004), Xanthomonas (Young et al., 2008), Streptomyces (Guo et al., 2008) and Bradyrhizobium (Rivas et al., 2009). In the current work, MLSA has been used to differentiate strains of Fe(II)-oxidizing acidithiobacilli and has provided firm evidence of the existence of multiple species within this bacterial group.

    Collectively, the results of the rrs, ITS1, recA, atpD, nifH, rusA/B and hip/iro phylogenetic analyses showed that the 21 strains analysed fell into four distinct groups (Figs 2⇑–4⇑), which currently comprise only two recognized species. Group I includes strains of At. ferroxidans while Group III is composed exclusively of At. ferrivorans strains. Group II includes ATCC 33020, a strain that has previously been suggested to be a distinct species (Selenska-Pobell et al., 1998). This grouping is similar to that of Karavaiko et al. (2003), who found that 17 strains of ‘At. ferrooxidans’ also fell into four distinct phylogenetic groups based on 16S DNA gene sequences, though Groups III and IV are reversed in the present study. DNA–DNA hybridization data confirmed that strains of Groups II and IV, like those of At. ferrivorans (Group III), do not belong to the species At. ferrooxidans (Group I) (Table 1⇑). Furthermore, MLSA (Figs 2⇑–4⇑) suggested that Groups III and IV are related and distinct from Groups I and II. Group IV strains, however, were different from Group III bacteria in some physiological traits, including growth at low temperatures, sensitivity to low pH, cell size and motility (Table 2⇑). We therefore suggest that phylogenetic Groups II and IV represent novel species of Acidithiobacillus, though more in-depth physiological analysis is necessary before these can be officially designated novel species.

    One of the most significant findings from this work concerns the observed distribution of rusticyanin- and HiPIP-encoding genes in these four groups. rusA and hip genes were found only in Groups I and II, while rusB and iro were present only in Groups III and IV, which correlates with the close phylogenetic relationship between these respective groups (Figs 2⇑–4⇑). These data further support the view that Group II bacteria belong to a species different from those in Groups III and IV, and also that Group IV bacteria are distinct from At. ferrooxidans (Group I strains) (Figs 2⇑–4⇑). Also, the failure to detect any strain that harboured copies of both rusA and rusB, coupled with the finding that one of these cultures contained both Group I and Group IV strains, implies that the results of Sasaki et al. (2003) were erroneous and due to the use of mixed cultures. However, the existence of two rusB genes in strain JCM 3865 was confirmed. The differences in the distribution of rusA/B and hip/iro genes suggest that pathways of ferrous iron oxidation in bacteria in Groups I and II are different from those in Groups III and IV, which probably accounts for the disparate results obtained previously by different research groups who have investigated this subject (e.g. Fukumori et al., 1988; Appia-Ayme et al., 1999; Sasaki et al., 2003; Bruscella et al., 2005). The upstream and downstream regions of the two rusticyanin genes are also completely different. The rusA gene (in Groups I and II) belongs to the rus operon (Fig. 1b⇑; Bengrine et al., 1998; Appia-Ayme et al., 1999; Valdés et al., 2008), which is more highly expressed when cells are grown with ferrous iron than with sulfur (Yarzábal et al., 2004; Quatrini et al., 2006, 2009). In contrast, analysis of the DNA sequence data from JCM 3865 (Sasaki et al., 2003) revealed that upstream of rusB there is a gene that encodes a putative VagC toxin component of a toxin–antitoxin system (Fig. 1b⇑). The location of the rusB gene in strains of At. ferrivorans (Group III) is currently unknown, and no information is available about the regulation of the expression of this gene in any Acidithiobacillus strain. The absence of rusB (as well as rusA) in At. ferrivorans strain CF27, which nonetheless grows by iron oxidation, suggests strongly that rusticyanin is not a core component of the Fe(II)-oxidizing apparatus in Group III or (by inference) Group IV strains.

    All strains in Groups III and IV have been shown to contain the iro gene, which encodes a HiPIP proposed to be an iron oxidase (Fukumori et al., 1988; Kusano et al., 1992), and which is monocistronic and located between the purA and leucyl tRNA genes (Kusano et al., 1992) (Fig. 1a⇑). The iro gene was not detected in the genome of At. ferrooxidans ATCC 23270T and ATCC 53993 (Bruscella et al., 2005; Valdés et al., 2008; this study), and could not be amplified from any strain in Groups I and II (Bruscella et al., 2005; this study). While neither At. ferrooxidans ATCC 23270T nor Group II strain ATCC 33020 contains the iro gene, they both possess a homologue of iro, that is the hip gene. The hip gene has been shown to be located in a genetic context different from that of the iro gene, and is part of the petII operon encoding the bc1 complex (Fig. 1a⇑). Transcription of this gene is higher when cells are grown with sulfur than with Fe(II) (Quatrini et al., 2006, 2009; Bruscella et al., 2007), implying that, in contrast to iro, hip participates in the oxidation of RISCs rather than Fe(II).

    The results of this study strongly suggest that at least two different pathways for Fe(II) oxidation have evolved in the four highly related taxa of Fe(II)-oxidizing acidithiobacilli identified, and reconcile the models (Fig. 1⇑) that had previously been considered to be conflicting. In At. ferrooxidans ATCC 23270T, other Group I strains and Group II strains, proteins encoded by rusA and the corresponding operon have a central role, while in Group III (At. ferrivorans) and Group IV strains, the iron oxidase encoded by iro is probably a key component. Other questions remain, such as the role of rusB in At. ferrivorans (in those strains which possess it) and Acidithiobacillus Group IV strains, and this will be the addressed in future work.

    Acknowledgments

    We gratefully acknowledge M. Dopson (Linnaeus University, Sweden) for providing the SS3 culture and O. Bruneel (Montpellier University, France) for strains B20 and CB5. We owe special thanks to W. Sand (Duisburg-Essen University, Germany) for strains R1, A1 and A2, and for giving us access to unpublished results. We thank D. Moinier (CNRS, Marseille) for her excellent technical assistance. Part of this work was financed by the EU framework 6 project ‘BioMinE’ ( °NM2.ct, 2005.500329), and we thank our various partners on the project for their contributions to the work reported in this paper. A. A. was supported by a CNRS fellowship. D. B. J. is grateful to the Royal Society (UK) for the award of an Industrial Fellowship. C. B-A. is supported by an Action Thématique et Incitative sur Programme (ATIP) of the Centre National de la Recherche Scientifique.

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