International Journal of Systematic and Evolutionary Microbiology (2000), 50, 511–516 Printed in Great Britain Reclassification of some species of Thiobacillus NOTE to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov. Donovan P. Kelly1 and Ann P. Wood2 Author for correspondence: Donovan P. Kelly. Tel: j44 24 7657 2907. Fax: j44 24 7652 3701. e-mail: mmza!dna.bio.warwick.ac.uk 1 Department of Biological The species of the genus ‘Thiobacillus’ fall into the α-, β- and γ-subclasses of Sciences, University of the Proteobacteria, the type species Thiobacillus thioparus being located in the Warwick, Coventry CV4 7AL, UK β-subclass. ‘Thiobacillus’ species exhibit almost as much diversity in DNA composition and physiology as is found collectively in all other proteobacterial 2 Microbiology Research Group, Division of Life groups. On the basis of physiological characters and 16S rRNA gene sequence Sciences, King’s College comparisons, eight of the existing Thiobacillus species are proposed for London, Franklin–Wilkins reassignment to three newly designated genera within the γ-subclass of the Building, 150 Stamford Street, London Proteobacteria, namely Acidithiobacillus, Halothiobacillus and SE1 8WA, UK Thermithiobacillus. Keywords: Thiobacillus, Acidithiobacillus, Halothiobacillus, Thermithiobacillus, reclassification In the first edition of Bergey’s Manual of Systematic we (Kelly & Wood, 2000) have reassigned eight further Bacteriology, the heterogeneity of the genus Thio- species to three new genera. These genera are Acidi- bacillus was emphasized, but insufficiently compre- thiobacillus (containing Thiobacillus thiooxidans, hensive information was available to enable a securely Thiobacillus ferrooxidans, Thiobacillus caldus and Thio- based reclassification of the species into new or existing bacillus albertensis), Halothiobacillus (containing genera (Kelly & Harrison, 1989). Since then, the Thiobacillus neapolitanus, Thiobacillus halophilus and widespread application of 16S rRNA gene sequence Thiobacillus hydrothermalis) and Thermithiobacillus analysis and the use of DNA–DNA hybridization (containing Thiobacillus tepidarius). In Fig. 1, we illu- have provided tools for clarifying and dissecting the strate the locations of the principal members of these taxonomic infrastructure of the species currently classi- new genera within the subclasses of the Proteobacteria. fied as Thiobacillus, whose members fall into the α-, β- This reassignment and creation of appropriate genera and γ-subclasses of the Proteobacteria, as shown in was encouraged by the editors of the Manual; vali- Fig. 1 (Woese et al., 1984; Lane et al., 1985, 1992; dation by IJSB\IJSEM of names published in Bergey’s McDonald et al., 1997; Goebel et al., 1999). The Manual of Systematic Bacteriology is normal practice application of these tools, together with traditional (J. T. Staley, personal communication). It is the pur- taxonomic devices, has already led to the transfer of six pose of this paper to present, formally, the new genus species from Thiobacillus into Paracoccus (the former designations and new species combinations proposed Thiobacillus versutus; Katayama et al., 1995; Rainey in the Manual. et al., 1999), Acidiphilium (the former Thiobacillus acidophilus; Hiraishi et al., 1998) and the new genus Status of the genus Thiobacillus Beijerinck (1904) Thiomonas [the former Thiobacillus intermedius, Thio- bacillus perometabolis, Thiobacillus thermosulfatus and The currently recognized species of Thiobacillus exhibit ‘Thiobacillus cuprinus’ (Moreira & Amils, 1997)]. We a wide range of physical growth conditions, e.g. have assessed the phylogenetic diversity of the re- tolerance of pH values from around 0 to above 8n5 maining species and shown how some form acceptable (with pH and temperature optima of ! 2–8 and taxonomic groupings (Kelly et al., 1998; McDonald 20–50 mC, respectively), diverse GjC content of the et al., 1997). DNA (50–68 mol%), diversity of DNA homology and a range of ubiquinones and fatty acids. All are small, In writing the Thiobacillus section for the second Gram-negative, rod-shaped bacteria (0n3–0n5i0n7– edition of Bergey’s Manual of Systematic Bacteriology, 4n0 µm), some species being motile by means of polar 01230 # 2000 IUMS 511 D. P. Kelly and A. P. Wood Proteobacteria Thiobacillus thiooxidansT 100 γ Acidithiobacillus gen. nov. subclass Thiobacillus ferrooxidansT Thiobacillus caldusT Thiobacillus tepidariusT} Thermithiobacillus gen. nov. 100 100 Nitrosococcus oceanus 100 Escherichia coli Thiobacillus neapolitanusT 100 Thiobacillus halophilusT Halothiobacillus 100 gen. nov. Thiobacillus hydrothermalisT Chromatium vinosum Methylococcus capsulatusT T 78 Azoarcus indigens b subclass 45 Rhodocyclus purpureusT 100 T 99 Thiobacillus thioparus 66 Thiobacillus aquaesulisT Neisseria gonorrhoeae T 89 Thiomonas perometabolis Comamonas testosteroni T Acidiphilium acidophilum a 1 % 100 Azospirillum lipoferumT subclass 100 T 69 Methylobacterium extorquens T 94 Agrobacterium tumefaciens Thiobacillus novellusT ................................................................................................................................................................................................................................................................................................................. Fig. 1. Phylogenetic tree based on 16S rRNA gene-sequence data analysis of members of the Proteobacteria, showing ‘Thiobacillus’ species in each of the α-, β- and γ-subclasses. The type species (Thiobacillus thioparus) is located in the β- subclass. The new genus designations for seven of the Thiobacillus species are also shown. Note that Thiomonas perometabolis and Acidiphilium acidophilum were originally also described as Thiobacillus species. The dendrogram shows the results from analyses using DNADIST, giving the bootstrap values (McDonald et al., 1997) from 100 replicates. Bar, 1% sequence divergence, determined by comparing the lengths of the horizontal lines connecting any two species. T, Type strain. flagella; no resting stages are known. Energy is derived substrates. Their distribution is seemingly ubiquitous from the oxidation of one or more reduced sulfur across marine, freshwater and soil environments, compounds, including sulfides, sulfur, thiosulfate, especially where oxidizable sulfur is abundant (e.g. polythionates and thiocyanate. Sulfate is the end sulfur springs, sulfide minerals, sulfur deposits, product of sulfur-compound oxidation, but sulfur, sewage-treatment areas and sources of sulfur gases, sulfite or polythionates may be accumulated, some- such as H#S from sediments or anaerobic soils). times transiently, by most species. Some species also Analysis of 16S rRNA sequences has revealed a derive energy by oxidizing organosulfur compounds or diversity and misclassification among the thiobacilli at by oxidizing ferrous iron to ferric iron. All species can least as profound as that shown many years ago, by fix carbon dioxide by means of the Benson–Calvin classical means, for the ‘hydrogen bacteria’, which cycle and are capable of autotrophic growth; some were subsequently reclassified into diverse genera species are obligately chemolithotrophic, while others (Davis et al., 1969). are also able to grow chemo-organotrophically. The genus currently includes obligate aerobes and fac- The genus Thiobacillus after revision in 1999 ultative denitrifying types. This diversity of properties among species is indicative of an extremely hetero- The type species, Thiobacillus thioparus (Beijerinck, geneous group, judged in terms of genetic and physio- 1904), is a member of the β-subclass of the Proteo- logical similarity. Indeed, the only historical criterion bacteria, so the original genus and species name must for concentrating all the species into one genus was be retained for this organism. Other members of the β- that all are rod-shaped eubacteria able to obtain energy subclass currently retained as Thiobacillus are Thio- for autotrophic growth by oxidizing inorganic sulfur bacillus denitrificans, which is very closely related to 512 International Journal of Systematic and Evolutionary Microbiology 50 New genera for some thiobacilli the type species, and Thiobacillus aquaesulis (Fig. 1) ferrous iron or use natural and synthetic metal sulfides and ‘Thiobacillus plumbophilus’. ‘Thiobacillus plumbo- for energy generation; some species oxidize hydrogen. philus’ (Drobner et al., 1992) has never been validated Optimum temperature 30–35 mC for mesophilic species and is in need of more detailed comparative study to and 45 mC for moderately thermophilic species. Con- determine if its retention as a Thiobacillus species is tain ubiquinone Q-8. The GjC content of the DNA is justified. Thiobacillus novellus, in the α-subclass, and 52–64 mol%. Other general characteristics are those ‘Thiobacillus prosperus’ (Huber & Stetter, 1989), in the of Thiobacillus. Members of the γ-subclass of the γ-subclass, have not yet been removed from the genus Proteobacteria. The type species is Acidithiobacillus as insufficient data are currently available to assign thiooxidans (formerly Thiobacillus thiooxidans). them to existing or new genera (Kelly & Wood, 2000). Similarly, Thiobacillus delicatus (Katayama-Fujimura et al., 1984) currently remains in the Thiobacillus genus Description of Acidithiobacillus thiooxidans (as its 16S rRNA sequence has not been reported) but (Waksman and Joffe 1922) comb. nov. its mixotrophy