International Journal of Systematic Bacteriology (1 998), 48, 1389-1 398 Printed in Great Britain Phylogeny and photosynthetic features of Thiobacillus acidophilus and related acidophilic bacteria : its transfer to the genus Acidiphilium as Acidiphilium acidophilum comb. nov. Akira Hiraishi,’ Kenji V. P. Nagashima,’ Katsumi Matsuura,’ Keizo Shimada,’ Shinichi Takai~hi,~Norio Wakao4 and Yoko Katayama5 Author for correspondence: Akira Hiraishi. Tel: +81 532 44 6913. Fax: +81 532 44 6929. e-mail : hiraishiialeco.tut.ac.jp Department of Ecological Phylogenetic analyses based on 16s rDNA sequences and genomic DNA-DNA Engineering, Toyohashi relatedness showed that the sulphur-oxidizing facultative chemolithotroph University of Technology, Toyohashi 441, Japan Thiobacillus acidophilus was closely related to members of the genus Acidiphilium, which is a group of strictly aerobic, heterotrophic acidophiles Department of Biology, Tokyo Metropol itan now categorized into aerobic photosynthetic bacteria. Lipophilic pigment University, Hachioji 192-03, analyses revealed that zinc-chelated bacteriochlorophyll a and carotenoids Japan occurred in appreciable amounts in T. acidophilus and all established species of B iolog ica I Laboratory, the genus Acidiphilium. PCR experiments showed that T. acidophilus as well as Nippon Medical School, Acidiphilium species contained puf genes, encoding the photosynthetic Kawasaki 21 1, Japan reaction centre proteins and the core light-harvesting complex of the purple Department of Bioscience bacteria. There were high similarities between T. acidophilus and Acidiphilium and Technology, lwate University, Morioka 020, species in the primary structure of their reaction centre proteins deduced from Japan the nucleotide sequence data. The phylogenetic tree of the reaction centre Department of proteins was in agreement with the 165 rDNA sequence-based phylogenetic Environmental and Natural tree in the relationship between T. acidophilus and Acidiphilium species and Resources Science, Tokyo between the Acidiphilium cluster and other purple photosynthetic bacteria. University of Agriculture and Technology, Fuchu Based on these results, together with previous phylogenetic and phenotypic 183, Japan information, it is proposed to reclassify T. acidophilus (Guay and Silver) Harrison 1983 as Acidiphilium acidophilum comb. nov. The type strain is ATCC 27807T (= DSM 700T). Keywords : Thiobacillus ncidophilus, zinc-bacteriochlorophyll, photosynthetic reaction cent re, phylogeny , Acidiphilium acidophilum corn b. nov. INTRODUCTION number of acidophilic species (Harrison, 1984 ; Pronk et a/., 1990), including Thiobacillus acidophilus, in The genus Thiobacillus is a group of obligately or addition to neutrophilic thiobacilli. The name T. facultatively chemolithotrophic aerobic proteo- acidophilus was proposed by Guay & Silver (1975) for bacteria that are capable of growing with reduced some strains of facultatively chemolithotrophic acido- inorganic sulphur compounds as sole energy source, philic thiobacilli, but did not appear on the Approved but, at this time, is quite heterogeneous with members Lists of Bacterial Names (Skerman et a/., 1980). exhibiting a wide range of physiological, chemo- Harrison (1983) revived the name T. acidophilus taxonomic and genetic characteristics (Katayama- following his confirmation that this organism grew Fujimura et a/., 1982, 1983; Kelly & Harrison, 1989; equally well with elemental sulphur or glucose as a sole Lane et ul., 1985, 1992). This group encompasses a energy source. Phylogenetic analyses based on 5s rRNA sequences (Lane et a/., 1985) and partial 16s .. .. ., .. .. .. ,. .. , . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. Abbreviations: BChl, bacteriochlorophyll; BPhe, bacteriopheophytin. rRNA sequences (Lane et a/., 1992), however, indi- The DDBJ accession numbersforthe 165 rDNAsequences determined in this cated that T.acidophilus was far distant from any other paper are D86508, D86509, D86511, D86513 and AB006712; for the puf Thiobacillus species and was closely related to species gene the number is AB013379. of the genus Acic~@kilium,which includes aerobic ~- 00819 0 1998 IUMS 1389 A. Hiraishi and others acidophilic chemo-organotrophic bacteria that are American Type Culture Collection (Rockville, MD, USA). unable to use reduced sulphur compounds as energy A. multivorum AIU30 1 was isolated from acidic mine source (Harrison, 1989; Kishimoto et al., 1995b). drainage (Wakao et al., 1994). All other strains were iso- Despite the dissimilarity in sulphur metabolism be- lated newly by us from acidic mine water. All test organisms were grown aerobically at 30 "C in GYS medium, a chemic- tween and species, the T. acidophilus Acidiphiliuvlz ally defined medium (pH 3.5) which consisted of a mineral available phylogenetic information has strongly base RM2 (Hiraishi & Kitamura, 1984), 15 mM glucose as suggested that the former species should be positioned the sole carbon source and 0.03 % (w/v) yeast extract as the among members of the genus Acidiphilium. growth factor. T. acidophilus was also grown chemolitho- trophically with elemental sulphur as the energy source as Recent research has shown that Acidiphilium species described by Harrison (1983). For chemical and genetic can be categorized into a group of aerobic photo- testing, cells were harvested by centrifugation from a culture synthetic bacteria (Shimada, 1995) because of their at the late-exponential phase of growth, washed twice with production of bacteriochlorophyll (BChl) only under sterile 50 mM phosphate buffer (pH 6-8) and pelleted. The aerobic growth conditions and their photosynthetic cell pellets were used immediately for analysis or stored at activity (Kishimoto et al., 1995a; Wakao et al., 1993, -20 "C until they were analysed. 1994). Interestingly, it has been shown more recently Analysis of BChls. Lipophilic pigments were extracted from that representative member a of Acidiphilium, fresh wet cells with acetone-methanol (7 : 2, v/v), evaporated Acidiphilium ruhrum, contains a fully active photo- in vacuum and analysed by reverse-phase HPLC with a synthetic system with zinc-chelated bacteriochloro- Shimadzu Liquid Chromatograph LC- 1OA equipped with a phyll (Zn-BChl) a as the major pigment (Wakao et al., Beckman Ultrasphere ODS column (4-6 i.d. x 250 mm) in a 1996). This was the first demonstration of the existence column oven at 30 "C. Samples were eluted with methanol at of natural photosynthesis using (bacterio-)chloro- a flow rate of 1 ml min-l and monitored with a photodiode phylls containing a metal other than magnesium. array detector, Shimadzu SPD-IOA, in a wavelength range Further studies have shown that all established species of 350-800 nm. Post-run data analysis was performed with of the genus Acidiphilium contain the puf operon the Shimadzu CLASS-M 1 OA program. For the identi- (Nagashima et al., 1997b), which is an assemblage fication and quantification of BChls, parameters for peak of genes encoding the proteins of the photosynthetic identification and calibration of detector response factors were set in the program on the basis of HPLC data on a reaction centre (L, M and C subunits) and the core known concentration of A. rubrum Zn-BChl a which had light-harvesting complex subunits) the (a and ,!3 of been determined spectrophotometrically (Wakao et al., purple photosynthetic bacteria. The structure of the 1996). BChl a and bacteriopheophytin (BPhe) purified from puf' operon is well-conserved among species of these a purple phototrophic bacterium, Rhodobacter sphaeroides bacteria, and thus the PCR technique is applicable DSM 158', were also used as the standard pigments. for detection of a conserved region of puf genes Although zinc-chelated BChl should be called zinc-BPhe (Nagashima et al., 1997a, b). more precisely, we used herein the term Zn-BChl for convenience. These recent findings motivated us to re-evaluate the phylogenetic relationships between T. acidophilus and Analysis of carotenoids. Pigments extracted as noted above members of the genus Acidiphilium and to determine were analysed by HPLC equipped with a pBondapak C18 whet her T. acidop hilus has photosynthetic properties . column (8 mm i.d. x 100 mm) (Waters). Carotenoid com- ponents were eluted with methanol at flow rate of 2 ml min previous study undertaken to detect photopigments ' A and detected with a MCPD-3600 photodiode array detector in T. acidophilus gave negative results (Kishimoto et (Otsuka Electronics) in a wavelength range of 250-600 nm al., 1995a), but our attempts to find Zn-BChl a andpgf (Takaichi & Shimada, 1992). For spectrophotometric genes in T. acidophilus, as well as in all Acidiphilium measurement of carotenoids, the following extinction species, have been successful. Phylogenetic analyses coefficients in methanol were used: 150 mM-' cm-l at 492 based on 16s rDNA sequences, genomic DNA-DNA and 480 nm for spirilloxanthin and rhodovibrin, respect- relatedness and the L and M subunit proteins of the ively. Major carotenoids were also purified by column reaction centre demonstrated that there were close chromatography on silica gel 60 (Merck). Molecular masses relationships between T. acidophilus and Acidiphiliuni were determined by field-desorption mass spectrometry with species. These results led us to conclude that T. a double-focusing gas chromatograph-mass spectrometer acidophilus should be transferred to the genus equipped with a field desorption apparatus (Hitachi) Acidiphilium as Acidiphilium