Transfer of Acidiphilium facilis and Acidiphilium aminolytica to the Genus Acidocella gen. nov., and Emendation of the Genus Acidiphilium

System. Appl. Microbiol. 18,85-91 (1995) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Transfer of Acidiphilium facilis and Acidiphilium aminolytica to the Genus Acidocella gen. nov., and Emendation of the Genus Acidiphilium NORIAKI KISHIMOT0 1, YOSHIMASA KOSAK0 2 , NO RIO WAKAO\ TATSUO TANO\ and AKIRA HIRAISHI 5 .. Mimasaka Women's Junior College, Tsuyama, Okayama 708 The Institute of Phvsical and Chemical Research (RIKEN), Wako, Saitama 351-01 3 Faculty of Agricult~re, (wate University, Morioka, Iwate 020 4 Faculty of Agriculture, Okayama University, Okayama 700 5 Laboratory of Environmental Biorechnology, Konishi., Sumida-ku, Tokyo 130, JAPAN • Present Address; Central Research Laboratories, Ajinomoto Co., Inc. Kawasaki-ku, Kawasaki 210, JAPAN I

2

Received December 13, 1994

Summary Phylogenetic relationships among members of the genus Acidiphilium and related genera were elucidated by studying 16S rRNA gene sequence information. Approximately 1.5 kbp fragments of 16S rDNAs from the test organisms were amplified by the polymerase chain reaction and sequenced directly by the combined method consisting of cycle sequencing and automated fluorescence detection. Sequence comparisons and evolutionary distance analyses with the sequence from some reference species showed that all members of the genus Acidiphilium formed a cluster within the alpha-! subclass of the Proteobacteria, with Acidomonas methanolica, Acetobacter aceti, and Rhodopila globiformis as the nearest phylogenetic neighbors. The Acidiphi/ium cluster was divided into two subclusters whose monophyly was supported by high levels of bootstrap confidence: one included A. facilis and A. aminolytica, and the other consisted of all other Acidiphilillm species. The levels of sequence similarity between species of the different subclusters were less than 94.2%. The A. fadlis-A. aminolytica group is also distinguishable from the other group in a number of phenotypic and chemotaxonomic characteristics. Thus we propose [0 transfer A. aminolytica and A. facjlis to a new genus, Acidocella gen. nov., with Acidocella facilis as [he type species. Following this proposal, the description of the genus Acidiphilium was emended.

Key words: Acidiphilium - Acidocella - Acidophilic bacteria - 16S rRNA - Phylogeny

Introduction Some physiological groups of bacteria are able to grow heterotrophically in highly acidic environments. Harrison (1981) isolated acidophilic heterotrophic bacteria from a culture of Thiobacillus ferrooxidans and classified these bacteria as a new genus, Acidiphilium, comprising the species A. cryptum. Urakami et al. (1989) proposed to create the second new genus of acidophilic chemoheterotrophs, Acidomonas, for some strains from septic culture of methylotrophic yeast. We isolated orange-pigmented acidophilic strains from acidic mineral environments and created the third new genus of the acidophiles, Acidobacterium, for these strains (Kishimoto et aI., 1991).

The genus Acidiphilium now consists of six validly named species as follows: A. angustum (Wichlacz et aI., 1986), A. cryptum (Harrison, 1981), A. facilis (Wichlacz et aI., 1986), A. organovorum (Lobos et aI., 1986), A. rubrum (Wichlacz et aI., 1986), and the recently described species A. aminolytica (Kishimoto et aI., 1993a). On the other hand, the genera Acidomonas and Acidobacterium are monotypic with the species Acidomonas methanolica and Acidobacterium capsulatum, respectively. Although members of the genus Acidiphilium share many common characteristics and have similar ecological niche, they differ from each other in some imponant traits.

86

N. Kishimo[O cr al.

For example, A.
Materials and Methods Bacterial straills .md cllltiat'atiOlz. Nine strains of acidophilic bacteria which consisted of six established and one unknown species of Acidiphililtm, Acetobacter ,zceri, and Addomonas methanolica were studied. The unknown Acidiphilium sp., strain C-I, was recently isolated by one of us (N.\~'.). In addition, Rhodosl,iri/lum rubrtlm and Rhodospirillum photometricllm, the phototrophic bacteria belonging to alpha. I subcldss of the Proteobacteria (W'oese, 1987), were used for comparison. Details of the test strains and their sources are shown in Table 1. The acidophilic bacteria were grown aerobicall~' at '>O°C in GYE medium, consisting of mineral salts, glucose, and yeast extract (Kishimoto and rallO, 1987). Acetobacter aceti was cultivated in the following medium; glucose 100 g, yeast extract 10 g, CaC0 3 30 g, and distilled water 1,000 ml. The phototrophic bacteria were grown phototrophically at 30°C in MYS medium (Hiraishi and HoshitlO, 1984). Cells were harvesred by centrifugation fror;n a culture at the mid-exponential phase of growth, washed with EDTA-saline, and resuspended in pure water, followed by storing at -20°C until they were used. PCR amplification alld sequellcing of 16S rONA. 16S rONA fragments were amplified by PCR from crude cell lysates and sequenced directly by the combined method consisting of linear PCR sequencing and automated fluorescence detection with a Pharmacia A.L.F. DNA sequencer. Detailed information concerning the PCR and sequencing procedures used has been given elsewhere (Hiraishi, 1992; Hiraishi et aI., 1994). Phylogelletic analysis. Sequences were compiled and binary sequence similarities were calculated with the GENETYX computer program package (Software Development Co., Tokyo, Japan). The CLUSTAL V program (Higgins et aI., 1992) was used for multiple alignment of sequence, calculation of nucleotide substiturion rates (KnuJ (Kimura, 1980), construcrion of neighborjoining phylogenetic trees (Satiolt and Nei, 1987), and evaluation of the topology of the tree by bootstrapping (Felsenstein, 1985). Alignment positions that included gaps and/or unidentified bases were ot taken into consideration for the calculations. Nucleotide sequence accession lIumbers. The sequence determined in this study has been deposited in the DDBJ, GSDB, EMBL, and NCB) nucleotide sequence data bases under the accession numbers 030768 to D30778 (Table 1).

Table 1. List of tcst organisms and accession numbers for 16S rONA sequences Species

Strain

Acidiphilill/1l cryptzlnz Addiphilirmz aminolytica Acidiphilill/1l angllstum Acidiphilillm faei/is Acidiphilill/1l organouorum Acidil,hilillm mbrum AcidiphilizlI1r sp. Acetob.,cter aceti AcidOl.IOII.,S lI1ethallo/ica Rhodospirillrmr rubrzlnr Rhodospirillzmz photometricllm

ATCC 101 T ATCC ATCC ATCC ATCC

J

C-J

33463' 35903 T 35904T 43141 1 35905 T

JCM 7641 T IMET 10945 T ATCC 11170T E-l1

Source' and reference

Accession number

ATCC, Harrisoll (1981) Kishimoro et al. (1993) ATCC, Wichlacz (1986) ATCC, \Vichlacz (1986) ATCC, Lobos (1986) ATCC, Wichlacz (1986) N. Wakao, isolated from mine drainage (1981) JCM )MET, Urakami et al. (1989) ATCC K. Shimada, Tokyo Metropolitan University

030773 D30771 030772 D30774 D30775 D30776 D30769 030768 030770 030778 030777

ATCC, American Type Culture Collection, Rockville, USA; JCM, Japan Collection of Microorganisms, RIKEN, Wako, Japan; IMET, Zentralinstitut iiir Mikrobiologie und Experimenrelle Therapie, Jena, Germany.

Acidocella gen. nov.

87

Results The 165 rONA segments that corresponded to positions 8 to 1510 of the Escherichia coli numbering system (Brosius et aI., 1978) were amplified from six known species of Acidiphilium, Acidiphilium sp. C-1, Acetobacter ace!i, Acidomonas methanolica, and two species of the phototrophic bacteria, and sequenced directly by the cycle sequencing method followed by automated fluorescence detection. The sequences we determined ranged from 1,406 to 1,417 bases, except the PCR primer-annealing regions (positions 8 to 27 and 1492 to 1510), representing ca. 95% of the entire 165 rRNA gene. All of the sequences exhibited large deletions in loop helices around positions 80, 210, and 450 of 165 rRNA in E. coli numbering, which are rypical of the alpha subclass of the Proteobaeteria. 5ince Lane et al. (1992) have already determined partial 165 rRNA sequences by reverse transcriptase sequencing for A. angustum, A. (aeilis, A. eryptum, and A. rubrum, we compared our sequence data with those published data. We found that there were a number of differences in identified bases and gaps berween the previous results and ours, i.e., 5 differences in 943 compared positions for A. angustum, 9 differences in 948 positions for A. eryptum, 10 differences in 888 positions for A. (acilis, and 16 differences in 1,052 positions for A. rubrum. This discrepancy may be due to the accuracy of the sequencing methods used. Our sequencing strategies involved direct automated sequencing of the PCR products and on-line data processing with manual correction, and thus, should provide more accurate data and minimize human errors. We also compared the 165 rONA sequence of A. aceti determined in this study to the sequence of the same strain (GeneBank accession no. X74066) recently reported by Sievers et al. (1994). There was only one base difference in 1,410 positions compared berween the rwo, and this level of difference seems to be within the range of sequencing errors. Binary sequence similarity and distance matrixes comparing the 11 tested species with 2 related species of the alpha-l subclass of the Proteobacteria and with E. coli are shown in Table 2. The evolutionary distance (Knuc values) matrix was based on the alignable 1,345 positions of all sequences of the entire set. For members within a single genus, Acidiphilium species showed a relatively broad range of similarity levels which varied berween 92.7 and 100% (Knuc = 0.0000 to 0.0684), suggesting their phylogenetic heterogeneiry. Of particular importance was that Acidiphilium species appeared to be divided into twO groups on the basis of the levels of sequence similarity. One of the groups included A. cryptum, A. organovorum, A. angustum, A. rub rum, and Aeidiphilium sp. C-l, and the other consisted of A. facilis and A. aminolytica. The levels of sequence similariry berween members within each group were above 95.0%, whereas those berween members of different groups were less 94.2%. A neighbor-joining phylogenetic tree was constructed on the basis of the distance matrix data on the 1,345 positions (Fig. 1). All species of the genus Acidiphilium formed a cluster with Acetobaeter aeeti, Aeidomonas

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ml.'thallolica, and R. globiformis as phylogenetic neighbors within the alpha-l subclass of the Proteobacteria. Similar topologies of the tree has been reported previously (Lalle et aI., 1992; Sievers et aI., 1994). As suggested from the sequence similarity data, the Acidiphilium cluster consisted of two major lines of descent: one of which included the type species A. cryptum, 3 related known species, and the unknown Acidiphilium strain C-\ (termed here the A. cryptum group) and one of which encompassed A. faeilis and A. amillolytiea (termed the A. faeilis group). A bootstrap analysis with 1,000 resamplings supported the monophyly of each group (100% suPPOrt), as shown in Fig. 2.

Discussion The results of the present study have confirmed and expanded the previous results for the phylogeny of Addiphilium species and related acidophilic bacteria (Lane et aI., 1992; Bulygina et al., 1992; Sievers et al., 1994). Phylogenetic analyses based on the 16S rRNA and rDNA sequence information show that Aeidiphilium species form a cluster within the alpha-l subclass of the Prote()bacteria, with Aeetobaeter aceti, Addomonas methanolica, and R. globiformis as the nearest phylogenetic

A. cryptum group

relatives. Within the alpha-l subclass, this major cluster is deeply branched off from the lineage that includes the phototrophic and chemotrophic species with a spiral cell morphology. These results are interesting, since members of the former cluster, including the phototrophic bacterium R. globiformis (Pfennig, 1974), are all acidophilic. Gluconobacter species and Thiobacillus acidophilus, which were not included in the present study but have been shown by 5S rRNA sequencing to be phylogenetic relatives -to Acetobaeter, Acidiphilium, and Acidomonas (Bulygina et al., 1992), are also acidophiles. Recent research by 16S rDNA sequencing (Sievers et aI., 1994) has confirmed that members of the genera Acetobacter and Gluconobacter, together with Aeidiphilium and Rhodopila species, represent a major branch of acidophiles in the alpha subclass of the Proteobacteria. Thus, it can be speculated that these acidophilic bacteria were adapted to acidic environments after branching off from the common ancestor of the alpha-l sublcass of the Proteobaeteria; otherwise all members of the alpha subclass were originally acidophilic, and the members of the above-noted cluster have retained this property. Our molecular data also provide phylogenetic evidence that the lineage encompassing Aeidiphilium species are divided into two major groups, the A. cryptum and A. faeilis

. facilis gr oup

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Fig. 2. Distance matrix tree showing phylogenetic relationships of Acidiphifium species and related acidophiles. The figure shows a magnification of the tree shown in Fig. 1, with bootstrap confidence values given at the branching points.

Acidocella gen. nov.

groups, both of which exist as a monophyletic unit supported by a 100% level of bootstrap confidence. The levels of sequence similarity between members of different clusters are less than 94.2%. These levels appear [0 be low enough to justify generic separation of the A. (acilis group from the A. cryptul1l group provided that major phenotypic differences exist between the two. The phylogenetic results presented here are in good agreement with phenotypic and chemotaxonomic data reported previously (Kishimoto et aI., 1993a, Wakao et aI., 1993). Species of the genus Acidiphiliul1l can be classified into two groups on the basis of these data. One of the groups comprises A. cryptllffl, A. mtgllstum, A. organoVOrlll1l, and A. rIIiJrIIl1l, and is characterized by high sensitivity to certain organic acids, production of BChl a, carotenoids, and bisphosphatidylglycerol (BPG), and the absence of 2-hydroxy fatty acids. Previous work showed A. organovorum to contain no BChl a (Wakao et aI., 1993), but in a concurrent study, we have found that prolonged cultivation resulted in detection of appreciable amounts of the photopigments from this organism. However, no BChl pigments were detected in Acidiphiliul1l strain C-I (data not shown). The recently described new species Acidiphililll1l mliitivorum also produces BChl a (Wakao et aI., 1994), and has been found, by partial 16S rONA sequencing (ca. 600 bases), to have a 100% level of similarity [0 A. cryptllm (unpublished data), indicating that A. multivoTtlm is a member of the A. cryptum group. The occurrence of BChl a in these aerobic acidophiles is quite interesting but of unknown biological significance. The other group consists of A. (aeilis and A. aminolytiea, and is characterized by low sensitivity to the organic acids, absence of BChl a, carotenoids, and BPG, and the presence of 2-hydroxy fatty acids. These twO groups also differ from each other in carbon source utilization and other phenotypic properties (Kishimoto et aI., 1993a).

!l9

In view of these phylogenetic and phenotypic data, it is reasonable to conclude that A. (aeilis and A. amino/ytica should be removed from the genus Acidiphilium and be transferred into a new genus. We propose the name Acidocella gen. nov. for this genus with the designation of Acidocella faeilis comb. nov. as the type species. Following this proposal, we emend the description of the genus Acidiphilium as described below. Differential characteristics of the new genus Acidocella, Aeidiphilium emend., and related genera of acidophiles based on the descriptions given by Wiehlacz et al. (1986) and Kishil1loto et al. (1993a) are shown in Table 3. The results of the present study provide important information concerning the taxonomic positions of some Acidiphilillm species. The molecular data demonstrate that the new isolate Col belongs to the genus Acidiphilium but differs genetically from the current species of this genus. Detailed descriptions and a nomenclatural proposal for this bacterium as a new species will be reported elsewhere. Also, the finding that the level of sequence similarity between A. angustum and A. rubrum is 100% suggests the need to reinvestigate their relationships at the species level, together with T. acidophilus, which has been shown to be closely related to these species on the basis of partial 16S rRNA sequences (Lane et aI., 1992). Description o( Acidocella gen. nov. Acidocella (A.ci.do-cel'la. Gr.n. aeidum an acid; L.N.fem. cella cell; Acidocella, acid (-requiring) cell). The descriptions below are based on the information from Wichlaez et al. (1986), Kishimoto et al. (1993), and this study. Cells are gram-negative asporogenous rods, 0.5-0.8 Ilm wide and 1.0-1.8 lAm long. They often occur in chains and small flocs. Motile by means of polar Hagella. Strictly aerobic chemoorganotroph having a respiratory

Table 3. Differential characteristics among Acidocella gen. nov. and some other genera of acidophilic chemoorganotrophic bacteria. (data from Wichlacz et aI., 1986, and Kishimoto et aI., 1993a) Acidocella gen. nov.

Motility Pigment Bacteriochlorophyll a B-galactosidase Growth inhibition by: 0.25 mM Acetate 2 ml..! Lactate 4 mM Succinate Assimilation of: Diaminobutane DL-5-aminovalerate Methanol fI·gentiobiose .\L1jor quinone Fanv acids Major nonpolar component 2·Hydroxy component Bisphosphatidylglycerol ~lo1% G+C of DNA

+

Acidiphilium emend.

Acidomonas

+

+

Pale pink, violet +/-

Orange

+ + +

+

+ +

+

+

+

Q-JO

Q-10

Q-10

CIM,I C14 ,o

C 18 '1

C 18 ,1 C I4 ,O, C I6 ,O + 64.7

58.7 -64.4

Acidobacterium

+ 63.2-67.5

+ MK-8 iso-Cls,o + 60.8

90

N. Kishimoto et al.

type of metabolism with oxygen as the terminal electron acceptor. Mesophilic and acidophilic. Grows at 25-37°C and pH 3.0-6.0. Catalase positive. Oxidase negative. Growth occurs in the presence of 0.25 mM acetate and 4 mM succinate. Neither elemental sulfur nor ferrous iron is used as an energy source. Photopigments such as bacteriochlorophyll and carotenoids are absent. The major isoprenoid quinone is ubiquinone with ten isoprene units. The major fatty acid is C 1H : 1 • 2-Hydroxymyristic acid and .~-hydroxypalmitic acid are present. Ornithine amide lipids, C IH: I fatty acid esters of a-N-3-hydroxystearylornithyltaurine and u-N-3-hydroxystearylornithine, are present as polar aminolipids. BPG is absent as membrane lipid. The DNA base ratios are 58.7-64.4 mol% G+c. Phylogenetic position is the alpha (a-I) subclass of the Proteo/lacteria. Type species; Aeidocella faeilis.

Description of Acidocella facilis comb. nov. Acidocella (aeitis (fa 'ci.lis. L. adj. facitis ready, quick, with respect to growth). The characteristics in terms of morphology, physiology, biochemistry, and chemotaxonomy are as described for the genus. Additional properties arc as follows. Colonies on GYE gellan gum plate (GYE medium solidified by 0.6% gellan gum, Wako Pure Chern. Ind. Ltd., Japan) are circular, smooth, opaque, with an entire margin, and white to pale light brown, becoming 4-5 mm after 3 days incubation. Growth occurs with 3.5% NaC!. Urease is produced. f)-Galactosidase is absent. Hydrolysis of gelatin, esculin, and hippuric acid is negative. The following organic compounds serve as carbon and energy sources: Larabinose, o-xylose, o-ribose, o-fructose, o-glucose, 0galactose, o-arabitol, mannitol, glycerol, ethanol, gluconate, 4-aminobutyrate, 5-aminovalerate, diaminobutane, glutamate, arginine, ornithine, and histidine. Not utilized are: man nose, maltose, cellobiose, lactose, trehalose, sorbitol, myo-inositol, starch, arbutin, and amygdalin. The DNA base rato is 62.4 to 64.4 mol% G+c. Sources: acidic coal mine drainage. Type strain: ATCC 35904 (Wichlacz PW2). Description of Acidocella aminolytica comb. nov. Acidocella aminolytica (a.mi.no.lyti.ca.M.L.n. aminum amine; Gr.adj. Iytica dissolving; M.L.adj. aminolytica amine dissolving). The characteristics in terms of morphology, physiology, biochemistry, and chemotaxonomy are as described for the genus. Additional properties are as follows. Colonies on GYE gellan gum plate (GYE medium solidified by 0.6% gellan gum, Wako Pure Chern. Ind. Ltd., Japan) are circular, smooth, opaque, with entire margin, and white to pale light brown, becoming 4-5 mm after 3 days incubation. Urease is produced. f)-Galactosidase is absent. Hydrolysis of hippuric acid is positive. Hydrolysis of gelatin and esculin is negative. The following organic compounds serve as carbon and energy sources: l-arabinose, D-xylose, o-ribose, D-fructose, o-glucose, o-galactose, o-arabitol, mannitol, sorbitol, myo-inositol, gluconate, 4-aminobuty-

rate, 5-aminovalerate, diaminobutane, glutamate, arginine, ornithine, spermine, lysine, f)-alanine, creatine, and histidine. Not utilized are: man nose, maltose, cellobiose, lactose, trehalose, ethanol, starch, arbutin, and amygdalin. The DNA base ratio is 58.7 to 59.2 mol% G+c. Sources: acidic coal mine drainage. Type strain: jCM 8796 (ATCC 51361, Kishimoto 101).

Emended description of the genus Acidiphilillm The descriptions below arc based on the information from Harrison (1981); Wichlacz et al. (1986), Wakao et al. (1993); Kishimoto et al. (1993b) and this study. Cells are gram-negative asporogenous rods, 0.3-1.2 f.l.m wide and 0.6 to 4.2 f.l.m long. Motile by means of polar flagella or two lateral flagella, or nonmotile. Colonies are cell suspensions are light brown, pale pink, red, or violet due to the presence of bacteriochlorophyll a and carotenoids. Some strains produce no bacteriochlorophyll. Strictly aerobic chemoorganotroph having a respiratory type of metabolism with oxygen as the terminal electron acceptor. Mesophilic and acidophilic. Grows at 25-37°C and pH 2.5-6.0. Catalase positive. Oxidase negative. No growth occurs in the presence of 0.25 mM acetate and 4 mM succinate. Neither elemental sulfur nor ferrous iron is used as an energy source. The major isoprenoid quinone is ubiquinone with ten isoprene units. The major fatty acid is C IS : 1' 3-Hydroxypalmitic acid is also present. 2-Hydroxy fatty acids are absent. Ornithine amide lipids, C 18 : 1 fatty acid esters of a-N-3-hydroxystearylornithyltaurine and a -N-3-hydroxystearylornithine, are present as polar aminolipids. BPG occurs as membrane lipid. The DNA base ratio is 63.2-67.5 mol% G+c. Phylogenetic position is the alpha (a-I) subclass of the Proteobacteria. Habitats: acidic mineral environments. Type species: Acidiphilium cryptum.

References Brosius, j., Palmer, j. L., Kenned)', J. P., Noller, H. F.: Complete

nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc. Natl. Acad. Sci. USA. 75, 4801-4805 (1978)

Bulygina, E. S., GlI/ikova, O. M., Dikanskaya, E. M., Netrusov, A. I., TOl/rova, T. P., Chumakov, K. M.: Taxonomic studies of the genera Acidomonas, Acetobacter, and Gluconobacter by

5S ribosomal sequencing.). Gen. Microbiol. 138,2283-2286 (1992) Felsenstein, j.: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39,783-791 (1985) Harrison, ir. A. P.: Acidiphilium cryptum gen. nov., sp. nov., heterotrophic bacterium from acidic mineral environments. Int. J. Syst. Bacteriol. 31,327-332 (1981) Higgins, D. G., Bleasby, A.j., Fuchs, R.: CLUSTAL Y: improved software for multiple sequence alignment. Comput. Appl. Biosci. 8, 189-191 (1992) Hiraishi, A., Hoshino, Y.: Distribution of rhodoquinone in Rhodospirillaceae and its taxonomic implications. ]. Gen. Appl. Microbiol., 30, 435-448 (1984)

Acidocella gen. nov. Hir"ishi, A.: Direct automated sequencing of 165 rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification. Lett. Appl. Microbiol. IS, 210-213 (1992) Hiraishi. A .. Shill, Y. K., Veda, Y., Sugiyama, j.: Automated sequencing of PeR-amplified 165 rDNA on "Hydrolink~ gels. ]. 1\licrobiol. Methods 19, 145-154 (1994) Kimllra, M.: A simple method for estimating evolutionary rates of base substitution through comparative studies of nucleotide sequences.]. Mol. Evol. 16.111-120 (1980) KishilllotO, N., Tallo, 1'.: Acidophilic heterotrophic bacteria isolated from acidic mine drainage, sewage, and soils. J. Gen. Appl. Microbiol. 33: 11-25 (1987) Kishimoto, N., Illagaki, K., SlIgio, T.. Tano, T.: Growth inhibition of Acidiphilillm species by organic acids contained in yeast extract. J. Ferment. Bioeng. 70, 7-10 (1990) Kishimoto, N., Kosako, Y., TallO, T.: Acidobacterium capsulatulII gen. nov., sp. nov.: an acidophilic chemoorganotrophic bacterium containing menaquinone from acidic mineral environment. Curr. Microbiol. 22, 1-7 (1991) KishilllOtO, N .. Kasako, Y., TallO, T.: Acidiphilium aminolytica sp. nov.: an acidophilic chemoorganotrophic bacterium isolated from acidic mineral environment. Curr. Microbiol. 27, 131-136 (1993a) Kishimoto, N .. Adachi, K., Talllura, S., Nishihara, M., Inagaki, K., Sugio, T., TallO, T.: Lipoamino acids isolated from Acidiphilil/lll organOvOrtllll. System. Appl. Microbiol. 16, 17-21 (1993b) Lalle, D. J., Stahl, D. A., Olsen, G. j., Heller, D. J., Pace, N. R.: Phylogenetic analysis of the genera Thiabacillus and Thialllicrospira by 55 rRNA sequences. ]. Bacteriol. 163, 75-81 (1985) Lane, D. J., Harrisal', ir. A. P., Stahl, D., Pace, B., Giovannoni, S. j., Olsell, C. J., Pace, N. R.: Evolutionary relationships

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Dr. Noriaki Kishimoto, Mimaska Women's Junior College, Tsuyama, Okayama 708, Japan