INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1997, p. 217-219 Vol. 47, No. 1 0020-7713/97/$04.00+ 0 Copyright 0 1997, International Union of Microbiological Societies Transfer of the Bacteriochlorophyll &Containing Phototrophic Bacteria Rhodopseudomonas viridis and Rhodopseudomonas sulfoviridis to the Genus Blastochloris gen. nov. AKIRA HIMISHI* Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441, and Laboratory of Environmental Biotechnology, Konishi Co., Tokyo 130, Japan The phylogenetic positions of the bacteriochlorophyll (BChl) b-producing budding phototrophic bacteria Rhodopseudomonas viridis and Rhodopseudomonas sulfoviridis were studied on the basis of 16s rRNA gene sequence information. These bacteria formed a tight cluster with the genus Rhodoplanes as a sister group within the alpha-2 subgroup of the Proteobacteria. Genomic DNA-DNA hybridization assays showed that R. viridis and R. sulfoviridis were closely related but were different species. Creation of the genus Blastochloris gen. nov. is proposed to accommodate these BChl b-producing species of phototrophic bacteria. In 1984 some species of the classically defined genus Rho- tion of the organisms. For Rhodopseudomonas sulfoviridis, the dopseudomonas were reclassified into new genera, including medium was supplemented with 10 mM glucose, 2 mM sodium the new genera Rhodobacter and Rhodopila, on the basis of sulfide (neutralized), and 2 mM thiosulfate. The medium was modern taxonomic criteria (17). In recent years, the taxonomy supplemented with 10 mM pyruvate (filter sterilized) for Rho- of the genus Rhodopseudomonas has been further reevaluated doplanes roseus. The organisms were grown at 30°C in screw- on the basis of increasing molecular and chemotaxonomic in- cap test tubes under anaerobic conditions in the light. Cells formation, and this has led to rejection of the name Rhodo- were harvested by centrifugation from cultures at the mid- pseudomonas rutila (10) and the transfer of some Rhodopseu- exponential phase of growth, washed, resuspended in pure domonas species to the genus Rhodobacter (19) and to the new water or EDTA-saline, and stored at -20°C until analysis. genera Rhodoplanes (12) and Rhodobium (13). Despite these Genomic DNA was extracted and purified by the method of taxonomic changes, the species currently in the genus Rhodo- Marmur (24). DNA base ratios were determined by the high- pseudomonas are still heterogeneous phylogenetically, and performance liquid chromatography method (7, IS), and both molecular and phenotypic data strongly suggest that only DNA-DNA hybridization assays were performed by photobi- the type species, Rhodopseudomonas palustris, should be otin labeling and colorimetric detection as described previ- placed in this genus. The proper taxonomic placement of the ously (7, 10). 16s rDNA fragments were amplified by PCR bacteriochlorophyll (BChl) b-containing Rhodopseudomonas from the crude cell lysate (11) and were purified by polyeth- species (i.e., Rhodopseudomonas viridis and Rhodopseudomo- ylene glycol precipitation (8, 22). The PCR products were nas sulfoviridis) is a subject for consideration in light of their sequenced directly with fluorescent primers and a SequiTherm characteristic features. It has been suggested that one of the Long-Read cycle sequencing kit (Epicentre Technologies, BChl b-producing species, Rhodopseudomonas viridis, is phy- Madison, Wis.) and analyzed with a Pharmacia automated logenetically distant from Rhodopseudomonas palustris (30) DNA sequencer. Sequences were compiled and similarities within the alpha-2 subgroup of the Proteobacteria and that this were calculated with the GENETYX-MAC computer program organism is more closely related to members of the genus (Software Development Co., Tokyo, Japan). Multiple align- Rhodoplanes (12). In the present study the 16s ribosomal DNA ments of sequences, calculation of nucleotide substitution rates (rDNA) sequence similarity of and genomic DNA relatedness (21), and construction of neighbor-joining phylogenetic trees between Rhodopseudomonas viridis and Rhodopseudomonas (27) were performed with the CLUSTAL W program (28). sulfoviridis were investigated. The results obtained indicate Alignment positions that included gaps and unidentified bases that these species should be placed in a distinct new genus of were not taken into consideration for the calculations. phototrophic bacteria, for which the name Blastochloris gen. The 16s rDNA sequences determined covered a continuous nov. is proposed. nucleotide stretch from positions 28 to 1,492 in the Escherichia The organisms investigated were Rhodopseudomonas viridis coli numbering system (1). The level of sequence similarity ATCC 19567T (T = type strain), Rhodopseudomonas sulfoviri- between the 16s rDNAs of Rhodopseudomonas viridis and dis DSM 729T, Rhodoplanes roseus DSM 5909T, and Rhodo- Rhodopseudomonas sulfoviridis was 98.7% (corrected distance, pseudomonas palustris ATCC 17001T, all of which were ob- 0.0123). This binary similarity value suggests that the two spe- tained either from the American Type Culture Collection, cies are phylogenetically closely related organisms which be- Rockville, Md., or from the DSM-Deutsche Sammlung von long to a single genus but different genospecies. The results of Mikroorganismen und Zellkulturen GmbH, Braunschweig, DNA-DNA hybridization assays supported this suggestion, as Germany. MYS medium (9), which contained mineral base the genomic DNAs of Rhodopseudomonas viridis and Rhodo- RM2, 20 mM sodium DL-malate as the carbon source, and pseudomonas sulfoviridis were related to each other at an av- 0.05% yeast extract as the growth factor, was used for cultiva- erage hybridization level of 38% (Table 1). A distance matrix tree was constructed on the basis of the 16s rDNA sequences of Rhodopseudomonas viridis, Rhodopseudomonas sulfoviridis, * Mailing address: Department of Ecological Engineering, Toyo- and related species of phototrophic and nonphototrophic bac- hashi University of Technology, Tenpaku-cho, Toyohashi 441, Japan. teria that belong to group 2 of the alpha subclass of the Pro- Phone: 81 (532) 44-6913. Fax: 81 (532) 44-6547. teobacteria (Fig. 1). The two species of BChl b-producing pho- 217 218 NOTES INT.J. SYST.BACTERIOL. TABLE 1. Genomic DNA relatedness among Rhodopseudomonas based on amino acid sequences of L and M subunit proteins of viridis, Rhodopseudornonas sulfoviridis, and some other photosynthetic reaction centers showed that Rhodopseudomo- species of budding phototrophic bacteria nus viridis represents a unique line of descent within the divi- % Hybridiza- sion of purple bacteria (25). Rhodopseudomonas viridis is one tion with la- of the best characterized organisms with respect to structures G+C content beled DNA of the reaction center, as X-ray crystal structures of the reac- Organism of DNA from: (mol%) tion center of this organism have been studied (2). In light of ATCC DSM the importance of Rhodopseudomonas viridis as a key organism 19567T 729T in photosynthetic research and the phylogenetic and pheno- typic evidence that the BChl b-producing budding phototrophs Rhodopseudomonas viridis ATCC 19567T 66.5“ 100 32 Rhodopseudomonas sulfoviridis DSM 729T 67.9 44 100 are members of a distinct taxonomic group, these organisms Rhodoplanes roseus DSM 5909T 66.8’ 11 7 should no longer be included in the genus Rhodopseudomonas. Rhodopseudomonas palustris ATCC 17001T 65.0” 64 Thus, I propose that Rhodopseudomonas viridis and Rhodo- pseudomonas sulfoviridis should be transferred to the genus ‘‘ Data from reference 10. Blastochloris gen. nov., as Blastochloris viridis comb. nov. and ’Data from reference 12. Blastochloris sulfoviridis comb. nov., respectively. Description of Blastochloris gen. nov. Blastochloris (Blas.to. chlo’ris. Gr. n. blustos, bud shoot; Gr. adj. chloros, green; M. L. totrophs formed a tight cluster with the genus Rhodoplanes as fem. n. Blastochloris, green bud shoot). The characteristics a sister group within the alpha-2 subgroup of the Proteobacte- described below are based on information from previous re- ria. ports (3, 14, 20, 23, 29) and this study. Cells are rod shaped to BChl b was first discovered in Rhodopseudomonas virids (3, ovoid and exhibit polar growth, budding, and asymmetric cell 5) and was later found in some other species of phototrophic division. Rosette-like aggregates sometimes occur. Motile by purple bacteria, including members of the genera Thiocapsa means of subpolar flagella. Gram negative. Photoheterotro- (4) and Ectothiorhodospira (15, 16). Among the species of the phic, growing anaerobically in the presence of light. Micro- genus Rhodopseudomonas and all other genera of purple non- aerophilic growth in the dark is also possible. Phototrophically sulfur bacteria so far recognized, however, Rhodopseudomonas growing cells contain intracytoplasmic membranes that are viridis and Rhodopseudomonas sulfoviridis are the only species present as lamellae underlying and parallel to the cytoplasmic that produce BChl b as the photosynthetic pigment. In addition membrane. Photosynthetic pigments are BChl b and carot- to these species, a BChl b-producing budding phototrophic enoids. The color of photosynthetic cultures is green to olive bacterium which may be related to the former species has been green. Photoorganotrophy with a number of simple organic isolated from a hot spring (26). The evolutionary and taxo- compounds as carbon and electron donors is the preferred nomic significance