Distribution of Rhodoquinone Rhodospirillaceae and Its Taxonomic

Home , Rhodocyclus, Rhodomicrobium, Rhodomicrobium vannielii, Rhodopseudomonas, Rhodospirillaceae, Rhodospirillum rubrum

J. Gen. Appl. Microbiol., 30, 435-448 (1984)

DISTRIBUTION OF RHODOQUINONE IN RHODOSPIRILLACEAE AND ITS TAXONOMIC IMPLICATIONS

AKIRA HIRAISHI ANDYASUO HOSHINO Department of Biology, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo 158, Japan

(Received November 27, 1984)

The rhodoquinone (RQ) composition was studied in representatives of 20 species of Rhodospirillaceae. Thin-layer chromatography, ultraviolet spectrophotometry, mass spectrometry, and high-performance liquid chromatography revealed that six of the 20 test species contained RQ as a major quinone in average amounts of 0.28 to 2.22 umol per g dry weight of cells. The predominant homologue of RQ in the six species was as follows: RQ-8-Rhodospirillum photometricum and an unknown species resembling Rhodocyclus gelatinosus; RQ-9+ RQ-10-Rhodopila globiformis; RQ-l0-Rhodospirillum rubrum, Rhodopseudomonas acidophila, and Rhodomicrobium vannielii. The taxonomic significance of RQ and other isoprenoid quinones is discussed in comparison with the new classification system for the phototrophic bacteria recently proposed by IMHOFFet al.

Isoprenoid quinones in eubacteria can be divided into two major structural groups, the benzoquinones and naphtoquinones represented by ubiquinone (Q) and menaquinone (MK), respectively. Some derivatives of Q and MK also occur in certain species. These lipoquinones play important roles in respiratory or photosynthetic electron transport in bacterial plasma membranes, and have been extensively studied not only from biochemical viewpoints but also in their taxonomic aspects (1, 2). Rhodoquinone (RQ) is a derivative of Q in which one of the methoxyl groups is replaced by an amino group (3, 4). This compound was first isolated from Rhodospirillum rubrum by GLOVERand THRELFALL(5). Later, this aminoquinone was shown to be present in the eucaryotic organisms Euglena gracilis var, bacillaris (6, 7), Astasia longa (8), and Ascaris lumbricoides var. suis (9-I1). In contrast to the ubiquitous distribution of Q and MK in nature, the mention of the natural occurrence of RQ in literature has hitherto been confined to the above species, and thus little is known about its physiological functions and its significance in microbial taxonomy. 435 436 HIRAISHI and HosHINo VOL. 30

In a previous study, we reported Q and MK composition to be of value in the classification of phototrophic bacteria of the family Rhodospirillaceae and reclas- sified them into several groups based on a combination of the quinone system and other taxonomic properties (12). This study was undertaken to determine more about the quinone system in these bacteria with special reference to RQ. Recently, a drastic rearrangement of the species and genera of Rhodospirillaceae has been proposed by IMHOFFet al. (13). This paper deals in particular with the taxonomic significance of RQ in Rhodospirillaceae in comparison with this new classification system.

MATERIALSAND METHODS Organisms and cultivation. Thirty-four strains of 20 species of Rhodospiril- laceae were studied (Table 1). Rhodomicrobium vannielii AV4 was newly isolated from the water of Lake Vanda, Antarctica (TAKIIet al., in preparation). Detailed information on the sources of all other test strains has been given elsewhere (12, 14). The names of the bacterial taxa were used according to the proposal for the reas- signment of the phototrophic bacteria (13)-that is, the species previously referred to as Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides, Rhodopseudo- monas sufdophila, Rhodopseudomonas adriatica, and Rhodopseudomonas globi formis are placed in the new genera Rhodobacter and Rhodopila as Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodobacter sulfidophilus, Rhodobacter adriaticus, and Rhodopila globiformis, respectively; the species formerly designated as Rhodospiril- lum tenue and Rhodopseudomonas gelatinosa are united with Rhodocyclus purpreus in the genus Rhodocyclus as Rhodocyclus tenuis and Rhodocyclus gelatinosus, respectively. The strains called "atypical Rhodopseudomonas gelatinosa" in the preceding papers (12,14) are included in an unknown species as the "Rhodocyclus gelatinosus-like (RGL)" group in this study. Unless otherwise indicated, MYS medium (mineral base RM2+20 mM malate +0.05 % yeast extract) (12,14) was used for culturing organisms. For certain species, modifications of the medium were used as described previously (12). For Rhodobacter adriaticus (15), the medium was supplemented with 500 mM NaCI, 2 mM thiosulfate, and 2.5 mM ascorbate (filter-sterilized). For Rhodocyclus purpreus (16), the medium was modified by addition of 10 mM acetate and cyanocobal- amin (30 , tg/l). Rhodopila globiformis was cultured according to PFENNIG(17) with 10 mM malate and 10 mM mannitol as carbon sources. All organisms were anaerobically grown in screw-capped bottles in light, harvested by centrifugation, and stored at 0-4° until assayed as described earlier (12). Extraction and purification of quinones. Quinones were extracted from fresh wet cells with chloroform-methanol (2: 1, v/v) and purified by thin-layer chromatography (TLC) (12) with developing solvents of 100 % benzene for Q and RQ and petroleum benzine-diethyl ether (85: 15, v/v) for Q and MK. For large-scale isola- tion of RQ, the lipid extracts were dissolved in petroleum ether, chromatographed 1984 Rhodoquinone in Rhodospirillaceae 437 on alumina columns (3 x 15 cm) built with Merck's acidic aluminium oxide, and eluted stepwise with 4, 8, and 16 % ether in petroleum ether, followed by TLC in 100% benzene. Quantification and identification of RQ. All spectrophotometric experiments were carried out with a Hitachi 200-20 spectrophotometer. RQ concentration was determined by the difference spectrum of the reduced minus the oxidized state in ethanolic solution kept under an atmosphere of argon, using the following extinc- tion coefficient : 4x283=7.9mM~ 1 cm- 1. Reduction was made by addition of 10 ul of 100 mM KBH4 to 2.5 ml of the sample. Components of RQ samples were separated by reversed-phase TLC using Whatman LKC18F plates and a developing mixture of acetone-water (18: 1, v/v), on which RQ homologues were detected as purple-colored bands in visual light and as fluorescence-quenched regions in 254 nm ultraviolet light. The chromatograms turned grey-blue on treatment with a 10% ethanolic solution of molybdophosphoric acid followed by heating at 120° for a few minutes. RQ homologues were also separated by high-performance liquid chromatography (HPLC) which was performed on a Shimadzu Liquid Chromatograph LC-3A equipped with a Zobax ODS pre-packed column and a 283 nm spectrophotometric detector; RQ samples were eluted with methanol-isopropyl ether (4: 1, v/v) at a flow rate of 1 ml/min at 25° and the relative percentage of elution peak areas to the total peak area was cal- culated on a microcomputer, Shimadzu Chromatopac C-RIA. Mass spectra were recorded on a JEOL JMS-DX300 mass spectrometer; ionization voltage, 70 eV; temperature, room temperature-300°; scan range of molecular weight, 50-1,000. Analysis of Q and MK. These quinones were quantified and identified by ultraviolet spectrophotometry and HPLC as described previously (12, 18). Standard quinones. Q and MK standards were prepared as described earlier (12). Reference RQ samples wer3 synthesized from authentic Q preparations by ammonolysis (3, 4). The published data have shown that synthetic RQ is in- distinguishable from natural RQ by TLC, ultraviolet spectrophotometry, mass spectrometry, and nuclear magnetic resonance spectrometry (3, 4, 6).

RESULTS Preliminary experiments with Rhodospirillum rubrum To observe the chromatographic and spectrophotometric behavior of RQ, preliminary experiments were performed with R, rubrum, which was thought to be the only RQ-containing phototrophic bacterium before this study. When the lipid extract from R. rubrum IFO 3986 was subjected to TLC with the developing solvent of 100% benzene, RQ could be easily detected as a visual purple spot (Rf 0.4) migrating just behind Q (Rf 0.5). Upon TLC with petroleum benzene-diethyl ether, the solvent system which has been commonly used for 438 HIRAISHI and HosHINo VOL. 30

vvaveiengrn ~nm)

Fig. 1. Difference spectrum of RQ from Rhodospirillum rubrum IFO 3986. The R. rubrum RQ was present at a concentration of 26.02 ~tgf ml ethanol in the sample (with KBH4) and the reference (without KBH4) cuvette. 4A~Rs=0.241

2 min Fig. 2. Redox responses of RQ from Rhodospirillum rubrum IFO 3986 to the addition of KBH4 under aerobic and anaerobic conditions. The R. rubrum RQ was present at a concentration of 35.12 µg/ml ethanol in the aerobic (A) and the anaerobic (B) cuvette (control, ethanol). KBH4 was added to the sample at the point indicated by arrows. separating Q and MK (2,12), the RQ fraction did not appear clearly even under ultraviolet light, due to masking by other lipid components. This was also the case with other Rhodospirillaceae species that contain RQ and carotenoids of the spirilloxanthin group. The RQ fraction obtained by TLC was eluted with acetone, evaporated in vacuo, and redissolved in ethanol for spectrophotometric analysis. The RQ solution had absorption maxima at 283 nm with a shoulder at 320 nm in ultraviolet light and at 500 to 510 nm in visual light. The addition of KBH4 to an anaerobic 1984 Rhodoquinone in Rhodospirillaceae 439

Table 1. RQ content of Rhodospirillaceae species. 440 HIRAISHI and HosHINo VoL. 30

r'1 V V V r w Fig. 3. Reversed-phase TLC of synthetic and natural RQ preparations. RQ samples : (A), synthetic samples (RQ-6 through RQ-10); (B), Rhodospirillum rubrum ATCC 11170; (C), Rhodospirillum photometricum IL256; (D), Rhodopseudo- monas acidophila ATCC 25092; (F), Rhodomicrobium vannielii AV4; (F), Rhodopila globiformis DSM 161; (G), RGL group strain SA34. The chromatograms were photographed after treatment with the molybdophosphoric acid reagent.

solution of RQ resulted in the maximum peak at 283 nm being replaced by a new peak at 290 nm. These spectrophotometric features are characteristic of RQ (3-6,11,19, 20). The ultraviolet difference spectrum of the R. rubrum RQ reduced versus oxidized exhibited a maximum decrease in absorption at 280 nm (Fig. 1). The 4Em m at 283 nm for this RQ (RQ-10) was 93, from which the following extinc- tion coefficient for RQ is obtained: d~~ss=7.9 m u'cm-'. This value is slightly greater than that given by PARSONand RUDNEY(19). When the RQ solution was treated with KBH4 in air, a rapid decrease and then a gradual increase in absorption at 283 nm was observed (Fig. 2), as reported by other workers (5, 6,19). This increase passed through two phases. A rapid but short-lived reduction was found upon the second addition of KBH4 to the solution. These findings suggest that the phenomenon of the rise in intensity at 283 nm is more than a simple reoxidation of quinol to quinone. There was no difference between aerobic and anaerobic RQ solutions in the decrease in absorption at 283 nm caused by the first addition of KBH4. Therefore, it may be possible to determine RQ concentration by spectrophotometric analysis under aerobic conditions if the maximum decrease in absorption at 283 nm after the first addition of KBH4 is carefully monitored.

RQ content of Rhodospirillaceae Based on the above experimental results, we determined the RQ content of the 34 test strains of the Rhodospirillaceae species by chromatographic and spectro- 1984 Rhodoquinone in Rhodospirillaceae 441

m/z Fig. 4. Mass spectra of RQ from Rhodospirillum rubrum IFO 3986 (A) and the RGL group strain SA34 (B). photometric methods. RQ occurred as a natural product in all tested strains of Rhodospirillum photometricum, Rhodopseudomonas acidophila, Rhodomicrobium vannielii, and Rodopila globiformis, in the RGL group, and in the strains of Rhodo- spirillum rubrum (Table 1). The averages of the concentrations of RQ in each strain of these organisms ranged from 0.28 to 2.22 ,umol per g dry weight of cells, corresponding to 9 to 68 % of the amount of Q concurrently present. In Rhodo- pseudomonas acidophila and Rhodopila globiformis, both of which have MK in addition, similar or higher amounts of RQ occurred compared with the MK content. None of the other test species produced RQ in appreciable amounts. 442 HIRAIsHI and HosHINo VOL. 30

Reversed phase TLC The reversed-phase TLC system facilitated the separation of components of the natural and synthetic RQ preparations as shown in Fig. 3. In this system, the synthetic RQ homologues gave the following Rf values : RQ-6, 0.60; RQ-7, 0.54; RQ-8, 0.48; RQ-9, 0.42; RQ-10, 0.36. The chromatographic behavior of the RQ samples purified from the six Rhodospirillaceae species differed from species to species. The Rhodospirillum rubrum, Rhodopseudomonas acidophila, and Rhodo- microbium vannielii RQ samples showed a single major band with the same Rf value as RQ-10. The mobility of the RQ preparations from Rhodospirillum photometricum and the RGL group was the same as that of RQ-8. Interestingly, the Rhodopilaglobiformis RQ gave two major bands corresponding to RQ-9 and RQ-10.

Mass spectrometry The natural RQ samples were further subjected to mass spectrometry to ac- curately determine their structure and molecular weight. Figure 4 shows the mass spectra of the RQ preparations from representative strains of Rhodospirillum rubrum and the RGL group, which had RQ-10 and RQ-8, respectively, as the predominant homologue as shown by reversed-phase TLC. Upon mass spectrometry, the R. rubrum RQ gave a prominent peak at m/z 849 and a series of peaks from m/z 780 to 302 indicating consecutive loss of isoprene units in the side chain (Fig. 4A). For the RQ from the RGL group, an intensive peak ion at m/z 711 was found and the cracking pattern exhibited by the polyprenyl side chain (m/z 643-302) was also clear (Fig. 4B). These observations confirm the results obtained with reversed- phase TLC. In all cases, the mass spectra of RQ showed strong peaks at m/z 220 and 182 that correspond to fragments (a) and (b), respectively, derived from the benzoquinone nucleus (6, 9).

HPLC experiments Information on the distribution of the predominant homologues and minor components of RQ in the Rhodospirillaceae species was provided in detail by the HPLC analysis. The results shown in Table 2 indicate that a predominant homologue constituting 88 to 98 % of the total RQ content occurred in all RQ-containing strains tested, with the exception of Rhodopila globiformis DSM 161, which had two major homologues, i.e., RQ-9 (45 % of the total content) and RQ-10 (49 1984 Rhodoquinone in Rho dospirillaceae 443

Table 2. RQ composition of Rhodospirillaceae species analyzed by HPLC.

of the total). In addition, two to three minor components that comprised 1 % and more of the total RQ content were found in these organisms. On the whole, the HPLC data support the results obtained with reversed-phase TLC and mass spectrometry.

Q and MK composition During the course of this study, we also investigated the Q and MK systems in the Rhodospirillaceae species in comparison with their RQ composition, and found that the length of the polyprenyl side chain of the predominant homologues was the same among these three quinone structural types within a species. This investi- gation confirmed our earlier results (12), with the exception of those for Rhodopila globiformis as described below, and provided further information on the Q and MK composition of several species not examined in the previous study (12). Namely, Q-10 was found as the major Q component in Rhodopseudomonas rutila (94 %), Rhodopseudomonas blastica (97 %), Rhodopseudomonas marina (98 %), Rhodomicro- bium vannielli (98 %), and Rhodobacter adriaticus (98 %), whereas Q-8 was the predominant homologue from Rhodocyclus purpreus (97 %). Rhodopseudomonas marina and Rhodocyclus purpreus also had MK-10 (97%) and MK-8 (97 %), respectively, as the predominant MK. The numbers in the parentheses refer to the relative percentage of the major quinone type to the total Q or MK content. The present results obtained with Rhodopseudomonas rutila, Rhodopseudomonas marina, and Rhodomicrobium vannielii are in agreement with those of AKIBAet al. ((21) and 444 HIRAISHI and HosHINo VOL. 30

T. AKIBA,personal communication), IMHOFF(22), and MAROCet al. (23), respectively. Although in the previous study, Q-9 and MK-9 but not Q-10 and MK-10 were found as the major quinone components in Rhodopila globiformis, this study showed both homologues of Q and MK to predominate in this organism, as would be ex- pected from the results for its RQ composition (Fig. 3 and Table 2). The reason for this discrepancy is not known, but might be due in part to partial decomposi- tion of the quinone samples in the previous analysis.

DISCUSSION

The results of this study have shown that RQ occurs as a major lipoquinone in the Rhodospirillaceae species Rhodospirillum photometricum, Rhodopseudomonas acidophila, Rhodomicrobium vannielii, Rhodopila globiformis, and the RGL group, as well as in Rhodospirillum rubrum. The predominant homologue of RQ in these organisms are as follows : RQ-8-Rhodospirillum photometricum and the RGL group; RQ-9+RQ-10-Rhodopila globiformis; RQ-10-Rhodospirillum rubrum, Rhodopseudomonas acidophila, and Rhodomicrobium vannielii. The data reported so far on this subject indicate that Rhodospirillum rubrum contains RQ-10 as the predominant homologue (3-5,19, 23), whereas RQ-9 is present in the eucaryotic organisms E. gracilis var. bacillaris (6, 7), A. longa (8), and A. lumbricoides var. suis (9-11). Further studies have demonstrated the natural occurrence of RQ in certain species of non-photosynthetic, heterotrophic bacteria (HIRAISHI,unpub- lished data). These findings suggest that RQ, as well as Q and MK, is widely distributed in nature. In view of the present results and the available information on the quinone system in the phototrophic bacteria (12, 13, 23), the Rhodospirillaceae species can be divided into four different categories according to their quinone structural types : (I) those species containing Q only; (II) those containing Q and RQ; (III) those containing Q, RQ, and MK; and (IV) those containing Q and MK. The species belonging to each category are further separated into several groups on the basis of the length of the polyprenyl side chain of their predominant quinone components. The distribution of these quinone structural variations in the Rhodospirillaceae species is summarized in Table 3, where the species and genera are arranged according to the new classification system proposed by IMHOFFet al. (13) for the phototrophic bacteria. The species now placed in the new genera Rhodobacter and Rhodocyclus are homogeneous with respect to the quinone system, belonging to categories I and IV, respectively. Also, the genera Rhodomicrobium and Rhodopila, each of which includes only one species, are characterized by their unique quinone composition (categories II and III, respectively). These traits and a number of other taxonomic and phylogenetic information (13) justify the establishment of the 1984 Rhodoquinone in Rho dospirillaceae 445

Table 3. Summary of the quinone system in Rho dospirillaceae. a

above four genera, although IMxoFF et al. (13) suggested that the species included in the genus Rhodocyclus might form different genera in a new family in the future because of the high degree of their dissimilarity in some phylogenetic properties compared with the other genera of Rhodospirillaceae. In contrast to the genera noted above, the remaining two genera Rhodospirillum and Rhodopseudomonas are still considerably heterogeneous in their quinone systems. The heterogeneity of the genus Rhodospirillum can be seen in other taxonomic aspects. R. rubrum is a facultatively aerobic species with intracytoplasmic membranes of the vesicular type, whereas the brown-colored species R. photometricum, Rhodospirillum fulvum, and Rhodospirillum molischianum are strictly anaerobic and have several stacks of lamellar membranes. In addition, Rhodospirillum salexigens, which was not examined in this study, is a facultatively aerobic, halophilic species that has the 446 HIRAISHI and HOsHINo VOL. 30 intracytoplasmic membrane structure of the lamellar type. With respect to the quinone system, however, R. photometricum is rather similar to R. rubrum (category II), and both species are clearly distinguishable from the other Rhodospirillum species lacking RQ (category IV). The similarity between R. rubrum and R. photometricum is also recognized in producing bacteriochlorophyll a of the geranyl- geranyol type (24) and having a "large" type of cytochromes c (25). IMHOFFet al. (13) themselves stated : "The heterogeneity of this genus may call for further separation into several genera. However, this seems inappropriate until more strains of each species are studied in detail and more data become available to justify the establishment of new genera." Based on the Q and MK composition and other taxonomic traits, we have already proposed that R. rubrum, R, photometricum, and the remaining brown-colored Rhodospirillum species should be placed in three different genera (12). The RQ composition of these species may also be helpful as a chemotaxonomic marker for their separation into several new genera in the future. Much the same case can be made for the genus Rhodopseudomonas. This genus includes species with different quinone types belonging to categories I, III, and IV, although it is uniform in showing asymmetrical cell division by budding and having intracytoplasmic membranes of the lamellar type. The RGL group of phototrophic bacteria is a taxon that resembles Rhodocyclus gelatinosus phenotypi- cally (14). However, this group differs from R. gelatinosus and the other species of Rhodocyclus in containing RQ and lacking MK (category II). This situation will lead us to describe elsewhere the RGL group as a new species of the purple nonsulfur bacteria. The occurrence of isoprenoid quinones in the Rhodospirillaceae species also attracts interest in conjunction with the physiological and functional significance of their structural variations. Apart from Q, which is well recognized as playing an important role in photosynthetic electron transport, RQ or MK in the phototrophic bacteria has not been extensively studied in their biochemical aspects (26). Since MK has a low redox potential compared with Q, it is capable of mediating anaerobic electron transport to low-potential acceptors, such as fumarate, in non- photosynthetic bacteria (27-30). Also, mitochondrial fumarate reduction in the eucaryotic organism A. lumbricoides var. suis is possibly associated with RQ (31), which was shown by ERABIet al. (32) to have a redox potential similar to that of MK rather than Q. Furthermore, RAMIREZ-PoNCEet al. (33, 34) have demonstrated the involvement of RQ in photooxidase and fumarate-reducing systems in Rhodo- spirillum rubrum. In this connection, we have studied further the relationship of fumarate reductase activity and quinone components in the phototrophic bacteria and have found that high activities of this enzyme with electron donors of reduced viologens or FMNH2 is detectable in the species containing RQ and/or MK (categories II, III, and IV) but not in those containing Q only (category I) (HIRAIsHI, in preparation). These observations suggest that the quinone system in the purple nonsulfur bacteria as well as in other microorganisms has phylogenetic implica- 1984 Rhodoquinone in Rhodospirillaceae 447 tions in the biochemical evolution of energy-transducing systems (35).

Note added in proof : The quinone composition of 33 species of phototrophic purple bacteria was recently reported by J. F. Imhoff (FEMS Microbiol. Lett., 25, 85 (1984)).

We are grateful to Dr. N. Pfennig, Universitat Konstanz, Dr. H. Takahashi, Tohoku Univer- sity, Dr. T. Akiba, Institute of Physical and Chemical Research, and Dr. T. A. Hansen, University of Groningen, for supplying the cultures. We also thank Dr. H. Kuraishi and colleagues of Tokyo University of Agriculture and Technology for making available their HPLC system and Drs. M, Kobayashi and H. Matsuyama of Tokyo Metropolitan University for mass spectrometry. Special thanks go to Drs. H. Kitamura, T. Satoh, and K. Shimada for support and helpful suggestions during this work.

REFERENCES

1) M. D. CoLLINS and D. JONES,Microbiol. Rev., 45, 316 (1981). 2) Y. YAMADAand H. KURAISHI,In Methods in Microbial Chemotaxonomy, ed. by K. KoMA- GATA,Japan Scientific Societies Press, Tokyo (1982), p. 143. 3) H. W. MOOREand K. FOLKERS,J. Am. Chem. Soc., 87,1410 (1965). 4) H. W. MOOREand K. FOLKERS,J. Am. Chem. Soc., 88, 567 (1966). 5) J. GLOVERand D. R. THRELFALL,Biochem. J., 85, 14P (1962). 6) P. PowLs and F. W. HEMMING,Phytochemistry, 5,1235 (1966). 7) P. PowLs and F. W. HEMMING,Phytochemistry, 5, 1249 (1966). 8) N. BEGIN-HEIKand J. J. BLUM,Biochem. J., 105, 813 (1967). 9) H. OGAWA,S. NATORI,M. SATOand H. OZAWA, Tetrahedron Lett., 24, 1969 (1969). 10) H. OZAWA, M. SATO, S. NATORIand H. OGAWA, Experientia, 25, 484 (1969). 11) M. SATOand H. OZAWA, J. Biochem., 65, 861 (1969). 12) A. HIRAISHI,Y. HOSHINOand H. KITAMURA,J. Gen. Appl. Microbiol., 30, 197 (1984). 13) J. F. IMHOFF,H. G. TRUPERand N. PFENNIG,Int. J. Syst. Bacteriol., 34, 340 (1984). 14) A. HIRAISHIand H. KITAMURA,Bull. Jpn. Soc. Sci. Fish., 50, 1929 (1984). 15) 0. NEUTZLING,J. F. IMHOFFand H. G. TRUPER, Arch. Microbiol., 137, 256 (1984). 16) N. PFENNIG,Int. J. Syst. Bacteriol., 28, 283 (1978). 17) N. PFENNIG,Arch. Mikrobiol., 100, 197 (1974). 18) J. TAMAOKA,Y. KATAYAMA-FUJIMURAand H. KURAISHI,J. Appl. Bacteriol., 54, 31(1983). 19) W. W. PARSONand H. RUDNEY,J. Biol. Chem., 240, 1855 (1965). 20) K. TAKAMIYA,M. NISHIMURAand H. TAKAMIYA,Plant Cell Physiol., 8, 79 (1967). 21) T. AKIBA, R. USAMIand K. H0RIK0SHI, Int. J. Syst. Bacteriol., 33, 551 (1983). 22) J. F. IMHOFF,Syst. Appl. Microbiol., 4, 512 (1983). 23) J. MAROC,H. DE KLERK and M. D. KAMEN,Biochim. Biophys. Acta,162, 621 (1968). 24) A. KUNZLERand N. PFENNIG,Arch. Mikrobiol., 91, 83 (1973). 25) R. E. DICKERSON,Nature, 283, 210 (1980). 26) W. W. PARSON,In The Photosynthetic Bacteria, ed, by R. K. CLAYTONand W. R. SISTROM, Plenum Press, New York (1978), p. 455. 27) R. K. THAUER,K. JUNGERMANNand K. DECKER,Bacteriol. Rev., 41,100 (1977). 28) A. KROGER,Biochim. Biophys. Acta, 505,129 (1978). 29) E. CALLIESand W. MANNHEIM,Int. J. Syst. Bacteriol., 28,14 (1978). 30) W. MANNHEIM,In The Flavobacterium-Cytophaga Group, ed, by H. REICHENBACHand 0. B. WEEKS,Verlag Chemie, Weinheim (1981), p. 115. 31) M. SATO,K. YAMADAand H. OZAWA,Biochem. Biophys. Res. Commun., 46, 578 (1972). 32) T. ERABI, T. HIGUCHI, T. KAKUNO,J. YAMASHITA,M. TANAKAand T. HORIO, J. Biochem., 78, 795 (1975). 448 HIRAISHIand HOSHINO VOL. 30

33) M. P. RAMIREZ-PONCE,G. GIMENEZ-GALLEGOand J. M. RAMIREZ, FEBS Lett., 114, 310 (1980). 34) M. P. RAMIREZ-PONCE,J. M. RAMIREZand G. GIMENEZ-GALLEGO,FEBS Lett., 119, 137 (1980). 35) H. LEST, FEMS Microbiol. Lett., 7, 73 (1980).