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Rhodopseudomonas Acidophila, Sp. N., a New Species JOURNAL OP BACrERiOLOGY, Aug. 1969, p. 597-602 Vol. 99, No. 2 Copyright @ 1969 American Society for Microbiology Printed In U.S.A. Rhodopseudomonas acidophila, sp. n., a New Species of the Budding Purple Nonsulfur Bacteria NORBERT PFENNIG Department of Microbiology, University of Illinois, Urbana, Illinois, and Institutfur Mikrobiologie der Universitdt Gottingen, Gottingen, Germany Received for publication 24 May 1969 A succinate-mineral salts medium ofpH 5.2 provided selective enrichment condi- tions for Rhodomicrobium vannielii and for a new species belonging to the Athiorho- daceae, described herein as Rhodopseudomonas acidophila. Sev'en strains of the new species have been isolated from different soUirces in the United States and Ger- many. The cells are rod-shaped or ovoid, 1.0 to 1.3 lum wide and 2 to 5 ,um long, and motile by means of polar flagella. Multiplication occurs by budding. The photopigments consist of bacteriochlorophyll a and carotenoids of the spirilloxan- thin series, together with new carotenoids. All strains can grow either under anaero- bic conditions in the light or under microaerophilic to aerobic conditions in the dark. No growth factors are required. The range of simple organic substrates photo- assimilated resembles that characteristic of Rhodomicrobium. Good photolitho- trophic growth is possible at the expense of molecular hydrogen; thiosulfate and sulfide are not utilized. In his monograph on the purple nonsulfur bac- the following natural sources: strain 7150, Lake teria, van Niel (4) showed that the use of different Monroe near Bloomington, Ind.; strain 7250, cypress substrates provides a convenient means for the swamp, Okeefenokee State Park, Ga.; strain 77550, selective enrichment of different members of the mud vulcano (pH: 4.5), Yellowstone National Park, Wyo.; strain 7750, farm pond near Athens, Ga.; Athiorhodaceae. He noted that extensive experi- strain 2751, forest pond near Grunenplan, Germany; ments on the effect of pH on the enrichment of strain 3251, sphagnum peat bog near Kolshorn/ these organisms had not been performed. His Hanover, Germany. enrichment experiments were usually conducted Media and conditions of cultivation. Unless other- at a pH between 7 and 8, and yeast extract was wise indicated, the following mineral base was used added to the medium. The pH range for growth of for all media: KH2PO4, 1 g; NH4Cl, 0.5 g; MgSOi- the resulting pure cultures obtained by such 7H20, 0.4 g; NaCl, 0.4 g; CaCl2.2H20, 0.05 g; trace methods is generally between pH 6 and 8, the pH element solution of Pfennig and Lippert (3), 10 ml; optimum being somewhat above 7. water to 1,000 ml. For enrichment cultures, agar- We examined the effect of low pH values (5.1) shake cultures and stock cultures, 1.5 g of disodium succinate per liter was added as the carbon source. In on the enrichment of the Athiorhodaceae, with solid media, the agar concentration was 1%. The pH succinate as a carbon source. From such enrich- of the enrichment was adjusted to give 5.1 to 5.2 ments, seven closely similar strains which grow after autoclaving. Screw-cap bottles (50 ml) were best at pH 5.5 and 6 were isolated. The new used as culture vessels. Pure cultures were obtained strains differ from all known species of the Athio- by repeated application of the agar-shake culture rhodaceae and are described here as a new species method, with the succinate medium used at a pH of the genus Rhodopseudomonas: R. acidophila of 5.6. Tests for the utilization of simple organic sub- sp. n. strates (sodium salts were used in case of organic acids) were performed by adding the substrates from MATERIALS AND METHODS sterile stock solutions (final concentration, 0.1% Sources of strains. The first strain of the new species w/v) to the autoclaved mineral medium; tests were (strain 7050) was isolated by enrichment from a water carried out in triplicate at a pH of 5.8 to 6.0. Growth sample from Crystal Lake, Urbana, Ill. Many water was estimated from measurements of the optical and mud samples from different localities have been density at 650 nm in 1-cm cuvettes by using a Unicam studied subsequently in order to find out how com- Spectrophotometer SP700, final readings being made mon and widespread the new species is. The additional after incubation for 2 weeks. Tests for photolithn- six strains described in this paper were isolated from trophic growth with molecular hydrogen and carbon 597 598 PFENNIG J. BACrERIOL. dioxide were performed in cotton-plugged Erlen- as much as 7 ,um long (see Table 1 and Fig. 1). A meyer flasks placed in desiccators containing a mixture tendency to form somewhat irregular and swollen of 80% H2 and 20% CO2. If not otherwise stated, the cells is apparent in all strains. The formation of incubation temperature was about 25 C, and the light rosette-like clusters, typical for R. palustris, has intensity was 50 to 100 ft-c from a 100 w tungsten been observed only in strains 7050, 7250, and lamp. Tests for ability to grow in the dark under 2751. All strains show polar cell growth and aerobic or microaerophilic conditions were performed in several different ways, by using the mineral medium multiply by budding (Fig. 2). In this respect, the (pH 5.8) with 0.1% pyruvate or lactate as the carbon new species is similar to R. palustris (6). In con- source. Cultures were prepared in 100 ml of liquid trast to Rhodomicrobium, the strains of R. acido- medium in 300-ml Erlenmeyer flasks and incubated phila do not form a tube or filament between without agitation; and agar-shake cultures (1% agar) mother cell and bud; the bud is sessile on the were prepared with 10 ml of medium per tube, control mother cell and separates by constriction when tubes being sealed with sterile Vaspar to exclude air. the bud reaches the size of the mother cell. The ability to grow on agar slants was also determined. After separation, both mother and daughter cell The absorption spectra of intact cells were measured again bud at the newly formed poles (see Fig. 2). on 3.5-ml samples of cell suspensions of freshly grown of polar flagella cultures in which 5 g of sucrose had been dissolved, All strains are motile by means in order to reduce light scattering. They were deter- and are gram-negative. The photosynthetic mem- mined with a Unicam Spectrophotometer SP700. brane system has been studied in thin sections by Deoxyribonucleic acid (DNA) base composition. The electron microscopy. As in R. palustris and Rho- moles per cent guanine plus cytosine of the DNA of domicrobium, the membrane system consists of all strains was determined by Manley Mandel, parallel lamellae which underlie and are possibly Houston, Texas, by the caesium chloride density continuous with the cytoplasmic membrane gradient method (5). [strains 7150, 7250, 7550, 7750, 2751, and 3251 (G. Cohen-Bazire, personal communication); RESULTS strain 7050 (D. S. Hoare, personal communica- Enrichment and isolation. The members of the tion)]. Athiorhodaceae which developed in anaerobic Physiological and biochemical characteristics. R. illuminated enrichment cultures in a succinate- vannielii is the only purple nonsulfur bacterium so mineral medium with an initial of pH 5.1 to 5.2 far described which does not require growth fac- depended primarily on the natural sample used as tors. All enrichment cultures in which R. acido- inoculum. In three cases, R. acidophila was the phila was detected microscopically yielded pure only phototrophic bacterium which developed in cultures of this organism which grew well in suc- the enrichments (strains 7050, 7150, and 3251). cinate mineral medium. R. acidophila is accord- Other enrichment cultures yielded Rhodomicro- ingly a second species of the Athiorhodaceae bium vannielii, either alone or together with R. which does not require organic growth factors. acidophila (strain 7250). The low pH-succinate The addition of Casamino Acids or yeast extract mineral medium evidently provides excellent en- to the succinate mineral medium does not increase richment conditions for R. vannielii, an organ- the growth rate of R. acidophila, and may even ism for which a specific enrichment method has inhibit growth at higher concentrations. The not so far been available. A large number of en- shortest doubling times measured with strain 7050 richments yielded, after several weeks of incuba- using the succinate-mineral medium in stationary tion, one or two of the well-known Rhodopseudo- bottle cultures were between 3.5 and 5.0 hr. monas species (R. palustris, R. capsulata, or R. All R. acidophila strains are capable of good gelatinosa). These species all require growth fac- photolithotrophic growth with molecular hydro- tors, and developed only as members of the mixed gen and carbon dioxide (80:20, v/v). Thiosulfate population of the enrichment culture. Strains is not utilized, and hydrogen sulfide strongly in- 7550, 7750, and 2751 of R. acidophila developed hibits growth. in enrichment cultures together with R. palustris The results of tests for phototrophic growth of or R. capsulata; by repeated transfer to fresh R. acidophila strains on single organic carbon enrichment media, it was possible in every case to sources are summarized in Table 2. The species is obtain R. acidophila as the predominant member characterized by the inability to utilize amino of the population. acids, sugars, higher fatty acids, cyclohexane Morphology. As a result of their large size and carboxylate, and benzoate for growth. characteristic shape, individual cells of R. acido- Three different methods were used to test for phila can be readily recognized among the cells of the ability of R. acidophila strains to grow in the other phototrophs in enrichment cultures.
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