Blastobacter aggregatus sp. nov., Blastobacter capsulatus sp. nov., and Blastobacter denitrificans sp. nov., New Budding Bacteria from Freshwater Habitats

System. App!. Microbio!. 6, 281-286 (1985)

Blastobacter aggregatus sp. nov., Blastobacter capsulatus sp. nov., and Blastobacter denitrificans sp. nov., New Budding Bacteria from Freshwater Habitats PETER HIRSCHI and MICHAEL MULLER 2 1

Institut fUr Allgemeine Mikrobiologie, Universitiit Kiel (Biozentrum), 2300 Kiel, Federal Republic of Germany

Received July 17, 1985

Summary Three Blastobacter strains from lake- or pondwater are described. They differ in initial bud morphology, capsule or rosette formation, and motility. There were also differences in their utilization of methanol, formate, formamide, ethanol, succinate, glutarate, L-proline, and urea. The temperature optima were 36°C (strain IFAM 3 1003), 27°C (IFAM 1004), and 41°C (IFAM 1005). A comparison with data from the literature for "Blastobacter viscosus" strain 7d, "B. aminooxidans" strain 14A, "B. natatorius" strain UQM 2507, and the type species (B. henricii) revealed species-specific differences with all three new strains. These are, therefore, described as new species: Blastobacter aggregatus sp. nov. (IFAM 1003 = DSM4 111), B. capsulatus sp. nov. (IFAM 1004 = DSM 112), and B. denitrificans sp. nov. (lFAM 1005 = DSM 113). A key is given for the determination of Blastobacter species.

Key words: Blastobacter - Budding bacteria - Freshwater bacteria

Introduction Budding as a mode of new cell formation appears to be widely spread among microorganisms and especially gram-negative bacteria (Hirsch, 1974). Some of these forms produce buds at the tips of hyphae ("prosthecae"): Hyphomicrobium, Hyphomonas, Rhodomicrobium, Pedomicrobium (Kingma-Boltjes, 1936; Pongratz, 1957; Duchow and Douglas, 1949; Aristovskaya, 1961). Other budding and non-prosthecate bacteria of oval or spherical shape excrete stalks and often form rosettes (Planctomyces; Gimesi, 1924; Hirsch and Muller, 1985b). Some pearshaped, budding forms attach to submerged surfaces (pirella; Schlesner and Hirsch, 1984). Budding of rod-shaped bacteria has rarely been observed. Henrici and Johnson (1935) provided a morphological description of an attaching, budding rod from freshwater (their Fig. 10). Similar organisms were found

2

3 4

by Zavarzin (1961), who named his slightly bent, often wedge-shaped and rosette-forming bacteria Blastobacter henricii on the basis of an enrichment culture. Although pure cultures were not obtained, B. henrici? was included in Bergey's Manual of Determinative Bacteriology (8th, ed., Buchanan and Gibbons, 1974). Later, this organism was incorrectly named "Methylosinus trichosporium" (Namsaraev and Zavarzin, 1972). Recently, pure cultures of rod-shaped budding bacteria were obtained from activated sludge; they were described as "Blastobacter viscosus" because of extensive exopolymer formation (Loginova and Trotsenko, 1979). These bacteria grew well on methanol or glucose. Autotrophic CO 2 fixation of methanol-grown cells was also observed (Loginova et al., 1981). Another new species from sewage, "B. aminooxidans", was found to be a facultative hydro-

Present address: Landes-Hygiene-Institut Oldenburg, Am Damm 46, D-2900 Oldenburg Institut fur Allgemeine Mikrobiologie, Univ. Kiel Deutsche Sammlung fur Mikroorganismen, Gottingen

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gen autotroph (Doronina et al., 1983). A third species, named "B. natatorius", came from a swimming pool (Sly, 1985). During a search for budding bacteria in various freshwater habitats and employing many different methods, Blastobacter-like organisms were found in several instances (M. Muller, 1977; Hirsch et al., 1977; Hirsch, 1978; Hirsch and Muller, 1985a). Pure cultures were obtained from lakes and ponds. The present publication provides detailed descriptions of three strains representing new Blastobacter species. Some properties of these isolates have already been mentioned by Loginova and Trotsenko (1979). Additional and similar Blastobacter isolates from a eutrophic Michigan forest pond, from the Solar Lake (Elat, Sinai), and from Antarctic rock samples have been obtained (Hirsch, 1978, 1980).

Materials and Methods Bacteria. Strains IFAM 1003, 1004, and 1005 came from our departmental culture collection; they were isolated from lake water (l003 and 1005) or pondwater (1004; Hirsch et al., 1977; Hirsch and Muller, 198sa). For media see Hirsch and Muller (198sb).

Morphological observations and physiological tests. Slide cultures, size measurements, light- and electron microscopy as well as media and physiological tests have all been described previously (Hirsch and Muller, 1985a). DNA base ratio determinations. Logarithmically grown cells were harvested by centrifugation (16000 x g, 4°q, resuspended and washed with 20 ml EDTA (0.1 M) in 0.15 M NaCl, and frozen for storage. For DNA extraction the cells were treated with lysozyme (0.1 mg/ml; 2h), pronase (0.036 mg/ml, 2h), and sodium dodecylsulfate (0.019 mg/ml, 3h) - all at 37°C and shaken. The cell lysate was then further treated as described by Marmur (1961), and the mol % G+C were calculated from thermal denaturation profiles according to Mandel and Marmur (1961).

Results Morphology. Strains 1003, 1004, and 1005 grew well on medium PYG at 30°C. Budding cells were readily detected in all cultures, and there was no indication of cross septation without prior budding. The cells were essentially rod-shaped and varied in length; shorter cells were often ovoid (Figs. 1-3). Cells of strain 1004 had a tendency to bend or even twist helically. Rosette formation was only observed with strain 1003. Two strains (1003 and 1005)

Table 1. Morphological properties of the Blastobacter isolates and other Blastobacter spp. of the literature Property

B. aggregatus B. capsulatus B. denitri"B. viscosus {ieans strain 7d IFAM 1003 T IFAM 1004T IFAM 1005 T (1)

"B. aminooxidans" 14A (2)

Cell shape

ovoid to rods

rods, often bent and tapering on budding pole

rods with rounded cell poles

pleomorphic rods, often bent a. branched

Initial bud shape

rods

ovoid

rods

ovoid 4

pleomorphic rods s. t. multiply by budding a. with minute appendages ovoid

Bud origin

narrow pole

slightly subpolar

Capsule formation Flagellation/motility Rosette formation Cell diameter (f,lm) Mature length (f,lm) Colony pigmentation

narrow pole or lateral +

polar or subpolar +

polar or lateral +5

+ + 0.6-0.8 1.5-2.3 colorless 7

Growth in liquids

turbidity

Origin

lake water

1 2

3 4

5 6

7

B. henrieii Zavarzin (1961) rods, wedge- or club-shaped, often curved

Rods with rounded ends, straight or slightly curved, or wedge-shaped

spherical or oblong polar

spherical or ovoid polar

n.d. 6

+ 0.7-0.9 1.5-2.3 colorless 7

0.6-0.8 1.5-2.3 colorless 7

0.5-0.9 1.0-3.2 yellow

turbidity or pellicle pond water

turbidity

turbidity, pellicle activated sludge

lake water

0.8-1.0 1.5-3.0 yellow, orange turbidity activated sludge

"B. natatorius" UQM 2507 (3)

+ 0.7-1.0 2.0-4.5 n.d. n.d. pond water

Loginova and Trotsenko (1979). Doronina et al. (1983). Sly (1985). The bud may arise as a gonidium, i. e. part of the rod-shaped cell is pinched off (Loginova and Trotsenko, 1979). A fibrillar microcapsule. Not determined. Old colonies in transparent illumination brownish.

+ + 0.5-0.8 1.0-3.0 (10) yellow or pale pink pellicle a. deposit swimming pool (freshwater)

Three New Blastobaeter spp. from Freshwater

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Table 2. Physiological properties of the isolates and other Blastobacter spp. of the literature

Property

B. aggregatus B. capsulatus sp. nov. sp. nov. 1004 1003

Utilization o( C-sourees (lOT 10 gilt Saccharose, cellobiose + Salicin Glucuronic acid lactone, N-acetyl- glucosamine Succinate, malate, formate, formamide Tartrate, glutarate + L-arginine + L-proline Utilization of methanol (0.5 vol %) + Utilization of ethanol (0.4 vol % ) + Aerobically: acid from D( + )glucose Aerobically: acid from mannitol + Utzilization of urea as N-source (4.6 gil ) NO] reduction (assimilatory) NO] reduction (dissimilatory) + CO : air tolerance (8 : 2) < 36 Tolerance of NaCl (gil) 13-43 Temperature range for growth (0C) 36 Temperature optimum (0C) 6.9 pH optimum for growth 60.4±0.4 DNA base ratio (mol % G + C) 1-3 4

B. denitri(ieans sp. nov. 1005

+ + + + +

"B. vis casus

7d l

n.d. n.d. n.d. +

+ + + + +

+ + + n.d.

+ + + < 27 14-35 27 7.3-7.8 58.9±1.3

+ + <27 13-46 41 6.8-7.2 64.5±0.8

+ n. d. n.d. n. d. 10-34

28-30 6.8-7.2 66.3±0.9

"B. aminooxidans" 14A2

"B. natatonus "

+ n.d. n.d. + n.d. n.d. n.d. n.d. +

n. d. n.d. n.d. n.d. n.d. n.d. n. d.

n.d. n.d. + n.d. n.d. n.d. 10-34 29-32 7.2-7.8 69.0±0.9

n.d. +

n.d. n.d. 11-39

25-30 n.d. 65.0

See references Table 1. Growth was tested after 18 d. Stimulation (+) was determined by an increase in OD 65o of significantly >0.015.

were motile, and 1004 produced a capsule. The morphological features are listed in Table 1. Utilization of carbon compounds. All three strains grew with 1-10 gil of D-glucose, D-fructose, lactose, D-ribose, D-galactose, and with 1-2 gil of mannitol, glycerol, pyruvate, a-oxoglutarate, acetate, L-glutamate, and L-histidine. None of the three strains utilized 1-2 gil of maltose, starch, dextrin, inulin, fumarate, lactate, caproate, citrate, propionate, indole, phthalate, D,L-phenylalanine, L-aspartate, leucine, L-serine, glycine, alanine, or a gas mixture of methane and air (1: 1). The strains did not grow oligocarbophilically. All three strains produced acid anaerobically from D( - )fructose, and D( + )galactose in Hugh-Leifson fermentation tests; aerobic acid production varied among these isolates. There was no gas production aerobically or anaerobically from D( + )glucose, D( - )fructose, D( +)galactose, saccharose, maltose, or mannitol. Differences in the spectrum of carbon utilization are given in Table 2. Utilization of nitrogen compounds. All three strains grew well with 0.5 gil NHt or 2 gil NO], but not with 2-6.9 gil nitrite or 8.5 gil nitrate. Several organic complex nitrogen sources supported excellent growth (1 gil): Bacto Peptone, Casein hydrolysate (vitamin-free), and Bacto Yeast Extract. There was no growth with 6.75 gil methylamine. HC!. Urea supported growth of strains IFAM 1003 and 1005.

Additional physiological tests. The three strains were aerobic, mesophilic, vitamin-independent, and had their pH optimum around 7.0 (Table 2). They were positive for catalase, oxidase, and peroxidase. They did not decarboxylate arginine or lysine, deaminate lysine or phenylalanine, liquefy gelatin or produce acetoin, indole, or H 2S. Cellulose was not hydrolyzed, NHt or NOi not oxidized, and litmus milk was neither acidified, peptonized or coagulated. Strain 1005 produced gas from nitrate under anaerobic conditions; strain 1004 showed assimilatory as well as dissimilatory nitrate reduction. The base ratios of strains 1003 and 1004 did not differ significantly, but that of strain 1005 was slightly higher (Table 2). Discussion A comparison of the three isolates clearly shows that there are differences, for example in their carbon utilization spectrum. But there were similarities, too. A taxonomic placement in a common genus seems to be justified. Only very few budding bacteria with similar morphology have been described so far. Rhodopseudomonas spp. and Chamaesiphon spp. are photosynthetic, but our strains were not pigmented. Nitrobacter spp. oxidize nitrite, but nitrate formation from nitrite has not been observed with our strains. "Methylosinus trichosporium" oxidizes methane (Namsaraev and Zavarzin, 1972), a

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2um

property which was absent from our strains. A budding, rod-shaped bacterium living attached to diatoms has been described on purely morphological grounds as "Chamaesiphonosira cymbellicola" (Geitler, 1975). Unfortunately, the complete lack of any information on this organism's physiology and the lack of cultures do not alIowa comparison. Our isolates show closest resemblance to members of the genus Blastohacter. Up to date, four species are known within this genus: B. henrici;T (Zavarzin, 1961) is the type species, but it was not obtained in pure culture. More recently, three other species have been described: "B. viscosus" (Loginova and Trotsenko, 1979), "B. aminooxidans" (Doronina et aI., 1983), and "E. natatorius" (Sly, 1985). Information on the type species is rather scanty, but the other species have been studied in more detail. The formation of rosettes by B. henricii resembles that of strain 1003, but the latter organism is definitely motile. Also, the initial bud morphology and cell length are quite different (Table 1). Strain 1003 can be easily differentiated from "B. viscosus" and "B. aminooxidans" on the basis of capsule and rosette formation, motility, utilization of carbon sources, and DNA base ratios (Table 2). It differs from "B. natatorius" in cell shape, colony pigmentation, utilization of urea, and DNA base ratio. Strain 1003 represents a new species, and the name Blastobacter aggregatus sp. nov. is proposed. Strain IFAM 1004 resembles "B. viscosus" and "B. aminooxidans" by its capsule formation. However, differences in colony pigmentation, carbon utilization spectrum, and DNA base ratios clearly indicate that it represents a separate new taxon. Also, capsule formation, colony pigmentation, rosette formation, nitrate reduction, and lack of motility as well as the DNA base ratio separate this strain from "B. natatorius". The name Blastobacter capsulatus sp. nov. is proposed for strain 1004. Strain IFAM 1005 differs from "E. viscosus" and "B. aminooxidans" by its motility, by the colony pigmentation and growth on acetate, L-glutamate, L-histidine, or glutarate. It does not utilize fumarate or methylamine. HCI, and it does not produce acid from glucose, as is the case with "E. viscosus" and "B. natatorius". Strain 1005 also differs distinctly from B. henricii in flagellation, rosette formation, and cell length. We propose for this strain the name Blastobacter denitrificans sp. nov. because of its anaerobic gas formation from nitrate. Formal description of the three new species. Blastobacter aggregatus sp. nov.

c

lum

Fig. 1. Blastobacter aggregatus strain IFAM 1003; electron micrographs. a) Young rosette with one budding cell. Negative stain. b) Two cells attached, one cell flagellated; shadow-cast. c) Rosette showing central holdfast; negative stain.

Ag.gre.ga'tus. L. adj. aggregatus joined together, referring to the frequent formation of rosettes. Cells ovoid to rod-shaped, new cell formation by budding from the narrow cell pole. Cells 0.6-0.8 X 1.5-2.3 f.tm, motile swarmer cells form rosettes (Fig. 1). Colonies colorless, but slightly brownish when older and in transmittent light, dull to shiny, round with entire edges. Grow in liquid media as turbidity. Gram-negatie, non-acidfast, do not form spores or cysts. Grow well on dilute media with 0.25 gil Bacto Peptone, Yeast Extract, and glucose,

Three New Blastobacter spp. from Freshwater with 10 mIll of Hutner's basal salt solution added. Vitamins are not required. Strictly aerobic, heterotrophic. Further properties see Tables 1 and 2. Isolated from surface water of Lake H6ftsee (Holstein) in October. DNA base ratio: 60.4 ± 0.4 mol % G+c. Type strain: IFAM 1003 T (= Muller 161, = DSM 111).

285

O.5um

Blastobacter capsulatus sp. nov. Cap.su.la'tus. L. n. capsula a small chest, capsule; M. L. n. adj. capsulatus encapsulated. Cells rod-shaped, often bent and narrowing on one pole. Old cells frequently Y-shaped. Initial bud shape is ovoid (Fig. 2). Cells with capsules, non-motile, 0.7-0.9 X 1.5-2.3 !-tm, do not form rosettes. Cells gram-negative, non-acidfast, do not form spores or cysts. Grow in liquid media as turbidity at 30°C. Cultures grown at 25 °C form much exopolymer. A surface pellicle is produced which later sinks down. Aerobic, heterotrophic; assimilatory and dissimilatory nitrate reduction positive, but no gas formation from nitrate. Grow well on dilute media (see B. aggregatus), not vitamin-dependent. Further properties as described in Tables 1 and 2. Habitat: Isolated from a shallow eutrophic pond near Westensee, Kiel, Germany. DNA base ratio: 58.9 ± 1.3 mol % G+c. Type strain: IFAM 1004T (= Muller 216, = DSM 112).

Blastobacter denitrificans sp. nov. De.ni.tri'fi.cans. L. prep. de away from; L. n. nitrum soda; M. L. n. nitrum nitrate; M. L. v. denitrifico denitrify; M. L. part. adj. denitrificans de nitrifying. Cells rod-shaped with rounded poles. Motile buds rodshaped and originating subpolary (Fig. 3), with 1-3 subpolar flagella. Cells non-capsulated, gram-negative, not acid-fast, do not form spores or cysts, 0.6-0.8 x 1.5-2.3 !-tm. Grow in liquid media as turbidity. Colonies color less, later in transmittent light brownish, glistening, raised and round with entire edges. Grow well on dilute media (see B. aggregatus). Gas is formed anaerobically from nitrate. Grow well with C-l compounds such as methanol, formate, formamide. Aerobic, heterotrophic. Additional properties are listed in Tables 1 and 2. Habitat: Isolated in April from surface water of Lake Plussee (Holstein). DNA base ratio: 64.5 ± 0.8 mol % G+c. Type strain: IFAM lO05 T (= Muller 222, = DSM 113).

Fig. 2. Blastobacter capsulatus strain IFAM 1004: electron micrographs, negative stains. a) Two cells with nearly mature, terminal buds. b) Lateral budding; the capsule causes heavy negative stain accumulation. c) A short cell chain.

Key to the species of genus Blastobacter: A. Cells rod-shaped or spherical to oblong, aggregating to form rosettes. 1. Bud cells flagellated. a. Colonies colorless; urea utilized as N-source: Blastobacter aggregatus sp. nov. b. Colonies yellow or pale pink; urea not an N-source: "Blastobacter natatorius" Sly 1985 2. Bud cells non-motile: Blastobacter henricii Zavarzin 1961 B. Cells rod-shaped, straight or slightly bent, or pleomorphic and sometimes branching; do not aggregate to form rosettes.

a

b

c

lpm

Fig. 3. Blastobacter denitrificans strain IFAM 1005: electron micrographs, negative stains. a) Young subpolar bud. b) Rodshaped bud. c) Flagellated swarmer cell.

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1. Bud cells flagellated; urea utilized as an N-source: Blastobacter denitrificans sp. nov. 2. Bud cells non-motile, urea not a nitrogen source. a. Colony pigmentation yellow. Grow on methanol, ethanol, fumarate, succinate, malate, formate, methylamine. HCI, or formamide. No growth on acetate. aa. Cells with voluminous capsular materia!' Acid formation from glucose, no growth on maltose or starch. DNA base ratio 66.3 ± 0.9 mol % G+C: "Blastobacter viscosus" Loginova and Trotsenko 1979 bb. Cells only with microcapsule, no acid formation from glucose. Maltose and starch are utilized. DNA base ratio 69 mol % G+C: "Blastobacter aminooxidans" Doronina et a!., 1983 b. Colonies colorless to beige-brownish. No growth on methanol, ethanol, fumarate, succinate, malate, formate, formamide, or methylamine. HC!. Utilize acetate. DNA base ratio: 58.9 ± 1.3 mol % G+C: Blastobacter capsulatus sp. nov.

Acknowledgements. We gratefully acknowledge helpful discussions with Dr. G. A. Zavarzin (Moscow) and the kind interest and cooperation of Dr.]. Trotsenko (Pushchina). Skillful technical assistance was rendered by A. Grater, B. Hoffmann, and K. Lutter-Mohr, Kiel. Part of this work was supported by a grant from the Deutsche Forschungsgemeinschaft to P. Hirsch.

References Aristovskaya, T. V.: Accumulation of iron by decomposing mineral complexes of humic matter by microorganisms. Dok!. Acad. Nauk SSSR 136, 954-957 (1961) Buchanan, R. E., Gibbons, N. E.: Bergey's Manual of Determinative Bacteriology, 8th ed. Baltimore, The Williams and Wilkins Co., 1974 Doronina, N. V., Govorukhina, N. ]., Trotsenko, Yu. A.: Blastobacter aminooxidans, a new species of bacteria growing autotrophically on methylated amines. Mikrobiologiya 52, 709-715 (Trans!. 547-553) (1983) Duchow, E., Douglas, H. c.: Rhodomicrobium vannielii, a new photoheterotrophic bacterium. ]. Bact. 58, 409-416 (1949) Geitler, L.: Chamaesiphonosira cymbellicola, n. gen., n. sp., ein knospenbildendes Bakterium und sein spezialisierter Epiphytismus. Arch. Mikrobiol. 103, 231-236 (1975) Gimesi, N.: Hydrobiologicai talmanyok (Hydrobiologische Studien). 1. Planctomyces Bekefii, Gim. nov. gen. et sp., pp. 1-8. Budapest, Kiadja a Magyar Ciszterci. Rend. 1924

Henrici, A. T., Johnson, D.: Studies of freshwater bacteria. II. Stalked bacteria, a new order of Schizomycetes. J. Bact. 30, 61-92 (1935) Hirsch, P.: Budding bacteria. Ann. Rev. Microbio!. 28, 392-444 (1974) Hirsch, P.: Distribution and isolation of potential indicator bacteria in lakes and a pond. Abstracts, Amer. Soc. Microbiol. Proceed., N. 56 (1978) Hirsch, P.: Distribution and pure culture studies of morphologically distinct Solar Lake microorganisms. In: Hypersaline Brines and Evaporitic Environments, A. Nissenbaum (ed.), pp. 41-60. Amsterdam, Elsevier Sci. Pub!. Compo 1980 Hirsch, P., Muller, M.: Methods and sources for the enrichment and isolation of budding, non-prosthecate bacteria from freshwater. Microbial Ecol. in press (1985a) Hirsch, P., Muller, M.:Planctomyces limnophilus sp. nov., a stalked and budding bacterium from freshwater. System. App!. Microbio!. 6,276-280 (1985b) Hirsch, P., Muller, M., Schlesner, H.: New aquatic budding and prosthecate bacteria and their taxonomic position. Soc. Appl. Bacterio!' Sympos. Ser. No.6, 107-133. London, Academic Press 1977 Kingma Bolties, T. Y.: Uber Hyphomicrobium vulgare Stutzer et Hartleb. Arch. Mikrobiol. 7, 188-205 (1936) Loginova, N. V., Govorukhina, N. [., Trotsenko, Yu. A.: Autotrophic metabolism of methanol in Blastobacter viscosus. Mikrobiologiya (Trans!.) 50, 428-434 (1981) Loginova, N. V., Trotsenko, Yu. A.: Blastobacter viscosus - a new species of autotrophic bacteria utilizing methano!' Mikrobiologiya (Trans!.) 48, 644-651 (1979) Mandel, M., Marmur, J.: Use of ultra violett absorbance-temperature profile for determining the guanine plus cytosine base content of DNA. Meth. Enzymol. 109, 195-206 (1961) Marmur, ].: A procedure for the isolation of DNA from microorganisms. J. Molec. Bact. 3, 208-218 (1961) Namsaraev, B. B., Zavarzin, G. A.: Trophic relationship in a methane-oxidizing culture. Mikrobiologiya 41, 887-892 (Trans!.) 1972 Pongratz, E.: D'une bacterie pediculee isolee pus de sinus. Schweiz. Z. Allg. Path. Bakt. 20, 593-608 (1957) Schlesner, H., Hirsch, P.: Assignment of ATCC 27377 to Pirella gen. nov. as Pirella staleyi comb. nov. Int. ]. system. Bact. 34, 492-495 (1984) Skerman, V. B. D.: A guide to the identification of the genera of bacteria, 2nd. ed. Baltimore, The Williams and Wilkens Compo 1967 Sly, L. ].: Emendation of the genus Blastobacter Zavarzin 1961 and description of Blastobacter natatorius sp. nov. Int. J. system. Bact. 35, 40-45 (1985) Zavarzin, G. A.: Budding bacteria. Mikrobiologiya (Trans!.) 30, 952-975 (1961)

Professor Dr. Peter Hirsch, Institut fur Allgemeine Mikrobiologie, Universitat Kiel (Biozentrum), Olshausenstr. 40-60, D-2300 Kiel