Stephen H Zinder

Department of Microbiology, Cornell University, Ithaca, NY 14853-0001, USA

Class I. Alphaproteobacteria class. nov.

George M. Garrity, Julia A. Bell and Timothy Lilburn'ri.a. Gr. n. alpha name of first letter of Greek alphabet; Gr. n. Proteus ocean god able to change shape; Gr. n. bakterion a small rod; M.L. fem. pl. n. Alphaproteobacteria class of bacteria having 16S rRNA gene sequences related to those of the members of the order Caulobacterales.

The class Alphaproteobacteria was circumscribed for this volume biales, Rhodobacterales, Rhodospirillales, Rickettsiales, and Sphingomon-on the basis of phylogenetic analysis of 16S rRNA sequences; the adales.

class contains the orders Caulobacterales, "Parvularculales", Rhizo- Type order. Caulobacterales Henrici and Johnson 1935a, 4.

Order I. Rhodospirillales Pfennig and Truper 1971, 17AL

George M. Garrity, Julia A. Bell and Timothy Lilburn'les. M.L. neut. n. Rhodospirillum type genus of the order; -ales suffix to denote order; M.L. fem. n. Rhodospirillales the Rhodospirillum order.

The order Rhodospirillales was circumscribed for this volume on the basis of phylogenetic analysis of 16S rRNA sequences; the order contains the families Rhodospirillaceae and Acetobacteraceae.

Order is morphologically, metabolically, and ecologically diverse. Includes chemoorganotrophs, chemolithotrophs, and fac ultative photoheterotrophs; some of the latter are also able to grow photoautotrophically. Other species can grow methylo-trophically.

Type genus. Rhodospirillum Molisch 1907, 24 emend. Imhoff, Petri and Siiling 1998, 796.

Family I. Rhodospirillaceae Pfennig and Truper 1971, 17AL

George M. Garrity, Julia A. Bell and Timothy Lilburn' M.L. neut. n. Rhodospirillum type genus of the family; -aceae ending to denote family; M.L. fem. pl. n. Rhodospirillaceae the Rhodospirillum family.

The family Rhodospirillaceae was circumscribed for this volume on the basis of phylogenetic analysis of 16S rRNA sequences; the family contains the genera Rhodospirillum (type genus), Azospiril-lum, Inquilinus, Levispirillum, Magnetospirillum, Phaeospirillum, Rho-docista, Rhodospira, Rhodovibrio, Roseospira, Skermanella, Thalasso-spira, and Tistrella (type genus). Inquilinus, Thalassospira, and Tis-trella were proposed after the cut-off date for inclusion in this volume (June 30, 2001) and are not described here (see Coenye et al., 2002; Lopez-Lopez et al., 2002; and Shi et al., 2002, respectively).

Preferred mode of growth for most genera is photohetero-trophic under anoxic conditions in light. Grow chemotrophically in the dark. Azospirillum, Magnetospirillum, and Skermanella are chemoorganotrophic. Motile by means of polar flagella; may have lateral flagella.

Type genus: Rhodospirillum Molisch 1907, 24 emend. Imhoff, Petri and Siiling 1998, 796.

Genus I. Rhodospirillum Molisch 1907, 24AL emend. Imhoff, Petri and Suling 1998, 796

Johannes F. Imhoff'lum. Gr. n. rhodon the rose; M.L. neut. n. Spirillum a bacterial genus; M.L. neut. n. Rhodospirillum the rose Spirillum.

Cells are vibrioid to spiral shaped, are motile by means of bipolar flagella, and multiply by binary fission. Gram negative, belonging to the Alphaproteobacteria.. Internal photosynthetic membranes are present as vesicles or as lamellae forming a sharp angle to the cytoplasmic membrane. Photosynthetic pigments are bacte-riochlorophyll a (esterified with phytol or geranylgeraniol) and carotenoids of the spirilloxanthin series with spirilloxanthin itself lacking in some species. Ubiquinones and rhodoquinones with 8 or 10 isoprene units are present. Major cellular fatty acids are

C18:1, C16:1, and C16:0, with C18:1 as dominant component (5155% of total fatty acids).

Grow preferentially photoheterotrophically under anoxic conditions in the light. Photoautotrophic growth with molecular hydrogen and sulfide as photosynthetic electron donors may occur. Chemotrophic growth occurs under microoxic to oxic conditions in the dark. Some species are very sensitive to oxygen; others grow equally well aerobically in the dark. Fermentation and ox-idant-dependent growth may occur. Polysaccharides, poly-ß-hy-

droxybutyric acid and polyphosphates may be present as storage products. Growth factors required. Mesophilic freshwater bacteria with preference for neutral pH.

Type species: Rhodospirillum rubrum (Esmarch 1887) Molisch 1907, 25 (Spirillum rubrum Esmarch 1887, 230.)

Further descriptive information

Two species of Rhodospirillum are currently known. Rhodospirillum rubrum, the type species, is one of the most intensively studied of the phototrophic bacteria. Numerous investigations on the physiology and enzymology of this species have focused on its metabolic properties, in particular CO2 fixation and characterization of ribulose-1,5-bisphosphate carboxylase (Tabita, 1995), ATP generation and coupling-factor ATPase (Gromet-Elhanan, 1995), and nitrogenase and nitrogen fixation (Ludden and Roberts, 1995).

Ri. rubrum grows very well under photoheterotrophic conditions, but it can also grow under photoautotrophic conditions with molecular hydrogen (Klemme, 1968) or sulfide as the electron donor if supplied at low concentrations (Hansen and van Gemerden, 1972). Autotrophic CO2 fixation is well documented and occurs via ribulose-1,5-bisphosphate carboxylase (Anderson and Fuller, 1967a, b). This enzyme has been highly purified and is well characterized (e.g., Tabita and McFadden, 1974a, b; Tabita, 1995). Ribulose-1,5-bisphosphate carboxylase is derepressed only at low CO2 tensions (1.5-2.0% of the atmospheric tension) and can make up to 50% of the total soluble protein of the cells under such conditions (Sarles and Tabita, 1983). In the presence of malate or acetate, however, CO2 is not assimilated via the reductive pentose phosphate cycle, but by other carboxylating reactions.

Under anoxic dark conditions, Ri. rubrum is able to ferment sugars and pyruvate (Kohlmiller and Gest, 1951; Giirgiin et al., 1976; Gorrell and Uffen, 1977). Pyruvate is cleaved by pyruvate formate lyase, which is specifically induced under these growth conditions (Uffen, 1973; Jungermann and Schon, 1974). From formate, H2 and CO2 are formed by a CO-sensitive formic hydrogen lyase (Gorrell and Uffen, 1977). H2, CO2, acetate, and eventually propionate are produced as fermentation products from pyruvate. Ri. rubrum is able to gain energy from the coupling of CO oxidation and H2 evolution and induces the synthesis of a carbon monoxide dehydrogenase on exposure to CO under anoxic conditions (Bonam et al., 1989; Kerby et al., 1995).

Rhodospirillum rubrum is also able to perform an anaerobic dark metabolism with DMSO and trimethylamine-N-oxide as electron acceptors (Schultz and Weaver, 1982). Under these conditions, growth is possible with succinate, malate, and acetate as substrates, and CO2 and DMSO or trimethylamine are formed (Schultz and Weaver, 1982).

Rhodospirillum species can grow with ammonia or N2 as sole nitrogen source (Siefert, 1976; Madigan et al., 1984). The nitrogenase of Ri. rubrum is subject to post-translational inactivation by ADP-ribosylation under energy-limiting conditions and iffixed nitrogen compounds are available (see Ludden and Roberts, 1995). Ammonia assimilation is mediated by the glutamine syn-thetase/glutamate synthase reactions, which are NADPH-de-pendent in Ri. rubrum (Brown and Herbert, 1977). Although a nitrate reductase is present in R. rubrum, growth with nitrate as sole nitrogen source apparently is not possible (Katoh, 1963; Taniguchi and Kamen, 1963; Ketchum and Sevilla, 1973;

Klemme, 1979). Purines can be used as a nitrogen source under anoxic conditions in the light and under oxic conditions in the dark (Aretz et al., 1978).

Comparably few studies have been performed with Rhodospirillum photometricum (Pfennig et al., 1965; Lehmann, 1976; Sarkar and Banerjee, 1980), which is very sensitive to oxygen and does not grow under oxic conditions in the dark (as does R. rubrum; Pfennig, 1969b), but only under microoxic conditions, provided the oxygen tension is lower than 0.5 kPa (Lehmann, 1976). Like Phaeospirillum species, Ri. photometricum is presumably unable to induce a second electron transport chain in the presence of oxygen and therefore depends on microoxic conditions in which the internal membrane system and the light-driven electron transport chain are fully expressed. Accordingly, the cells are fully pigmented under these growth conditions (Lehmann, 1976).

Two different types of alcohols are esterified with the bacte-riochlorophyll a of Rhodospirillum species. Besides phytol, which is present as an alcohol in the majority of the Purple Nonsulfur Bacteria (PNSB), geranylgeraniol is the major component of R rubrum and of some strains of Ri. photometricum (Brockmann and Knobloch, 1972; Kiinzler and Pfennig, 1973). Carotenoids of the spirilloxanthin series are present in Rhodospirillum species, but R. photometricum is unable to synthesize the end product, spiril-loxanthin, and accumulates intermediates of this pathway, such as lycopene, rhodopin, anhydrorhodovibrin, and rhodovibrin (Schmidt, 1978).

Growth of Ri. photometricum is completely inhibited at penicillin concentrations of only 10 U/ml, whereas in R. rubrum, complete inhibition occurs at penicillin concentrations of more than 1000 U/ml (Weaver et al., 1975a).

Enrichment and Isolation Procedures

Media and growth conditions used for isolation and cultivation of freshwater PNSB in general can also be applied for Rhodospi-rillum species. Various recipes for appropriate media have been developed in different laboratories (see Biebl and Pfennig, 1981; Imhoff, 1988; Imhoff and Triiper, 1992). One of these, a mineral salts medium that has been used for cultivation of the great majority of PNSB over many years is given in the footnote below.1 Standard techniques for the isolation of anaerobic bacteria in agar dilution series and on agar plates can be applied for Rhodo-spirillum species (Biebl and Pfennig, 1981; Imhoff and Traper, 1992), if care is taken to establish and maintain oxygen-free conditions, especially for oxygen-sensitive species. This can be achieved by addition of 0.05% sodium ascorbate or 0.025% thio-glycolate to the growth media in completely filled screw-capped bottles.

1. AT medium contains (g/l): KH2PO4, 1.0; MgCl2-6H2O, 0.5; CaCl2-2H2O, 0.1; NH4Cl, 1.0; NaHCO3, 3.0; Na2SO4, 0.7; NaCl, 1.0; sulfate-free trace element solution SLA (Imhoff and Truper, 1977; Imhoff, 1992), 1 ml; and vitamin solution VA (Imhoff and Truper, 1977; 1992), 1 ml. Organic carbon sources (routinely 10 mM sodium malate, sodium succinate, sodium pyruvate, or sodium acetate) and, for oxygen-sensitive strains, 0.5 g sodium ascorbate or 0.25 g thioglycolate are added separately. The initial pH is adjusted to 6.9. Vitamin solution VA contains in 100 ml of double distilled water: biotin, 10 mg; niacin amide, 35 mg; thiamine dichlo-ride, 30 mg; p-aminobenzoic acid, 20 mg; pyridoxal hydrochloride, 10 mg; calcium pantothenate, 10 mg; and vitamin B12, 5 mg.

The trace element solution SLA has the following composition: FeCl2-4H2O, 1.8 g; CoCl2-6H2O, 250 mg; NiCl2-6H20,10 mg; CuCl2-5H20,10 mg; MnCl2-4H2O, 70 mg; ZnCl2,100 mg; H3BO3, 500 mg; Na2MoO4-2H20,30 mg; and Na2SeO3-5H2O, 10 mg. These components are dissolved in 1 liter of double distilled water. The pH of the solution is adjusted with HCl to 2-3.

Maintenance Procedures

Cultures can be preserved by standard techniques in liquid nitrogen or at — 80°C in a mechanical freezer.

Differentiation of the genus Rhodospirillum from other genera

A number of chemotaxonomic properties distinguish Rhodospirillum species from other spiral-shaped Purple Nonsulfur Bacteria (PNSB). They differ from Phaeospirillum species, their closest relatives, in major quinone components and cytochrome c structure. Large-type cytochromes c2 are present in Ri. rubrum and Ri. photometricum, whereas small-type cytochromes <2 were found in Phaeospirillum species (Ambler et al., 1979). Major differentiating properties for phototrophic bacteria in the Rhodospirillaceae are shown in Table BXII.a.1. The phylogenetic relationships of these bacteria based on 16S rDNA sequences are shown in Fig. 1 (p. 124) of the introductory chapter "Anoxygenic Phototrophic Purple Bacteria", Volume 2, Part A.

Taxonomic Comments

Traditionally all spiral-shaped phototrophic PNSB have been assigned to the genus Rhodospirillum (Pfennig and Trüper, 1974). Recognition of the large amount of chemotaxonomic and phy-logenetic diversity in the PNSB, and their presence in different groups of the Proteobacteria, initially led to the taxonomic reclassification of those species belonging to the Betaproteobacteria. "Rhodospirillum tenue" was assigned to Rhodocyclus tenuis (Imhoff et al., 1984) and this reclassification was later supported by 16S rDNA sequence analyses (Hiraishi et al., 1991a). After this reclassification, all species of the genus Rhodospirillum were Alpha-proteobacteria, though the group remained very heterogeneous in phenotypic properties and genetic relationships.

The description of new species, assigned to the genus Rhodo-spirillum, based merely on their spiral shape, continued until recently, although most of them were quite distinct from Rhodo-spirillum rubrum, the type species of this genus. Four halophilic species were classified together with several freshwater species of the genus Rhodospirillum. In addition, Rhodospirillum centenum (Fa-vinger et al., 1989) was assigned to this genus, though significant differences from Rhodospirillum rubrum had been stated in the species description. More recently, the great genetic distance among the spiral-shaped PNSB has been recognized in several proposals. Rhodospirillum centenum was transferred to a new genus as Rhodocista centenaria (Kawasaki et al., 1992). Another new spiral-shaped species, Rhodospira trueperi, was assigned to a new genus based on significant phenotypic and genotypic differences from Rhodospirillum rubrum and other known PNSB (Pfennig et al., 1997).

The anticipated heterogeneity of the genus Rhodospirillum became clearly apparent with the 16S rDNA sequence information of most of the known species (Kawasaki et al., 1993b; Imhoff et al., 1998), and these data implied that the spiral-shaped Alpha-proteobacteria are phylogenetically quite distantly related to each other and do not warrant classification in one and the same genus. Therefore, a reclassification of the spiral-shaped phototrophic Alphaproteobacteria, based on distinct phenotypic properties and 16S rDNA sequence similarities, has been proposed (Imhoff et al., 1998; see Table BXII.a.1 and Fig. 1 [p. 124] of the introductory chapter "Anoxygenic Phototrophic Purple Bacteria" , Volume 2, Part A).

Major quinone components and fatty acid composition, salt requirements, and phylogenetic relationships based on 16S rDNA sequences were considered of primary importance in defining and differentiating these genera. Several phylogenetic lines of salt dependent species were recognized and the salt-dependence was regarded as a genus-specific property (Imhoff et al., 1998). Four of the genera of spiral-shaped phototrophic Alphaproteobacteria were defined as salt-dependent and three are freshwater bacteria. Only Pi. rubrum and Pi. photometricum were maintained as species of the genus Rhodospirillum. All other species were transferred to the new genera Phaeospirillum, Phodovi-brio, Phodothalassium, and Poseospira (Imhoff et al., 1998) and are considered in the respective chapters of this volume.

Further Reading

Drews, G. andJ.F. Imhoff. 1991. Phototrophic purple bacteria. In Shively and Barton (Editors), Variations in Autotrophic Life, Academic Press, London. pp. 51-97. Imhoff, J.F. 1988. Anoxygenic phototrophic bacteria. In Austin (Editor), Methods in Aquatic Bacteriology, John Wiley & Sons Ltd., Chichester. pp. 207-240.

Imhoff, J.F. 1992. Taxonomy, phylogeny, and general ecology of anoxy-genic phototrophic bacteria. In Mann and Carr (Editors), Biotechnology Handbooks: Photosynthetic Prokaryotes, Vol. 6, Plenum Press, New York. pp. 53-92. Imhoff, J.F. 1999. A phylogenetically oriented taxonomy of anoxygenic phototrophic bacteria. In Pescheck, Loffelhardt and Schmetteter (Editors), The Phototrophic Prokaryotes, Plenum Publishing Corporation, New York. pp. 763-774. Imhoff, J.F., R. Petri and J. Silling. 1998. Reclassification of species of the spiral-shaped phototrophic purple non-sulfur bacteria of the a-Pro-teobacteria: description of the new genera Phaeospirillum gen. nov., Phodovibrio gen. nov., Phodothalassium gen. nov. and Poseospira gen. nov. as well as transfer of Phodospirillum fulvum to Phaeospirillumfulvum comb. nov., of Phodospirillum molischianum to Phaeospirillum molis-chianum comb. nov., of Phodospirillum salinarum to Phodovibrio sali-narum comb. nov., of Phodospirillum sodomense to Phodovibrio sodomensis comb. nov., of Phodospirillum salexigens to Phodothalassium salexigens comb. nov. and of Phodospirillum mediosalinum to Poseospira mediosalina comb. nov. Int. J. Syst. Bacteriol. 48: 793-798. Imhoff, J.F. and H.G. Truper. 1992. The genus Phodospirillum and related genera. In Balows, Truper, Dworkin, Harder and Schleifer (Editors), The Prokaryotes: A Handbook on the Biology of Bacteria. Ecophy-siology, Isolation, Identification, Applications, 2nd ed., Springer-Verlag, New York. pp. 2141-2155. Kawasaki, H., Y. Hoshino and K. Yamasato. 1993. Phylogenetic diversity of phototrophic purple non-sulfur bacteria in the alpha proteobacteria group. FEMS Microbiol. Lett. 112: 61-66. Rodriguez-Valera, F., A. Ventosa, G. Juez andJ.F. Imhoff. 1985. Variation of environmental features and microbial populations with salt concentrations in a multi-pond saltern. Microb. Ecol. 11: 107-116.

Differentiation of the species of the genus Rhodospirillum

Major differentiating properties between Phodospirillum species are shown in Tables BXII.a.1 and BXII.a.2.

TABLE BXII.a.1. Diagnostic properties of the spiral-shaped phototrophic Alphaproteobactericf


0 0

Post a comment