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1 Department of Biochemistry and Cell Physiology, Faculty of Biology and Soil Science, Voronezh State University, Universitetskaya pl. 1, Voronezh 394006, Russia
2 Bremen Institute for Materials Testing, Department of Microbiology, Paul-Feller-Straße 1, D-28199 Bremen, Germany
3 Winogradsky Institute of Microbiology of the Russian Academy of Sciences, Pr. 60-letiya Oktyabrya 7/2, Moscow 117312, Russia
4 Bioengineering Centre of the Russian Academy of Sciences, Pr. 60-letiya Oktyabrya 7/2, Moscow 117312, Russia
Correspondence
Margarita Grabovich
margarita_grabov{at}mail.ru
ABSTRACT
A novel obligately organotrophic, facultatively microaerophilic spirillum, designated strain D-427T, was isolated from sulfidic sludge of a municipal wastewater-treatment plant. Cells were Gram-negative, large and highly motile due to bipolar tufts of flagella covered with mucous sheaths. Coccoid cells were sometimes formed. Strain D-427T grew optimally at pH 7.5–7.8 and 28 °C in the presence of 2 % O2 in the gas phase. The organism showed oxidase and very low catalase activity. The isolate grew chemo-organotrophically with a limited number of organic acids as substrates. The DNA G+C content was 38.0 mol% (Tm). Phylogenetic analysis of the 16S rRNA gene sequence placed strain D-427T in the genus Spirillum within the class Betaproteobacteria. The 16S rRNA gene sequence similarity between strain D-427T and Spirillum volutans ATCC 19554T, the type strain of the single species of the genus, was 98.6 %. The low level of DNA–DNA hybridization and different phenotypic properties indicate that strain D-427T is clearly distinguishable from Spirillum volutans. No strain of S. volutans is available from any established culture collection or from the authors who described this species. Therefore, on the basis of phenotypic and genotypic data and the fact that the type and single species of the genus Spirillum cannot be included in any scientific study, since the type strain has been lost, we propose to assign strain D-427T as a novel species of the genus Spirillum, Spirillum winogradskyi sp. nov. (type strain D-427T =DSM 12756T =VKM B-2518T), and we request that the Judicial Commission place the name Spirillum volutans on the list of rejected names if a suitable type strain is not found or a neotype is not proposed within 2 years following the publication of this paper. An emended description of the genus Spirillum is also provided.
Phase-contrast and transmission electron micrographs of strain D-427T and its detailed fatty acid profile are available as supplementary material with the online version of this paper.
The genus Spirillum Ehrenberg 1832
is one of the oldest established bacterial genera. The formal description of the genus has been changed several times (Williams & Rittenberg, 1957). In 1973, the genus Spirillum was defined as encompassing large, obligately microaerophilic freshwater spirilla having a DNA base composition of 36–38 mol% G+C and included only a single species, the type species Spirillum volutans (Hylemon et al., 1973a, b). Phylogenetic analysis based on 16S rRNA gene sequences showed that the genus Spirillum falls within the family Spirillaceae of the Betaproteobacteria (Krieg, 2005). At present, the description of the genus is based mainly on the phenotypic properties of a single species, which was represented by two strains (Rittenberg & Rittenberg, 1962; Wells & Krieg, 1965). In spite of the fact that, according to ecological observations, large spirilla are widespread in freshwater and anthropogenic water habitats representing niches with low levels of oxygen and sulfide (Dubinina et al., 1993; Kuenen & Dubinina, 2005), other representatives of the genus Spirillum have not been obtained in pure culture.
We have isolated a heterotrophic, microaerophilic, spiral-shaped strain that was classified as a representative of the genus Spirillum (Podkopaeva et al., 2006). Strain D-427T was originally given the name ‘Spirillum winogradskii’, but this name has not been validly published. In this paper, we present the results of a polyphasic taxonomic study of strain D-427T and discuss the status of the type species of the genus Spirillum, S. volutans, the type strain (ATCC 19554T) and reference strain ATCC 19553 both having been lost.
Samples from aeration tanks of a municipal wastewater-treatment plant containing sulfide at 1–2 mg l–1 and oxygen at 2.0–3.5 mg l–1 were used for inoculation of an enrichment using modified semi-liquid MPSS medium (Caraway & Krieg, 1974) with a freshly prepared FeS suspension (Kucera & Wolfe, 1957) of the following composition (l–1): 1 g (NH4)2SO4, 1 g MgSO4, 0.03 g CaCl2 . 2H2O, 0.002 g FeCl3 . 6H2O, 0.002 g MnSO4, 1 g sodium succinate, 1 g casein hydrolysate and 1 g agar (Difco). The medium was adjusted to pH 7.5. Vitamins and trace elements (Pfennig & Lippert, 1966) were added before inoculation. The incubation was carried out at 28 °C, corresponding to the temperature in the aeration tanks. After 3 days of incubation, thin white bands consisting of large motile spirilla with intracellular inclusions of elemental sulfur appeared in the medium about 0.5–1.0 cm below the surface. These cells were collected with a capillary tube and transferred to a test tube. Enrichment cultures were obtained by using serial 10-fold dilutions in test tubes. The last positive tube was used for isolation on the same medium solidified with 1.5 % agar (Difco) in Petri dishes. After 5 days of incubation at 28 °C, small, semi-transparent colonies of large spiral cells appeared only below the agar surface. Colonies were flat, of irregular shape with fringed edges, with a diameter ranging from 0.2 to 0.5 mm. A single colony was transferred into liquid MPSS medium without FeS. The purity of the culture was tested using phase-contrast microscopy (NU-2; Zeiss) and transfers into nutrient-rich media. Routine cultivation was carried out in liquid MPSS medium without FeS, casein hydrolysate being replaced by peptone at a concentration of 5 g l–1. The purified organism was designated strain D-427T.
The morphology of cells grown for 18 h was studied with a phase-contrast microscope and in a transmission electron microscope (JEM-100C; JEOL) as described elsewhere (Dubinina et al., 1993). In order to study the effect of aeration conditions on the growth of spirilla, rubber-stoppered 50 ml vials with 10 ml MPSS medium were flushed with filter-sterilized argon and a calculated volume of sterile air was then injected to create final oxygen concentrations in the gas phase of 1, 2, 5, 10 and 20 %. Cultivation was performed in vials with agitation on a shaker at 28 °C. Physiological and biochemical properties were determined as described previously (Dubinina et al., 1993). The ability to use different carbon and nitrogen sources was tested by removing peptone and succinate from the medium. Carbon and nitrogen sources were added to the medium at a concentration of 1 g l–1 or, in the case of amino acids, 0.25 g l–1. All tests were performed in triplicate. Mucous polysaccharide capsules were revealed by staining with ruthenium red (Luft, 1971). The fatty acid content was determined from 4–6 mg lyophilized biomass after acid methanolysis. Fatty acid methyl esters were analysed on a specialized chromatograph from the Microbial Identification System (Sherlock; MIDI Inc.) (Stead et al., 1992). DNA was isolated according to the method of Marmur (1961) from 5 l batch cultures grown aerobically on liquid MPSS medium in which casein hydrolysate was replaced by peptone at a concentration of 10 g l–1. The DNA G+C content was determined by thermal denaturation as described previously (Owen & Lapage, 1976). DNA of Escherichia coli K-12 DSM 498 (51.7 mol%) was used as a reference. Levels of DNA–DNA binding were determined by measuring the renaturation rate of denatured DNA at the optimal renaturation temperature as recommended by De Ley et al. (1970). For the DNA–DNA hybridization analysis, lyophilized DNA from S. volutans ATCC 19553 was used.
The almost-complete 16S rRNA gene sequence of strain D-427T was amplified by PCR with the universal eubacterial primers 27f and 1492r (Medlin et al., 1988; Lane, 1991) and aligned with related sequences using CLUSTAL_X software (Thompson et al., 1997). An evolutionary-distance matrix was calculated using the algorithm of Jukes & Cantor (1969). The phylogenetic tree was constructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods. Bootstrap analyses were based on 1000 resamplings. The PAUP 4.0b10 (Swofford, 1998) and TREECON (Van de Peer & De Wachter, 1994) software packages were used for the analysis.
Cells of strain D-427T were spiral-shaped and actively motile, with polar tufts of flagella that were visible as a single flagellum under a phase-contrast microscope. Thin-section electron micrographic preparation revealed a Gram-negative cell-wall structure and that the flagella formed several tufts covered with sheaths. Cell morphology of the isolate is shown in Supplementary Fig. S1 (available in IJSEM Online). Spores were never observed. Coccoid bodies, 7–10 µm in diameter, appeared in cultures during the stationary growth phase. Intracellular polyhydroxyalkanoates and volutin were formed. In the presence of sulfide and polysulfide in the medium, elemental sulfur accumulated in the cells. Strain D-427T oxidized thiosulfate quantitatively to tetrathionate, which accumulated in the medium (Podkopaeva et al., 2005).
The isolate was facultatively microaerophilic. Although growth occurred under an air atmosphere (20 % O2), the optimum O2 concentration for growth, as determined by the maximum increase in cell biomass, was 2 %. When inoculated into liquid or semi-liquid MPSS medium under an air atmosphere, growth of strain D-427T occurred only in a zone located 0.5–1.0 cm below the surface, and massive cell lysis was observed microscopically during the exponential growth phase. Electron micrographs revealed that the cells were surrounded by polysaccharide capsules. The capsule size varied according to the oxygen regime used for incubation, and it was minimal at low oxygen partial pressure.
Strain D-427T grew within a temperature range of 10–35 °C, with optimal growth at 28 °C. The pH range for growth was pH 6.5–8.5, with an optimum at pH 7.5–7.8. No growth was observed above 0.5 % NaCl in the medium. The cells showed oxidase activity and very low catalase activity. A comparative characterization of all tested physiological properties of strain D-427T, as well as some other characteristics, is given in the species description and also in Table 1. The strain utilized a limited number of organic acids, mainly intermediates of the tricarboxylic acid cycle. The isolate did not grow with nitrate, nitrite or individual amino acids (glutamate, aspartate, serine, methionine or cysteine) as nitrogen sources. It had a strictly respiratory metabolism, using only oxygen as electron acceptor.
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Comparative analysis of 16S rRNA gene sequences revealed that strain D-427T belonged to the class Betaproteobacteria and was most closely related to the type strain of S. volutans, ATCC 19554T (98.6 % similarity) (Fig. 1). The level of DNA–DNA hybridization between strain D-427T and S. volutans ATCC 19553 was 12 %. The low value of DNA–DNA binding between these two strains together with some phenotypic properties clearly indicated that strain D-427T represents a novel species within the genus Spirillum, according to the criteria for differentiation of bacterial species (Wayne et al., 1987), although most physiological characteristics of strain D-427T were essentially the same as those displayed by S. volutans ATCC 19553. The isolate differed from the type species by several phenotypic characteristics, such as its non-obligate microaerophily, the formation of coccoid bodies, growth at 0.5 % NaCl and the ability to assimilate acetate and the inability to utilize lactate as a carbon source (Table 1
). The new isolate also differed from S. volutans by its ability to grow in liquid medium under an atmosphere of air without addition of catalase or superoxide dismutase to MPSS medium. The strain seems to achieve a suitable environment for microaerophilic growth through the production of extracellular polysaccharides during aerobic growth.
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Request for an Opinion regarding the status of the species Spirillum volutans Ehrenberg 1832
Since the type species of the genus Spirillum is not represented by a type strain which conforms to the description of the taxon, we suggest that the Judicial Commission consider the following points.
(i) Based on our findings that the type strain (ATCC 19554T) and the known reference strain (ATCC 19553) of S. volutans are not currently available, we propose that a search should be made for a suitable replacement strain, or a neotype should be designated according to Rule 18c of the International Code of Nomenclature of Bacteria (Lapage et al., 1992).
(ii) If a suitable replacement type strain or a neotype of S. volutans cannot be found or proposed, respectively, within 2 years of the publication of this paper, we suggest that the Judicial Commission should place the name Spirillum volutans Ehrenberg 1832 on the list of rejected names and designate Spirillum winogradskyi as a new type species and use the description of Spirillum winogradskyi as a description for the genus Spirillum.
Emended description of the genus Spirillum Ehrenberg 1832, 38AL
The description is as given by Krieg (2005) with the following additional features. Cells form coccoid bodies. Motile by bipolar tufts of sheathed flagella; each tuft is covered with an individual sheath and is easily visible as an apparently single thick polar flagellum by phase-contrast microscopy. During growth on sulfide or polysulfide, cells accumulate globules of elemental sulfur. Catalase-negative or exhibit very low catalase activity. Cells are obligately or facultatively microaerophilic. Growth is observed only at low NaCl concentrations (below 0.5 %). Peptone and casein hydrolysate are used as nitrogen sources. Growth factors and vitamins are required. Major fatty acids are 16 : 0, 16 : 1 and 18 : 1. Representatives of the genus are widespread in freshwater and anthropogenic aquatic habitats containing sulfide.
Description of Spirillum winogradskyi sp. nov.
Spirillum winogradskyi (wi.no.grad'sky.i. N.L. masc. gen. n. winogradskyi of Winogradsky, named after Sergey N. Winogradsky, a Russian microbiologist who made a great contribution to the study of chemolithoautotrophic micro-organisms).
Spiral cells, 1.7–2.1 µm in diameter, with one to three helices; helix diameter 6.1–10.3 µm. Cells are motile by means of bipolar tufts of flagella. Each tuft is covered with an individual sheath and looks like a thick polar flagellum under the phase-contrast microscope. Accumulates hydroxyalkanoates and volutin and forms globules of elemental sulfur in the presence of sulfide or polysulfide. Cells are facultatively microaerophilic, with optimal growth at an O2 concentration of 2 %. Activity of oxidase and very low activity of catalase are present. Growth occurs within a pH range of 6.5–8.5, with an optimum at pH 7.5–7.8. The optimum growth temperature is at 28 °C. Chemo-organotrophic. Utilizes the following organic acids as carbon and energy sources: 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, pyruvate and acetate. Citrate, aconitate and isocitrate are utilized in the presence of yeast extract. Does not utilize amino acids, sugars or alcohols. Vitamins are required. Uses ammonium salts, casein hydrolysate, yeast extract and peptone as nitrogen sources. Does not hydrolyse casein or starch. Does not use nitrate, fumarate, sulfate, thiosulfate or elemental sulfur as terminal electron acceptors. Forms sulfide from cysteine. Predominant cellular fatty acids are 16 : 0, 16 : 1 and 18 : 1. The DNA G+C content of the type strain is 38.0 mol% (Tm).
The type strain, D-427T (=DSM 12756T =VKM B-2518T), was isolated in Russia from sediments of an aeration tank for the treatment of municipal wastewater containing sulfide.
ACKNOWLEDGEMENTS
We greatly thank Professor Noel R. Krieg for providing DNA material of Spirillum volutans ATCC 19553 and discussing our results. We also thank Dr G. A. Osipov for performing fatty acid analysis and L. L. Mityushina for ultrathin sectioning of the cells. This work was supported by the Russian Foundation for Basic Research (01-04-06433, 05-04-48299).
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