Introduction

The family Rhodospirillaceae, affiliated to the subclass Alphaproteobacteria currently accommodates 34 bacterial genera that were isolated and described from various geographical locations. The members of nitrogen-fixing genus Azospirillum of the family Rhodospirillaceae are distributed in natural environments such as soils of tropical, subtropical and temperate regions, where they are frequently associated with grasses, cereals and crops (Bally et al. 1983; Döbereiner and Day 1976; Kirchhof et al. 1997; Ladha et al. 1987; Patriquin et al. 1983), discarded road tar, oil-contaminated soil and fermentative tanks (Young et al. 2008; Lin et al. 2009, 2012, 2013) and a sulfide spring (Lavrinenko et al. 2010). The other spiral-shaped phototrophic purple non-sulfur bacteria were included in the genus Rhodospirillum (Trüper and Imhoff 1989; Favinger et al. 1989; Kawasaki et al. 1993; Gest and Favinger 2001) before the proposal of the species Rhodocista centenaria (Kawasaki et al. 1992; Euzéby and Kudo 2001) and Rhodospira trueperi (Pfennig et al. 1997). The genus Rhodocista was established based on the results of 16S rRNA gene sequences, unique cyst-producing morphology and ubiquinone Q-9 as predominant respiratory quinone (Kawasaki et al. 1992). As new phototrophic bacterial strains were isolated and more information on the phenotypes and genotypes (particularly 16S rRNA gene sequences) of these and other previously described strains became available, it was evident that the original description of some of the genera within the family Rhodospirillaceae needed revision.

Materials and methods

Bacterial strains and growth conditions

While investigating bacterial diversity in a fermentative tank in central Taiwan (24°12′ N, 120°67′ E) located near a greenhouse set up at the National Chung Hsing University, Taichung. Bacteria were isolated from the fermentation medium; approximately 100 μl fermentation broth were spread on nutrient agar (NA, Hi-Media) by using the standard ten-fold dilution plating technique. After three days of aerobic incubation on nutrient agar (NA, Himedia) at 30 °C, strain CC-LY736T formed a cream-colored colony, which was isolated, purified and preserved at −80 °C as glycerol suspension.

The following type strains Azospirillum amazonense BCRC 14279T (= DSM 2787T) (Magalhães et al. 1983; Falk et al. 1985, 1986); Azospirillum brasilense BCRC 12270T (Tarrand et al. 1978); Azospirillum doebereinerae DSM 13131T (Eckert et al. 2001); Azospirillum fermentarium BCRC 80505T (Lin et al. 2013); Azospirillum formosense BCRC 80273T (Lin et al. 2012); Azospirillum irakense BCRC 15764T (= DSM 11586T) (Khammas et al. 1989); Azospirillum lipoferum BCRC 12213T (type species; Tarrand et al. 1978); Azospirillum oryzae JCM 21588T (Xie and Yokota 2005); Azospirillum picis DSM 19922T (Lin et al. 2009); Azospirillum rugosum DSM 19657T (Young et al. 2008); Azospirillum zeae LMG 23989T (Mehnaz et al. 2007b); Inquilinus limosus LMG 20952T (type species; Coenye et al. 2002); Rhodocista centenaria JCM 21060T (type species; Kawasaki et al. 1992); Rhodocista pekingensis JCM 11669T (Zhang et al. 2003) and Skermanella aerolata DSM 18479T (type species; Weon et al. 2007) were purchased for the purpose of direct comparative taxonomy. All these strains were grown on NA at 30 °C for 2 days, unless specified otherwise.

Morphological tests and biochemical characterization

Cell morphology of strain CC-LY736T, including presence of flagella was determined by placing the cells (1–2 days old) on a carbon-coated copper grid followed by staining with aqueous solution of 0.2 % (w/v) uranyl acetate for 5–10 s, brief air-drying and observation under a transmission electron microscope (JEOL JEM-1400). Gram-staining was performed as described by Murray et al. (1994). Growth was tested using nutrient broth (NB, Hi-Media) at temperature 20–50 °C (at 5 °C intervals) and at pH 5–10 (at 1 unit intervals). Salt tolerance was determined by cultivating the organism in nutrient broth (Hi-Media) supplemented with 0–5 % (w/v) NaCl (at 1 % intervals). Catalase activity was determined by assessing bubble production by cells in 3 % (v/v) H2O2 and oxidase activity was determined by using 1 % (w/v) N,N,N,N,-tetramethyl-1,4-phenylenediamine reagent (bioMérieux, France). Intracellular poly-β-hydroxybutyrate (PHB) granules were detected with different methods, including Sudan Black staining (Schlegel et al. 1970) and Nile blue A staining (Ostle and Holt, 1982), which result in dark blue or fluorescent granules. DNase test was conducted by using DNase test agar (Hi-Media). Carbon source utilization pattern were determined by using Biolog GN2 MicroPlate (bioMérieux, France). Nitrate reduction, indole production, activity of β-galactosidase and urease, hydrolysis of esculin and gelatin, and assimilation of 12 substrates were tested with API 20 NE strips (bioMérieux, France). The activities of various enzymes were determined by using API ZYM system (bioMérieux, France).

Nitrogen-fixing capability

Acetylene-reduction assay described by Hardy et al. (1973) was used to assay the nitrogen-fixing capability. Vials (30 ml) containing 10 ml semisolid nitrogen-free broth (NFB) medium (Reinhold et al. 1987) were inoculated with strain CC-LY736T, A. irakense BCRC 15764T and A. brasilense BCRC 12270T, sealed with rubber septa and incubated at 30 °C in the dark incubator. After 72 h, 10 % (v/v) of the air phase was replaced with acetylene (Koch and Evans, 1966) and the vials were re-incubated. The amount of ethylene was measured for a total of 24 h by using a gas chromatograph (FID Gas Chromatograph, HITACHI, Model 163) equipped with a flame-ionization detector and a packed column (1.0 m × 2.0 mm i.d., steel column packed with Porapak-T 80–100). Conditions of analysis were: carrier gas, nitrogen; flow rate, 35 ml h−1; temperature of the flame ionization detector, 110 °C; column temperature, 65 °C.

Genomic DNA preparation and PCR amplification

Bacterial genomic DNA was isolated by using UltraClean™ Microbial Genomic DNA Isolation Kit (MO BIO, USA) by following manufacturer’s instructions. An almost full-length of 16S rRNA gene was amplified by using bacterial universal primers 1F (5′-GAG TTT GAT CAT GGC TCA GA-3′) and 9R (5′-AAG GAG GTG ATC CAA CCG CA-3′); the primers 3F (5′-CCT ACG GGA GGC AGC AG-3′), 5F (5′-AAA CTC AAA TGA ATT GAC GGG G-3′) and 4R (5′-TTA CCG CGG CTG CTG GCA C-3′) were used for sequencing reaction (Edwards et al. 1989). Gene sequencing was performed by using the Bigdye terminator kit (Heiner et al. 1998), and nucleotide sequence of PCR product was determined by an automatic DNA sequencer (ABI PRISM 310, Applied Biosystems, CA, USA) (Watts and MacBeath 2001). Obtained DNA sequences were then assembled using the Fragment Assembly System program from the Wisconsin Package (GCG 1995).

Azospirillum-specific PCR and nifH gene detection

The specific primer pair for the genus Azospirillum was designed and discussed by Lin et al. (2011). The genomic DNA of novel strain CC-LY736T as well as reference strains (including A. oryzae JCM 21588T; A. brasilense BCRC 12270T; A. canadense LMG 23617T; A. doebereinerae DSM 13131T; A. fermentarium BCRC 80505T; A. formosense BCRC 80273T; A. halopraeferens DSM 3675T; 6, A. largimobile DSM 9441T; A. lipoferum BCRC 12213T; A. melinis LMG 23364T; A. picis DSM 19922T; A. rugosum DSM 19657T; A. thiophilum DSM 21654T; A. zeae LMG 23989T) were screened by using a primer pair Azo494-F (5′-GGC CYG WTY AGT CAG RAG TG-3′) and Azo756-R (5′-AAG TGC ATG CAC CCC RRC GTC TAG C-3′) that can selectively amplify an Azospirillum-specific DNA fragment of size 263 bp. On the other hand, Gene nifH (nitrogenase reductase) was amplified to confirm the existence of sym gene.

The nifH gene was amplified by PCR using the primer set PolF (5′-TGC GAY CCS AAR GCB GAC TC-3′)/PolR (5′-ATS GCC ATC ATY NTC RCC GGA-3′) and the conditions were described previously by Poly et al. (2001). The expected size of the product was about 360 bp. Another primer set Zehrf (5′-TGY GAY CCN AAR GCN GA-3′)/Zehrr (5′-AND GCC ATC ATY TCN CC-3′) was used as described by Zehr and McReynolds (1989). The amplified patterns were screened by electrophoresis in 1.0 % (w/v) agarose gel, stained with ethidium bromide, visualized under UV radiation, and photographed.

16S rRNA gene sequence analysis and phylogenetic analysis

The 16S rRNA gene sequence data were submitted to EzBioCloud server (EzTaxon-e Database, Kim et al. 2012) and NCBI GenBank for BLAST search. Subsequently, closely related 16S rRNA gene sequences were retrieved and aligned by using the CLUSTAL_X (1.83) program (Thompson et al. 1997). Unfortunately, sequence data of several type strains of Azospirillum that are available in GenBank were ambiguous due to many unidentified bases that leads to erroneous phylogenetic distinctions of some Rhodospirillaceae representatives Therefore, the 16S rRNA genes of several Azospirillum species were re-sequenced and submitted to NCBI earlier (Lin et al. 2011, 2012). The phylogenetic analysis was performed using MEGA 5 software (Molecular Evolutionary Genetics Analysis, version 5) (Tamura et al. 2011) and the topology of the resultant trees were evaluated by Neighbor-Joining (Saitou and Nei 1987), Maximum Likelihood (Felsenstein 1981) and Maximum Parsimony (Fitch 1971) methods. The bootstrap values (Felsenstein 1985) were computed after 1,000 replications.

Sequence deposition

Ambiguous 16S rRNA gene sequences of A. amazonense BCRC 14279T (X79735), A. canadense LMG 23617T (DQ393891), A. halopraeferens DSM 3675T (Z29618), A. irakense DSM 11586T (Z29583), A. lipoferum DSM 1691T (M59061) were replaced with newly obtained 16S rRNA gene sequences during current phylogenetic analysis.

DNA–DNA hybridization analysis

DNA–DNA hybridization assay was conducted between strain CC-LY736T and A. irakense BCRC 15764T using DIG DNA labeling and detection kit (Roche Diagnostics; Cat. No.11 093 657 910) according to the manufacturer’s protocol. Chromosomal DNA of strain CC-LY736T was used to construct hybridization probe by labelling with digoxigenin–11-dUTP (DIG). The experiment was carried out in triplicate for each sample.

Fatty acid methyl ester analysis

Fatty acid methyl esters (FAME) were prepared, separated and identified according to the standard protocol (Paisley 1996) of the Microbial Identification System (MIDI) (Sasser 1990) by gas chromatograph (Agilent 7890A) fitted with a flame ionization detector. The cultures were grown under the same condition on NA plates at 30 °C. After 48 h cells were harvested from the plate and subjected to saponification, methylation and extraction (Miller 1982). Identification and comparison were made by using the Aerobe (RTSBA6) database of the MIDI System (Sherlock version 6.0).

Cellular polyamines and polar lipids analyses

Polyamines were extracted as described by Scherer and Kneifel (1983), analyzed by high performance liquid chromatography (HPLC). The dansyl derivatives were separated by using a Hitachi L-2130 equipped with Hitachi L-2200 autosampler, Hitachi L-2485 fluorescence detector (excitation at 360 nm and emission at 520 nm), and a reverse-phase C18 column (Phenomenex® Synergi Fusion-RP80, 250 × 4.60 mm, 4 μm particle size). Polar lipids were extracted and analyzed by two-dimensional TLC, and isoprenoid quinones were purified by the methods according to Minnikin et al. (1984) and analyzed by HPLC as described by Collins (1985).

Determination of the predominant ubiquinone and DNA base composition

Ubiquinone is an essential component of electron transfer system in the plasma membrane of prokaryotes. The isoprenoid quinones were purified by the methods according to Minnikin et al. (1984). Extraction and assay of quinone was routinely performed according to the HPLC method described by Collins (1985). For the analysis of DNA G+C content, DNA samples were prepared and degraded enzymatically into nucleosides as described by Mesbah et al. (1989). The nucleoside mixtures obtained were then separated and analyzed via HPLC (Hitachi L-2130 chromatograph equipped with Hitachi L-2200 autosampler, Hitachi L-2455 diode array detector, and a reverse-phase C18 column (Phenomenex® Synergi 4 μ Fusion-RP80 250 × 4.60 mm)).

Results and discussion

Phenotypic and biochemical characterization

Strain CC-LY736T is Gram-stain negative, spiral shaped, 2.0–2.3 μm in length and 0.6–0.8 μm in diameter. PHB granules were observed under light microscopy and visualized by UV illumination after directly staining growing bacteria on plates containing Nile blue A. The cell morphology of strain CC-LY736T is shown in Fig. S1 and the phenotypic characteristics are given in the genus and species description. Strain CC-LY736T did not growth in nutrient broth supplemented with 3 % NaCl (w/v), which is different with strain Niveispirillum irakense BCRC 15764T. Nitrate reduction was observed in strain CC-LY736T, but Niveispirillum irakense BCRC 15764T and Nitrospirillum amazonense BCRC 14279T did not reduce this compound. Urea hydrolysis activity and indole production were negative. The new strain was able to utilize utilize α-d-lactose, d-sorbitol, acetic acid, cis-aconitic acid, γ-hydroxybutyric acid, succinic acid, succinamic acid, d-alanine, glycyl-l-aspartic acid, l-histidine, l-leucine and d-serine as carbon sources; assimilation of d-glucose, N-acetyl-glucosamine and d-maltose, which distinguished from other related type species. Distinctive phenotypic characteristics for the strains of the novel group and the type strains of the phylogenetically closest species are shown in Table 1.

Table 1 Physiological differences between strain CC-LY736T and other representatives of the family Rhodospirillaceae. Taxa: 1, CC-LY736T; 2, Niveispirillum irakense BCRC 15764T; 3, Nitrospirillum amazonense BCRC 14279T; 4, Azospirillum lipoferum BCRC 12213T; 5, Azospirillum fermentarium BCRC 80505T; 6, Azospirillum brasilense BCRC 12270T; 7, Azospirillum formosense BCRC 80273T; 8, Azospirillum picis DSM 19922T; 9, Rhodocista pekingensis JCM 11669T; 10, Rhodocista centenaria JCM 21060T; 11, Skermanella aerolata DSM 18479T; 12, Inquilinus limosus LMG 20952T
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Nitrogen-fixing capability and nifH gene detection

Strains CC-LY736T, A. irakense BCRC 15764T and A. brasilense BCRC 12270T were able to reduce acetylene to ethylene with a mean value of 0.4, 0.8 and 21.4 nmol ethylene h−1 (108 cells) at 30 °C, respectively. The free-living nitrogen fixing activity was also compared with our previously described novel bacteria namely A. formosense CC-Nfb-7T (Lin et al. 2012), A. picis IMMIB TAR-3T (Lin et al. 2009) and A. rugosum IMMIB AFH-6T (Young et al. 2008), and the free-living nitrogen fixing activities were 25, 93 and 18 nmol ethylene h−1, respectively. It demonstrates that the nitrogen-fixing ability of strains CC-LY736T and A. irakense BCRC 15764T was related lower than the other free-living nitrogen-fixing Azospirillum species. Furthermore, the PCR amplicon of nifH gene in strain CC-LY736T was not observed by using the PolF/PolR or Zehrf/Zehrr primer sets in this study.

16S rRNA gene sequence analysis

The 16S rRNA gene sequence of strain CC-LY736T showed highest pair-wise similarity to A. irakense DSM 11586T (97.2 %), R. centenaria JCM 21060T (96.3 %), R. pekingensis JCM 11669T (96.1 %), A. rugosum DSM 19657T (91.8 %), A. doebereinerae DSM 13131T (91.6 %), A. formosense BCRC 80273T (91.6 %) and A. picis DSM 19922T (91.6 %). The bacterial genera such as Skermanella and Ochrobactrum shared relatively lower (<91.1 %) sequence similarity to strain CC-LY736T. The Neighbor-Joining phylogenetic tree based on 16S rRNA gene sequences showed that the type strains of Azospirillum largely constituted one subcluster within the family Rhodospirillaceae, except for A. irakense with A. amazonense, same result was also emphasized with Maximum Likelihood and Maximum Parsimony methods (Fig. 1). The pairwise 16S rRNA gene sequence similarity data of strain CC-LY736T and other representatives of the family Rhodopsirillaceae are shown in Table 2. Low similarity data (<92 %) obtained for Azospirillum representatives and strain CC-LY736T, A. amazonense BCRC 14279T and A. irakense BCRC 15764T suggested that none of those strains belonged to the genus Azospirillum. Therefore, further detailed studies were carried out to investigate most likely taxonomic positions of strain CC-LY736T , A. amazonense DSM 2787T and A. irakense DSM 11586T.

Fig. 1
figure 1

Phylogenetic analysis of Niveispirillum, Nitrospirillum and other representatives of the family Rhodospirillaceae based on 16S rRNA gene sequences. Distances and clustering were performed by using Neighbor-Joining method with the software package MEGA 5. Filled circles indicate that the corresponding nodes were also recovered in the tree constructed based on Maximum Likelihood and Maximum Parsimony algorithm. Bootstrap values (>50 %) based on 1,000 replications are shown at the branching points. Rhizobium alkalisoli LMG 24763T and Burkholderia sabiae LMG 24235T were used as outgroups. Bar, 0.02 substitutions per nucleotide position

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Table 2 Pairwise 16S rRNA gene sequence similarity between strain CC-LY736T, Niveispirillum irakense DSM 11586T, Nitrospirillum amazonense DSM 2787T and other representatives of the family Rhodospirillaceae
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Azospirillum-specific PCR detection

The genus-specific primer pair (Azo494-F/Azo756-R) selectively amplified 263 bp Azospirillum-specific fragments from representative strains of large Azospirillum subcluster (including A. oryzae JCM 21588T; A. brasilense BCRC 12270T; A. canadense LMG 23617T; A. doebereinerae DSM 13131T; A. fermentarium BCRC 80505T; A. formosense BCRC 80273T; A. halopraeferens DSM 3675T; 6, A. largimobile DSM 9441T; A. lipoferum BCRC 12213T; A. melinis LMG 23364T; A. picis DSM 19922T; A. rugosum DSM 19657T; A. thiophilum DSM 21654T; A. zeae LMG 23989T). The 263 bp DNA fragment was not detected in strains CC-LY736T, A. amazonense BCRC 14279T and A. irakense BCRC 15764T. These data also clearly suggested a possible earlier misclassification of A. amazonense BCRC 14279T and A. irakense BCRC 15764T.

DNA–DNA hybridization analysis

The DNA–DNA relatedness values of strain CC-LY736T with A. irakense BCRC 15764T was 51.2 ± 2.3 %. Strain CC-LY736T showed less DNA–DNA homology with A. irakense BCRC 15764T. Using the established molecular criteria for species-level relatedness described by Wayne et al. (1987), strain CC-LY736T shows less DNA–DNA homology with other closest related species, which supports its genomic distinction as a separate species.

Cellular fatty acid analysis

The FAME profiles in strain CC-LY736T and A. irakense BCRC 15764T were similar, both strains contain C16:1ω5c, C19:0 cyclo ω8c, C16:0 3-OH, C18:1 2-OH and C18:1 ω7c/C18:1 ω6c as major (>5 % of the total) fatty acids. Strain CC-LY736T and A. irakense BCRC 15764T were distinguished from genera Azospirillum, Rhodocista and Skermanella based on the FAME features. A. amazonense BCRC 14279T also accommodated similar major fatty acids of strain CC-LY736T and A. irakense BCRC 15764T except for C16:1ω5c, which was found in minor amounts in the former. On the other hand, FAME profile of A. lipoferum BCRC 12213T was distinct when compared to strain CC-LY736T, A. amazonense BCRC 14279T and A. irakense BCRC 15764T as it accommodated C17:1ω6c, C18:1 2-OH, summed features 2, 3 and 8 in major amounts. Similarly, members of the genus Rhodocista possessed C14:0, C16:1ω5c and summed feature 8 in major amounts. There were clear qualitative and quantitative differences in terms of several fatty acids within Rhodospirillaceae members, irrespective of the presence of summed feature 8 in significant (>25 %) amounts. The details of fatty acid profiles of strain CC-LY736T and other Rhodospirillaceae representatives are given in Table 3.

Table 3 Comparison of the cellular fatty acid contents (%) of strain CC-LY736T and. other representatives of the family Rhodospirillaceae
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Cellular polar lipid analysis

Strain CC-LY736T contained phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylmethylethanolamine (PME), and phosphatidyldimethylethanolamine (PDE), which were in line with that of A. irakense BCRC 15764T. In addition, strain CC-LY736T contained two unidentified glycolipids (GL1–2) that were absent in A. irakense BCRC 15764T. Interestingly, A. amazonense BCRC 14279T possessed DPG and three unidentified aminolipids (AL1–3), three unidentified phospholipids (PL1–3), an unidentified glycolipid (GL) and an unidentified lipid (L) besides lacking other polar lipids that were found commonly in strain CC-LY736T and A. irakense BCRC 15764T. In contrast, other Azospirillum species (n = 4) possessed PC, PE, PG, PDE and AL in common, but consistently lacked PME. Similarly, members of the genera Rhodocista and Skermanella also lacked PME, but accommodated other polar lipids in significant amounts. Taken together, polar lipid profile clearly distinguished strain CC-LY736T, A. amazonense BCRC 14279T and A. irakense BCRC 15764T from each others as well as from other Rhodospirillaceae representatives. The details of polar lipid profiles of strain CC-LY736T and other Rhodospirillaceae representatives are given in Table 4 and Fig. S2.

Table 4 Comparison of the polar lipid profiles of strain CC-LY736T (= BCRC 80504T) and other representatives of the family Rhodospirillaceae
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Cellular polyamine analysis

The distribution of polyamines within various members of the family Rhodospirillaceae is given in Fig. S3. Irrespective of their quantitative variations, putrescine and sym-homospermidine were two important polyamines detected consistently in Rhodospirillaceae representatives. Strain CC-LY736T possessed major amounts of sym-homospermidine and moderate amounts of putrescine. However, A. irakense BCRC 15764T contained both sym-homospermidine and putrescine in predominant amounts. In contrast, A. amazonense BCRC 14279T accommodated major amounts of putrescine and moderate amounts of sym-homospermidine. The polyamine data presented here revealed that sym-homospermidine is most likely to be a predominant polyamine in Azospririllum species, whereas putrescine could be a major polyamine in the members of the genera Rhodocista and Skermanella.

Cellular respiratory quinone and DNA base composition

Strain CC-LY736T and the reference strains (except for Rhodocista, which possessed ubiquinone Q-9 as a major respiratory quinone) contained ubiquinone Q-10 as the predominant respiratory quinone. The DNA G+C content of strain CC-LY736T was 67.6 ± 0.1 mol %, a value that was well within the range (64.0–70.0 mol %) reported for the representatives of the family Rhodospirillaceae.

In conclusion, based on the phylogenetic, physiological, chemotaxonomic and phenotypic characteristics, strain CC-LY736T should be classified as a novel genus of the family Rhodospirillaceae, for which we propose the name Niveispirillum fermenti gen. nov. sp. nov.; the type strain of the type species is CC-LY736T (= BCRC 80504T = LMG 27263T). We also reclassify Azospirillum irakense as Niveispirillum irakense comb. nov., and Azospirillum amazonense as Nitrospirillum amazonense gen. nov., sp. nov.

Description of Niveispirillum gen. nov

Niveispirillum (Ni.ve.i.spi.ril’lum. L. adj. niveus snow-white; L. fem. n. spirillum spiral; N.L. neut. n. Niveispirillum snow-white spiral)

Cells are Gram-stain negative and show positive reaction for catalase and oxidase. The polyamine profile consists of sym-homospermidine and putrescine. The predominant (>30 %) fatty acids are C18:1 ω7c/C18:1 ω6c. The major polar lipids are DPG, PG, PC, PE, PME, PDE and an unidentified aminolipid (AL1). The predominant quinone is ubiquinone Q-10. The type species is Niveispirillum fermenti.

Description of Niveispirillum fermenti sp. nov

Niveispirillum fermenti (fer.men’ti. L. neut. gen. n. fermenti, of a fermentation process)

Cells are spiral-shaped, which are 0.6–0.8 μm in width and 2.0–2.3 μm in length and contain a monopolar flagellum. Colonies appear cream-colored, circular and smooth when grown on R2A agar. Growth occurs at temperature 20–40 °C, pH 5.0–9.0 and <2 % (w/v) NaCl. The accumulation of intracellular granules (PHB) is observed. In the GN2 Biolog MicroPlate, cells can utilize α-d-lactose, d-melibiose, β-methyl-d-glucoside, d-sorbitol, acetic acid, cis-aconitic acid, d-galactonic acid lactone, γ-hydroxybutyric acid, itaconic acid, succinic acid, succinamic acid, d-alanine, glycyl-l-aspartic acid, l-histidine, l-leucine, L-pyroglutamic acid, d-serine, l-serine, urocanic acid, putrescine, 2-aminoethanol, 2,3-butanediol, d,l-α-glycerol phosphate and α-d-glucose-1-phosphate as sole carbon source; other carbon sources are not utilized. Nitrate and nitrite are reduced. In the API ZYM strip, positive for alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, naphthol-AS-BI-phosphohydrolase and β-glucosidase; negative for other reactions. The major fatty acids are C16:0, C16:1 ω5c, C19:0 cyclo ω8c, C18:1 ω7c/C18:1 ω6c, C16:0 3-OH and C18:1 2-OH. The polar lipids are DPG, PG, PC, PE, PME, PDE, AL1, PL and GL1-2. The predominant quinone is ubiquinone Q-10 and DNA G+C content of the type strain CC-LY736T is 67.6 ± 0.1 mol %.

Type strain is CC-LY736T (= BCRC 80504T = LMG 27263T) isolated from a fermentor in Taiwan.

Description of Niveispirillum irakense comb. nov

Niveispirillum irakense (i.ra.ken’se. N.L. neut. adj. irakense, pertaining to Iraq, where the organism was first isolated)

Gram-stain negative, aerobic, curved rods or S-shaped, motile with winding or snake-like movements. Catalase- and oxidase-positive. The growth temperature ranges from 20 to 35 °C, pH 5.5–8.5 (optimum pH is 6.5) and tolerates up to 3 % (w/v) NaCl. A monopolar flagellum is observed when the cells are grown in a liquid medium. Intracellular granules (poly-β-hydroxybutyrate) are observed. The predominant quinone is Q-10. In the GN2 Biolog MicroPlate, cells can utilize dextrin, adonitol, l-arabinose, d-arabitol, d-cellobiose, d-fructose, l-fucose, d-galactose, α-d-glucose, maltose, d-mannitol, d-mannose, l-rhamnose, sucrose, D-trehalose, acetic acid, citric acid, l-histidine, l-leucine, l-ornithine, l-proline, and d-serine as sole carbon source; other substrates are not utilized. Nitrate is reduced to nitrite. In the API 20NE system, cells assimilate d-glucose, l-arabinose, d-mannose, d-mannitol, N-acetyl-glucosamine, d-maltose, potassium gluconate, capric acid and malic acid. In the API ZYM system, positive for alkaline phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase and trypsin. The DNA G+C content of the type strain KBC1T is 64–67 mol %.

Type strain is KBC1T (= ATCC 51182T = BCRC 15764T = CIP 103311T) isolated from rice rhizosphere in Iraq.

Description of Nitrospirillum gen. nov

Nitrospirillum (Ni.tro.spi.ril’lum. Gr. n. nitron soda; L. neut. n. spirillum spiral; N.L. neut. n. Nitrospirillum soda spiral)

Cells are Gram-stain negative, diazotrophic and show positive reaction for catalase and oxidase. The polyamine patterns include major amounts of putrescine and moderate amounts of sym-homospermidine. The predominant (>27 %) fatty acids are C18:1 ω7c/C18:1 ω6c. The polar lipid profile constitutes major amounts of DPG, three unidentified phospholipids (PL1–3) and two unidentified aminolipids (AL2–3). The predominant quinone is ubiquinone Q-10. The type species is Nitrospirillum amazonense.

Description of Nitrospirillum amazonense sp. nov

Nitrospirillum amazonense (a.ma.zon.en’se N.L. neut. adj. amazonense, pertaining to the Amazon region of Brazil)

Cells are spiral-shaped, which are 0.9–1.0 μm in width and 3.5 μm in length. Colonies appear cream-colored, circular and smooth when grown on R2A agar. Optimal growth occurs at temperature 35 °C, pH 5.7–7.8 and <3 % (w/v) NaCl. In the GN2 Biolog MicroPlate, cells are able to utilize α-cyclodextrin, dextrin, d-cellobiose, α-d-glucose, maltose and d-melibiose as sole carbon source; other carbon sources are not utilized. Nitrate and nitrite are not reduced. Assimilate d-glucose, l-arabinose and d-mannose. In the API ZYM strip, positive for esterase (C4), esterase lipase (C8), α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase and α-mannosidase; other reactions are negative. The major fatty acids are C16:0, C19:0 cyclo ω8c, C16:0 3-OH, C18:1 2-OH and C18:1 ω7c/C18:1 ω6c. The polar lipid profile constitutes DPG, three unidentified phospholipids (PL1–3) and three unidentified aminolipids (AL1–3), an unidentified lipid (L) and glucolipid (GL) each. The predominant quinone is ubiquinone Q-10 and DNA G+C content of the type strain Am14T is 67–68 mol %.

Type strain is Am14T (= ATCC 35119T = BCRC 14279T = DSM 3787T) was isolated from grass rhizosphere in Brazil.

Emended description of the genus Rhodocista Kawasaki et al. 1992

The genus description of Rhodocista is given by Kawasaki et al. (1992) with following amendments: both nitrate and nitrite are reduced. The predominant (>51 %) fatty acids are C18:1 ω7c/C18:1 ω6c. The major polar lipids are PC, PG, DPG, PE, PDE, PL and two unidentified aminolipids (AL2 and AL4). Polyamine profile contains major amounts of putrescine and moderate amounts of sym-homospermidine. The range of DNA G+C content is 68.8–70 mol %.