Reclassification of Alcaligenes latus strains IAM 12599T and IAM 12664 and Pseudomonas saccharophila as Azohydromonas lata gen. nov., comb. nov., Azohydromonas australica sp. nov. and Pelomonas saccharophila gen. nov., comb. nov., respectively

The nitrogen-fixing, aerobic, hydrogen-oxidizing bacteria Alcaligenes latus strains H-4^T (=IAM 12599^T) and H-1 (=IAM 12665) were originally identified by Palleroni & Palleroni (1978) based on phenotypic studies, and strain H-N (=IAM 12664) isolated in Australia was classified as a reference strain of Alcaligenes latus in their report, but no substantial taxonomic data were supplied to confirm this conclusion. Later work by Willems et al. (1991) questioned the taxonomic position of the type strain of Alcaligenes latus and suggested that this bacterium was not a member of the genus Alcaligenes Castellani and Chalmers 1919 (Kersters & De Ley, 1984). Moreover, taxonomic study of strains IAM 12664 (=ATCC 29713=DSM 1124) and IAM 12665 (=ATCC 29714=DSM 1123) has not been reported until now. The strains of Alcaligenes latus were identified as nitrogen-fixing bacteria by Malik et al. (1981). On the other hand, Pseudomonas saccharophila was described by Doudoroff (1940) as an aerobic, hydrogen-oxidizing bacterium and it was identified as a nitrogen-fixing bacterium by Barraquio et al. (1986). Studies on DNArRNA hybridization (Willems et al., 1991) and 16S rRNA gene sequence analysis (Anzai et al., 2000) revealed that Pseudomonas saccharophila belonged to the Betaproteobacteria and was remote from the genus Pseudomonas Migula 1894 (Palleroni, 1984). Historically, the genus Pseudomonas has been poorly investigated in terms of the phylogeny of its species. In recent years, some bacterial species that previously had unclear taxonomic positions within this genus have been reclassified using a polyphasic taxonomic approach (Anzai et al., 2000).

Based on substantial data from 16S rRNA and nifH gene sequence analyses, DNADNA hybridization, respiratory quinone and cellular fatty acid analyses and phenotypic characteristics, we believe that Alcaligenes latus and Pseudomonas saccharophila should be removed from the genera Alcaligenes and Pseudomonas, respectively. Alcaligenes latus IAM 12599^T (together with IAM 12665) and strain IAM 12664 are proposed to represent two novel species of a new genus, for which the names Azohydromonas lata gen. nov., comb. nov. and Azohydromonas australica sp. nov. are proposed. We also propose to reclassify Pseudomonas saccharophila as Pelomonas saccharophila gen. nov., comb. nov.

Bacterial strains.
Alcaligenes latus IAM 12599^T, IAM 12664 and IAM 12665, Pseudomonas saccharophila IAM 14368^T, Rubrivivax gelatinosus IAM 14808^T and Derxia gummosa IAM 13946^T were used in this study. Strains of Alcaligenes latus and Pseudomonas saccharophila were grown on B-1 medium (nutrient agar). Pseudomonas saccharophila was also incubated in a selective medium (KH₂PO₄, 4·4 g; Na₂HPO₄, 4·8 g; NH₄Cl, 1·0 g; MgSO₄.7H₂O, 0·5 g; ferric ammonium citrate, 50·0 mg; CaCl₂, 6·5 mg; sucrose, 1·0 g; distilled water, 1·0 l). Rubrivivax gelatinosus was cultured in a medium containing (l¹) 2·5 g yeast extract, 2·5 g peptone and 1·25 g NaCl. All of these strains can grow on nitrogen-free medium (glucose, 10·0 g; CaCl₂.2H₂O, 0·1 g; MgSO₄.7H₂O, 0·1 g; K₂HPO₄, 0·9 g; KH₂PO₄, 0·1 g; CaCO₃, 5·0 g; FeSO₄.7H₂O, 10·0 mg; Na₂MoO₄.2H₂O, 5·0 mg; distilled water, 1·0 l, pH 7·3). They were incubated at 29 °C.

Phenotypic characterization.
The colony morphology, colour and size of the bacteria were observed after 48 h cultured on nitrogen-free medium at 27 °C. API 20E and 50CHL microtest galleries (bioMérieux) were used to determine physiological and biochemical characteristics. The API strips were incubated for 2 days at 30 °C. Cellular fatty acid methyl esters were prepared, separated and identified by using the Microbial Identification system, the respiratory quinone system was extracted and characterized by HPLC (Shimadzu) as described by Xie & Yokota (2003) and genomic DNA extraction was carried out by the method of Marmur (1961). DNADNA hybridization was performed by the photobiotin-labelling method of Ezaki et al. (1989) using a Multi-well Plate Reader (CytoFluoR; Perseptive Biosystems). The hybridization temperature was 52 °C and reciprocal experiments were performed as follows: DNA of strain IAM 12599^T was used as a probe to hybridize DNA of strains IAM 12599^T, IAM 12665 and IAM 12664 and a negative control.

Phylogenetic analyses.
PCR-mediated amplification of 16S rRNA and nifH gene sequences and sequencing of the PCR products were carried out as described previously (Xie & Yokota, 2004). A 420 bp fragment of the nifH gene (encoding the iron protein of nitrogenase) was amplified from extracted DNA using the forward primer IGK (5'-TACGGYAARGGBGGYATCGG-3') and the reverse primer AQE (5'-GACGATGATYTCCTG-3') (Y=C/T; S=G/C; R=A/G; B=C/G/T; D=A/G/T) (Poly et al., 2001). A 716 bp fragment of the nifH gene of Derxia gummosa IAM 13946^T was determined with the forward primer IGK and the reverse primer R750 (5'-TCCATBGTGATCGGGDCGGGATG-3') (designed in this study). We compared the DNA sequences obtained in this study and sequences from the DNA Database of Japan (DDBJ). The sequences were aligned using the CLUSTAL W software package (Thompson et al., 1994) and evolutionary distances and K_nuc values (Kimura, 1980) were generated. Alignment gaps and ambiguous bases were excluded from the calculation. A phylogenetic tree based on the comparison of 1383 bases of the 16S rRNA gene sequences was constructed using the neighbour-joining method (Saitou & Nei, 1987). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates, while similarity values were calculated using PAUP 4.0b1 (Swofford, 1998). Using the same method, we aligned 408 bp nifH fragments and constructed a phylogenetic tree.

The 16S rRNA gene sequences of Alcaligenes latus strains IAM 12599^T, IAM 12664 and IAM 12665 and Pseudomonas saccharophila IAM 14368^T clearly indicated that they are related to the RubrivivaxRoseateles branch within the family Comamonadaceae and do not belong to the genus Alcaligenes (type species Alcaligenes faecalis, type strain IAM 12369^T) or the genus Pseudomonas (Gammaproteobacteria) (Fig. 1). The three strains of Alcaligenes latus constituted a strong cluster with 90 % bootstrap support and were a sister phyletic group of the RubrivivaxIdeonella cluster. Strain IAM 12599^T showed 96·1 % 16S rRNA gene sequence similarity to IAM 12664. The DNADNA hybridization value between them was 35·1 %, suggesting that they belong to two species (Stackebrandt & Goebel, 1994; Wayne et al., 1987). The 16S rRNA gene sequence similarity and DNADNA hybridization value between strains IAM 12599^T and IAM 12665 were 99·9 and 89·7 %, respectively, suggesting that they belong to a single species. Pseudomonas saccharophila IAM 14368^T was most closely related to Mitsuaria chitosanitabida (Amakata et al., 2005) and Roseateles depolymerans (Suyama et al., 1999), with 97·2 and 96·8 % similarity, respectively. Unfortunately, we could not study the former species further. However, Pseudomonas saccharophila and Roseateles depolymerans can be distinguished well by their morphology, metabolism and physiological characteristics (Table 1). Therefore, these two bacteria should not be included in the same genus.

(40K): Fig. 1. 16S rRNA gene sequence-based phylogenetic tree generated by the neighbour-joining method, showing relationships of Alcaligenes latus IAM 12599T and IAM 12664, Pseudomonas saccharophila IAM 14368T and related genera of the Betaproteobacteria. The type species of the genus Alcaligenes, Alcaligenes faecalis, was used as the outgroup. Numbers at nodes indicate percentages of occurrence in 100 bootstrapped trees; only values greater than 55 % are shown. Sequences indicated in bold were determined in this study.

Table 1. Differential characteristics of Azohydromonas gen. nov., Pelomonas gen. nov. and their phylogenetic neighbours Some data were obtained from Kersters & De Ley (1984), Suyama et al. (1999) and Willems et al. (1991). NT, Not tested; W/+, weak or positive reaction.

We detected the nifH gene in three strains of Alcaligenes latus and in Pseudomonas saccharophila IAM 14368^T in this study and confirmed that they are capable of nitrogen fixation. The nifH gene sequences were selected as another phylogenetic marker to elucidate their evolutionary relationship with other diazotrophic bacteria. nifH has been closely examined as part of a nitrogen-fixation gene group (nifHDK), showing strong conservation, and this analysis is largely consistent with the 16S rRNA gene phylogeny, except for discrepancies with a few taxa (Xie & Yokota, 2004; Moulin et al., 2001; Rosado et al., 1998; Young, 1992). Based on the nifH phylogenetic analyses, we found that the highest sequence similarity to the Alcaligenes latus strains was shown by Derxia gummosa (90 %), not the 16S rRNA gene phylogenetic neighbour Rubrivivax gelatinosus. The three strains of Alcaligenes latus constitute a tight cluster with 99 % bootstrap support and share 96·6100 % similarity, suggesting that the strains had the same origin in the evolutionary process of nitrogen fixation. Pseudomonas saccharophila had no close phylogenetic neighbour, with no more than 89 % similarity compared with other nitrogen-fixing bacteria (Fig. 2). Phylogenetic analysis of the nifH sequence indicated clearly that the strains of Alcaligenes latus and Pseudomonas saccharophila were distinct from their 16S rRNA gene phylogenetic neighbours. Therefore, the nifH sequence is more suitable for evaluating the close phylogenetic relationship of diazotrophic bacteria.

(42K): Fig. 2. nifH gene sequence-based phylogenetic tree generated by the neighbour-joining method. Numbers at nodes indicate percentages of occurrence in 1000 bootstrapped trees; only values greater than 50 % are shown. Sequences indicated in bold were determined in this study.

As members of the Betaproteobacteria, Alcaligenes latus IAM 12599^T, IAM 12664 and IAM 12665 and Pseudomonas saccharophila IAM 14368^T possess Q-8 as the predominant respiratory ubiquinone (Hiraishi et al., 1996). The G+C contents of the DNA were within the range, 66·072·5 mol%, found in their phylogenetic neighbours. Alcaligenes latus IAM 12599^T, IAM 12664 and IAM 12665 were distinct from their phylogenetic neighbours Rubrivivax and Ideonella in terms of the morphology of flagellation, the presence of non-phototrophic pigments, hydrogen autotrophy, nitrogen fixation, the presence of poly-β-hydroxybutyrate (PHB) granules and utilization of carbon sources (Table 1). Pseudomonas saccharophila IAM 14368^T can be also differentiated from the phylogenetically neighbouring genus Roseateles by phenotypic features (Table 1): colonies of Pseudomonas saccharophila IAM 14368^T are grey or white and the cells are monotrichously flagellated, non-phototrophic, nitrogen-fixing and capable of autotrophic growth with hydrogen. In contrast, members of Roseateles have pink colonies and the cells are peritrichously flagellated, phototrophic but not capable of autotrophic growth with hydrogen or nitrogen fixation. The major cellular fatty acids of Alcaligenes latus IAM 12599^T, IAM 12664 and IAM 12665 were 16 : 1ω7c, 16 : 0 and 18 : 1ω7c, whereas members of the phylogenetic neighbour Rubrivivax had the same major fatty acids but possessed less 18 : 1ω7c (Table 2). The major hydroxy fatty acids were 10 : 0 3-OH and 12 : 0 2-OH, while strains of Rubrivivax had only 10 : 0 3-OH. The major cellular fatty acids of Pseudomonas saccharophila IAM 14368^T were 16 : 1ω7c, 16 : 0 and 18 : 1ω7c; the major hydroxy fatty acids were 10 : 0 3-OH and 12 : 0 2-OH. Moreover, strain IAM 12664 can be categorized separately from Alcaligenes latus IAM 12599^T by phenotypic characteristics and hydroxy fatty acid profile (Table 3). Alcaligenes latus IAM 12664 contains 12 : 0 3-OH, while IAM 12599^T and IAM 12665 do not. Based on the genetic and phenotypic analyses, we conclude that strains IAM 12599^T and IAM 12664 are evenly distant from known genera, which is sufficient to indicate that they belong to two different species of a new genus. The names Azohydromonas lata gen. nov., comb. nov. and Azohydromonas australica sp. nov. are proposed for Alcaligenes latus strains IAM 12599^T and IAM 12664, respectively. Our studies also reveal that Pseudomonas saccharophila is remote from the genus Pseudomonas and could be separated from its phylogenetic neighbour Roseateles. We reclassify it here as Pelomonas saccharophila gen. nov., comb. nov.

Table 2. Cellular fatty acids of Azohydromonas lata strains IAM 12599T and IAM 12665, Azohydromonas australica IAM 12664T, Pelomonas saccharophila IAM 14368T and Rubrivivax gelatinosus IAM 14808T Values are percentages of total fatty acids. With the exception of IAM 14808T, data were determined twice at different times.

Table 3. Differentiating characteristics of Azohydromonas lata IAM 12599T and Azohydromonas australica IAM 12664T Both species can utilize glucose, fructose, sucrose and aesculin but not starch, citrate, L-arabinose, lactose or ribose as carbon sources. Both can produce acid from glucose, arabinose and sucrose but not from sorbitol. W, Weak.

Description of Azohydromonas gen. nov.
Azohydromonas (A'zo.hy.dro.mo'nas. French n. azote nitrogen; Gr. n. hydor water; Gr. n. monas a unit, monad; N.L. fem. n. Azohydromonas nitrogen-fixing and hydrogen-autotrophic monad).

Cells are Gram-negative, short to straight coccoid rods or rods, 1·62·4 µm long and 1·11·4 µm in diameter, motile by means of five to ten flagella arranged in a peritrichous fashion. Colonies are grey, round and opaque and they sometimes become wrinkled. Growth occurs at 1542 °C; the optimum temperature is 3035 °C. No growth occurs at more than 2·5 % NaCl. Cells accumulate PHB granules as a storage material. Acid is produced from glucose. Catalase, oxidase and arginine dihydrolase activities are present. D-Ribose, L-arabinose, lactose and starch cannot be utilized for growth. Able to fix nitrogen and to grow autotrophically with hydrogen but not capable of photoautotrophy. The G+C content of the DNA ranges from 69·1 to 71·1 mol%. 16 : 1ω7c, 16 : 0 and 18 : 1ω7c are the major components of the cellular fatty acids and 10 : 0 3-OH and 12 : 0 2-OH are the major hydroxy fatty acids. Ubiquinone-8 is the major component of the quinone system. The type species is Azohydromonas lata.

Description of Azohydromonas lata comb. nov.
Azohydromonas lata (la'ta. L. fem. adj. lata broad).

Basonym: Alcaligenes latus Palleroni and Palleroni 1978.

The description is based on that given by Palleroni & Palleroni (1978) and this study. The characteristics are the same as those given in the description of the genus, with the following additions. Urease and tyrosinase are present. Cells can hydrolyse gelatin and starch but not aesculin, Tween or chitin. Nitrate reduction is positive, but indole, H₂S production and β-galactosidase are negative. Acids are produced from glucose, sucrose and arabinose. D-Glucose, D-fructose, sucrose, maltose, gluconate, 2-ketogluconate, glycerol, betaine, trehalose, sebacate, citrate, ethanol, n-butanol, isobutanol, D-arabitol, mucate, formate, butyrate, isobutyrate, malonate, succinate, fumarate, suberate, lactate, L-malate, L-alanine, D-alanine, L-serine, L-leucine, L-aspartate, L-glutamate and L-proline are utilized for growth. The G+C content of the DNA is 69·4 mol%.

The type strain is IAM 12599^T (=LMG 3321^T=ATCC 29712^T=CIP 103458^T=DSM 1122^T); strain IAM 12665 (=LMG 3325=ATCC 29714=DSM 1123) is a reference strain. These strains were isolated from soil in California, USA.

Description of Azohydromonas australica sp. nov.
Azohydromonas australica (aus.tra'li.ca. N.L. fem. adj. australica pertaining to Australia, where the type strain was isolated).

The characteristics are the same as those given in the description of the genus, with the following additions. Acid is produced from glucose, mannitol, inositol, rhamnose, sucrose, melibiose and arabinose, but not from sorbitol. Galactose, D-glucose, D-fructose, mannitol, aesculin, sucrose, melezitose and D-turanose are utilized for growth, but not glycerol, D-arabinose, L-arabinose, ribose, maltose, lactose, trehalose, starch or 2-ketogluconate. Positive reactions for arginine dihydrolase and the VogesProskauer test but negative for citrate and gelatin liquefaction. The major hydroxy fatty acids are 10 : 0 3-OH, 12 : 0 2-OH and 12 : 0 3-OH. The G+C content of the DNA is 70·4 mol%.

The type strain is IAM 12644^T (=LMG 3324^T=ATCC 29713^T=DSM 1124^T), which was isolated from soil in Australia.

Description of Pelomonas gen. nov.
Pelomonas (Pe.lo.mo'nas. Gr. n. pelos mud; Gr. n. monas a unit, monad; N.L. fem. n. Pelomonas a monad isolated from mud).

Cells are Gram-negative rods, motile with one polar flagellum. Colonies are grey, round and opaque; old colonies (cultured for more than 2 weeks) are brown in nitrogen-free medium. Cells possess PHB granules as a storage material. Catalase and oxidase activities are present. Acid is produced from glucose. Positive reaction for gelatin liquefaction and starch hydrolysis but negative for denitrification, arginine dihydrolase and lipase. Able to fix nitrogen and show autotrophic growth with hydrogen but not photoautotrophy. The G+C content of the DNA is 69·1 mol%. Straight-chain 16 : 1ω7c, 16 : 0 and 18 : 1ω7c are the major components of cellular fatty acids; 10 : 0 3-OH and 12 : 0 2-OH are the major hydroxy fatty acids. Ubiquinone-8 is the major component of the quinone system. The type species is Pelomonas saccharophila.

Description of Pelomonas saccharophila comb. nov.
Pelomonas saccharophila (sac.cha.ro.phi'la. Gr. n. saccharon sugar; Gr. adj. philos loving; N.L. fem. adj. saccharophila sugar-loving).

Basonym: Pseudomonas saccharophila Doudoroff 1940.

The characteristics are the same as those in the description of the genus, with the following additions. Cells are 3·04·0 µm long and 0·5 µm in diameter. Growth occurs at 440 °C with an optimum at 2532 °C; unable to grow at 41 °C. The following enzymes are present: 2-keto-3-deoxy-6-phosphogalactonate aldolase, 2-keto-3-deoxy-6-phosphogluconate aldolase, D-galactose dehydrogenase, mannose isomerase, pullulanase, sucrose phosphorylase and α-amylase. D-Galactose, mannose, D-ribose, glucose, fructose, sucrose, D-xylose, rhamnose, glutarate, acetate, pyruvate, butyrate, lactate, L-malate, succinate, fumarate, citrate, L-proline and isobutanol are utilized as carbon sources for growth, but not glycerol, mannitol, sorbitol, ethanol, glycine, L-lysine, suberate, azelate or L-serine.

The type strain is IAM 14368^T (=ATCC 15946^T=CFBP 2433^T=CIP 59.18^T=DSM 654^T=HAMBI 373^T=LMG 2256^T=NCCB 46053^T=VKM B-902^T), which was isolated from mud of a stagnant pool.