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Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan
Correspondence
Cheng-Hui Xie
aa37116{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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7c and 16 : 0) and major hydroxy fatty acids (3-OH 10 : 0 and 3-OH 12 : 0), are similar to those of the genus Zoogloea. The novel strains showed positive results for floc formation which is accepted as confirmatory for species of the genus Zoogloea. However, the novel strains can be distinguished from the other species of Zoogloea by physiological characteristics. The name Zoogloea oryzae sp. nov. is therefore proposed for the novel strains with strain A-7T (=IAM 15218T=CCTCC AB 2052005T) as the type strain. Phylogenetic and chemotaxonomic analyses indicate that strain ATCC 19623, designated as a reference strain of Zoogloea ramigera, does not belong to the genus Zoogloea but to a new genus of Alphaproteobacteria. The name Crabtreella saccharophila gen. nov., sp. nov. is proposed for strain ATCC 19623T (=IAM 12669T).
-hydroxybutyratePublished online ahead of print on 28 October 2005 as DOI 10.1099/ijs.0.63755-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and nifH gene sequences of strains A-7T and A-4 are AB201043–AB201044 and AB201045–AB201046, respectively.
A phylogenetic tree based on nifH gene sequences and a table detailing the fatty acid content of the three Zoogloea species are available as supplementary material in IJSEM Online.
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). Historically, the criteria for taxonomic classification in this genus were based on phenotypic characteristics and this has resulted in confusion in identifying members of this genus. Based on their quinone system and fatty acid analyses, strains ATCC 25935 (=IAM 12670) and ATCC 19623 (=IAM 12669) of Zoogloea ramigera have been found to differ from the type strain of the species, strain ATCC 19544T (=IAM 15300T) (Hiraishi et al., 1992). This led to strain ATCC 25935 being transferred to Duganella zoogloeoides (Hiraishi et al., 1997), but the taxonomic position of strain ATCC 19623 (=IAM 12669) is still uncertain. To date, the genus Zoogloea comprises two species: the type species Z. ramigera (Crabtree & McCoy, 1967) and Zoogloea resiniphila (Mohn et al., 1999). In this study, the taxonomic position of strain ATCC 19623 was further investigated by phylogenetic analyses together with earlier chemotaxonomic studies. We found that strain ATCC 19623 was phylogenetically related to the Alphaproteobacteria and distant from the genus Zoogloea. Phenotypic and chemotaxonomic analyses consistently support the fact that strain ATCC 19623 belongs to a new genus and species of the Alphaproteobacteria, for which the name Crabtreella saccharophila gen. nov., sp. nov. is proposed.
Strains A-7T and A-4 were isolated from paddy soil associated with the roots of Oryza sativa in 1982 (Oyaizu-Masuchi & Komagata, 1988). Since molecular studies had not been performed, the taxonomic position of the strains was uncertain. In this study, the phylogenetic relationship of the strains was deduced from the 16S rRNA gene sequence and it was found that the novel strains were closely related to Z. ramigera ATCC 19544T, and the recently proposed species Z. resiniphila ATCC 700687T with 100 % bootstrap support. The acetylene-reduction assay and sequencing of a partial fragment of the nifH gene (encoding the iron protein of nitrogenase) confirmed that the novel strains are nitrogen-fixing bacteria, which distinguishes them from the two recognized species of the genus Zoogloea. The level of DNA–DNA relatedness between strains A-7T and A-4 was 89·5 %. This strongly supports the assignment of the two strains as representatives of a single novel species.
The novel bacterial strains were grown in a nitrogen-free medium containing (l–1), 10·0 g glucose, 0·1 g CaCl2.2H2O, 0·1 g MgSO4.7H2O, 0·9 g K2HPO4, 0·1 g KH2PO4, 5 g CaCO3, 10 mg FeSO4.7H2O, 5·0 mg Na2MoO4.2H2O at pH 7·3 or supplemented with 0·5 g yeast extract l–1 at 25 °C. Strains were also grown on a zoogloea medium, containing (l–1) 5·0 g casitone, 5·0 g glycerol, 0·5 g sodium lactate and 1·0 g yeast extract, or nutrient broth. Strains Z. ramigera IAM 15300T, Z. resiniphila CIP 106709T and Z. ramigera IAM 12669 were also included in this study for fatty acid and physiological investigations. The formation of flocs was determined by test tube growth on LYS medium [2·2 g sodium lactate, 1·0 g (NH4)2SO4, 0·6 g K2HPO4, 0·4 g KH2PO4, 0·5 g yeast extract, 0·05 g vitamin B12 and mineral base RM2 (IAM, 2004)] and was confirmed by examination with the naked eye and a phase-contrast microscope after 3 days incubation. DNA base composition, quinone system, acetylene-reduction assay and the presence of poly--hydroxybutyrate (PHB) granules were determined by the methods of Oyaizu-Masuchi & Komagata (1988). Physiological characteristics were determined according to Cowan (1974) and then confirmed by the API 20NE and 50CHB microtest galleries (bioMérieux). Salinity tolerance was determined by inoculation in nutrient broth supplemented with 0–4·0 % (w/v) NaCl. Cellular fatty acid methyl esters were prepared, separated and identified using the Microbial Identification System as described by Xie & Yokota (2003).
DNA–DNA 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 51 °C and reciprocal experiments were performed as follows: the DNA of strain A-7T was hybridized to itself, to strain A-4 and to the negative control. PCR-mediated amplification of the 16S rRNA gene and sequencing of the PCR products were carried out as described previously (Xie & Yokota, 2003). A 420-base fragment of the nifH gene sequence was amplified from the extracted DNA using the forward primer IGK, 5'-TACGGYAARGGBGGYATCGG-3' and the reverse primer AQE, 5'-GACGATGATYTCCTG-3' (Xie & Yokota, 2004). DNA sequences were compared with those obtained from GenBank and aligned using the CLUSTAL W software package (Thompson et al., 1994) and evolutionary distances and Knuc values (Kimura, 1980) were generated. Alignment gaps and ambiguous bases were excluded from the calculation. A phylogenetic tree based on a comparison of 1249 bases 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 and similarity values were calculated using PAUP 4.0b1 (Swofford, 1998). Using the same method, 316 bases of nifH sequence were also aligned and a phylogenetic tree was constructed.
Strain A-7T showed 99·9 % 16S rRNA gene sequence similarity and 89·5 % DNA–DNA relatedness to strain A-4, suggesting that they are members of a single species. Phylogenetic analysis of the 16S rRNA gene sequence revealed that the novel strains fell within the Zoogloea lineage with 100 % bootstrap support (Fig. 1). Sequence similarity also indicated that their closest relatives were Z. ramigera (96·7 %) and Z. resiniphila (95·8 %). The novel strains showed lower sequence similarities (<95·0 %) to all other recognized species of the Betaproteobacteria. A nifH gene fragment was detected in the novel isolates and it was confirmed that they are capable of fixing nitrogen. Phylogenetic analysis based on the nifH gene sequence revealed that the novel isolates are closely related to the diazotrophic members of the family ‘Rhodocyclaceae’ such as Dechloromonas sp. SIUL (GenBank accession number AJ563286), Azoarcus indigens VB32T (U97118) and Azoarcus communis S2 (U97117) (see Supplementary Fig. S1 in IJSEM Online). This cluster of bacteria showed phylogenetic discrepancies when compared with the 16S rRNA gene sequences and this could be due to horizontal gene transfer (Hurek et al., 1997). Generally, at the family level, the two gene chronometers consistently indicate that the novel bacteria fall within the family ‘Rhodocyclaceae’ lineage. The novel strains are the first nitrogen-fixing bacteria to be assigned to the genus Zoogloea. Strain A-7T showed less than 96·7 % 16S rRNA gene sequence similarity to Z. ramigera and Z. resiniphila, a level sufficient to allow the proposal of a novel species (Stackebrandt & Goebel, 1994). Moreover, the novel strains can be easily distinguished from Z. ramigera and Z. resiniphila by their phenotypic characteristics (Table 1).
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; Wen et al., 1999). The fatty acid profile of the novel strains is similar to those of the two recognized species of the genus Zoogloea, which possess large amounts of 16 : 17c and 16 : 0 and have 3-OH 10 : 0 and 3-OH 12 : 0 as the major hydroxy fatty acids. The fatty acids of Z. resiniphila are a little different from the others (see Supplementary Table S1 in IJSEM Online). Colonies of the novel strains are colourless and cells are Gram-negative rods that are motile by monotrichous flagella. The novel strains show accumulation of PHB, are catalase-, oxidase- and urease-positive, show denitrification to N2 and do not form acid from carbohydrates. These phenotypic characteristics are in excellent agreement with the general description of the genus Zoogloea (Unz, 1984). Strains A-7T and A-4 can grow well in nitrogen-free medium. Their nitrogen fixation capability was identified by the acetylene-reduction assay and nifH gene detection, but the other species of Zoogloea do not show this ability. Moreover, the novel isolates can be differentiated from the two recognized species of Zoogloea by the presence of catalase activity and the hydrolysis of gelatin, casein and other substances (Table 1
). Based on phylogenetic and phenotypic analyses, the two novel strains are therefore proposed as representing a novel bacterium of the genus Zoogloea.
Strain IAM 12669 was derived from strain ATCC 19623 and the 16S rRNA gene sequences of these strains have been deposited in the GenBank/EMBL/DDBJ databases with accession numbers X74915 and D14255. We found six nucleotide mismatches between the two strains, including ambiguous bases. We determined the 16S rRNA gene sequence of strain IAM 12669 (GenBank accession no. AB238789) and found that it had 100 % similarity with X74915, apart from two ambiguous bases (NN) that appear in the first section of X74915. A phylogenetic tree based on a 1302 bp alignment was constructed using the neighbour-joining method (Fig. 2). Strain ATCC 19623 (=IAM 12669) was distantly related to the typical strains of Z. ramigera (Betaproteobacteria). Furthermore, the quinone system (Q-10) supports the affiliation of strain ATCC 19623 to the Alphaproteobacteria (Lechner et al., 1995; Yokota et al., 1992). A 16S rRNA gene sequence search revealed that the closest relatives of strain ATCC 19623 were two uncultured bacteria, clone HP1B02 and clone 015B-H01 (McMahon et al., 2002), with 98·5 % sequence similarity, but strain ATCC 19623 showed no more than 95·9 % sequence similarity to the other taxa within the Alphaproteobacteria. Strain ATCC 19623 and the uncultured bacteria were isolated from activated sludge systems and uranium-contaminated groundwater. They formed a monophyletic cluster and were a sister phyletic group to Mycoplana dimorpha IAM 13154T with poor bootstrap support. Phylogenetic analyses using the maximum-likelihood method also strongly supported the suggestion that strain IAM 12669 and the uncultured bacteria form an independent cluster not related to other taxa (data not shown). Comparisons of the taxonomic characteristics support this conclusion. Strain IAM 12669 has a large amount of fatty acid 18 : 17c (78·1 %) with smaller amounts of 16 : 0 (6·2 %), 19 : 0 10-methyl (2·7 %), 16 : 17c (2·0 %), 13 : 1 at 12–13 (1·8 %) and 18 : 0 (1·6 %). The major hydroxy fatty acids of strain IAM 12669 are 3-OH 16 : 0 (5·4 %), 3-OH 14 : 0 (0·7 %) and 3-OH 18 : 0 (0·6 %), which clearly distinguishes the strain from its phylogenetic neighbours Mycoplana (Urakami et al., 1990), Rhizobium, Mesorhizobium and Ochrobactrum (Tighe et al., 2000). Moreover, phenotypic characteristics also provide evidence that strain IAM 12669 cannot be characterized as a member of any known genus within the family Rhizobiales of the Alphaproteobacteria (Table 2). These results are in agreement with the phenotype found by Unz (1971), who rejected this strain as a representative of Z. ramigera. Therefore, we propose that strain IAM 12669 represents a new genus and novel species, Crabtreella saccharophila gen. nov., sp. nov.
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Cells are straight or curved, 0·5–0·7x1·5–2·0 µm and motile with a polar flagellum. Single or pairs of cells are observed. Growth occurs from 10 to 37 °C, but not at 4 or 40 °C. Growth is inhibited by 0·5 % NaCl. Flocs are formed at late growth stages and cells become embedded in gelatinous matrices to form zoogloeae, which are distinguished by a ‘tree-like’ morphology. Colonies are <1 mm in diameter and colourless in nitrogen-free medium. PHB accumulates in cells. Viscid slime and water-soluble fluorescent pigments are not produced in King A and B medium or in mannitol-yeast extract medium. Nitrogen-fixing. Catalase, oxidase and urease activities are present, but not DNase or tyrosinase activity. Nitrate reduction is positive, but indole production and gelatin, starch and Tween 80 hydrolysis are negative. Acid is not produced from glucose oxidatively or fermentatively. Most carbohydrates, such as glucose, arabinose, fructose, sucrose, galactose, maltose, xylose, mannitol, rhamnose, ribose, mannose, lactose, acetate and citrate, cannot be used as a sole carbon source. The quinone system is Q-8. The major fatty acids are 16 : 17c and 16 : 0. The major hydroxy fatty acids are 3-OH 10 : 0 and 3-OH 12 : 0. The DNA G+C content is 65·1 mol%.
The type strain, A-7T (=IAM 15218T=CCTCC AB 2052005T), was isolated from soil from a rice paddy field.
Description of Crabtreella gen. nov.
Crabtreella (Crab.tre'el.la. N.L. fem. n. Crabtreella named after Dr K. Crabtree, the American microbiologist who isolated the type strain of the type species).
The description is based on the data compiled by Unz (1971) and this study. Cells are Gram-negative, straight rods with rounded, blunt or tapered ends. The cells are 0·6–0·9 µm wide and 1·5–2·0 µm long with one to several flagella that are attached laterally. Growth occurs on 3 % NaCl, but not 6 % NaCl. PHB is accumulated. Zoogloea formation is absent. Catalase and oxidase activities are present, but urease activity and nitrate reduction are absent. The quinone system is Q-10. The major fatty acids is 18 : 1. The major hydroxy fatty acids are 3-OH 16 : 0, 3-OH 14 : 0 and 3-OH 18 : 0. The type species is Crabtreella saccharophila.
Description of Crabtreella saccharophila sp. nov.
Crabtreella saccharophila (sacc.ha.ro.phi'la. Gr. n. saccharon sugar; Gr. adj. philos loving; N.L. fem. adj. saccharophila sugar-loving).
Displays the following properties in addition to those given in the genus description. Cells grow in CY medium (5·0 g casitone and 1·0 g yeast extract). Colonies are straw-coloured. The optimum pH for growth is 7·0–7·5 with no growth at pH 4·5 or 9·6. The optimum temperature is 28–30 °C, with slow growth at 10 °C and no growth at 45 °C. Tributyrin is hydrolysed. H2S is produced in a peptone-cysteine-sulfated medium. Arginine dihydrolase is present. Hydrolyses tyrosine agar and brown pigment appears. Utilizes acetate, pyruvate, malate, fumarate, oxalacetate, asparagine, aspartate, glutamate, citrulline, ornithine, arginine, histidine, tyrosine, ammonia, n-propanol, ethanol, n-butanol and ethanol, but not citrate, benzoate or m-toluate. Acid is formed oxidatively from arabinose, ethanol, rhamnose, ribose, xylose, fructose, glucose, glycerol, mannitol and sucrose, but no acid is formed from starch or inulin. Indole is not produced. Nitrate is not reduced to nitrite and nitrogen gas. The DNA G+C content is 65·3 mol%.
The type strain is IAM 12669T (=ATCC 19623T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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