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1 Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan
2 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
3 Institute of Hyperthermophiles, Motobu-Noge Hospital, Aza-Ohama 880-1, Motobu, Okinawa 905-0212, Japan
Correspondence
Akira Nakamura
a-nak{at}agbi.tsukuba.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and nifHD gene sequences of strain 6H33bT are respectively AB189452 and AB189453.
A phylogenetic tree based on the nifH gene sequence of 6H33bT and other representative members of the 
- and 
-Proteobacteria is available as a supplementary figure in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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), but recent studies have demonstrated that several strains, classified as Pseudomonas stutzeri, do have the ability to fix nitrogen (Krotzky & Werner, 1987; Vermeiren et al., 1999; Desnoues et al., 2003). Among them, P. stutzeri A15 (A1501), isolated from rice paddies in China, was studied in detail to determine the conditions under which nitrogen fixation occurred and the structure of the nitrogenase genes involved (Desnoues et al., 2003). A unique composting process has recently been developed, referred to as ‘hyperthermal composting’, which consists of rapid composting at high temperatures using a newly developed hyperthermal composting machine. When the final compost pile produced by this process was packed in polyethylene bags and sealed, several bags were found to be almost completely deflated after about 2 weeks at room temperature, similar to ‘vacuum-packing’. Repacking of this compost produced the same result, i.e. a loss of atmospheric gas pressure after 2 weeks. It was considered that this result might have been achieved through biological nitrogen fixation, because nitrogen gas (80 % of the atmosphere) must have been lost. Screening of the nitrogen-fixing micro-organism(s) from the ‘vacuum-packed’ compost was therefore undertaken. We report the isolation of a novel nitrogen-fixing species of the genus Pseudomonas sensu stricto from this sample.
Five grams of a sample from the ‘vacuum-packed’ compost was inoculated in 100 ml N-free minimum medium (NFMM; 0·18 g K2HPO4, 0·026 g KH2PO4, 0·16 g NaCl, 0·24 g MgSO4.7H2O, 2 mg CaCl2, 2 mg FeCl3, 0·2 mg ZnSO4.7H2O, 0·06 mg MnCl2.4H2O, 0·6 mg H3BO3, 0·4 mg CoCl2.6H2O, 0·02 mg CuCl2.2H2O, 0·04 mg NiCl2.6H2O, 10 mg Na2MoO4.2H2O, 5·0 ml acetic acid, 1000 ml distilled water, pH 7·0) and cultured for 2 weeks at 30 °C. A portion of this culture was transferred to fresh NFMM and cultivated again for 2 weeks. This treatment was repeated, and the final culture was then spread onto NFMM-agar plates to obtain single colonies. Many types of colonies were initially observed, but most did not grow when they were cultured again in liquid NFMM (data not shown). Only one colony, designated strain 6H33bT, grew when re-cultured. This strain was then further investigated. Strain 6H33bT grew in an aggregated form in NFMM and required several weeks to reach the stationary phase of growth. As 6H33bT also grew on Luria–Bertani (LB) medium, this medium was used to maintain the strain for further work. Cells of strain 6H33bT formed translucent, wrinkled, obscure-edged colonies on an LB agar plate. Microscopic observation revealed that cells were Gram-negative, straight rods, 2–5 µm long and 0·5 µm wide, and motile.
An acetylene reduction assay, a modification of the method of Desnoues et al. (2003), was used to detect the nitrogenase activity of 6H33bT. Cells cultured in LB medium overnight were washed with NFMM three times and suspended in NFMM to give an OD600 of 0·08. Five millilitres of this suspension was transferred to 20 ml tubes and sealed with rubber caps. After the tubes were degassed once, the headspace gas of the tubes was replaced by argon gas, and acetylene and oxygen gasses were added to final concentrations of 10 % (v/v) and a range from 0 to 6 % (v/v), respectively. The tubes were incubated at 30 °C with shaking, and 0·5 ml of the headspace gas was taken periodically for measurement of ethylene formation. Detection of ethylene was performed using GC with an HP 6890 series (Hewlett Packard) gas chromatograph equipped with a thermal conductivity detector. Injector and detector temperatures were set to 80 °C. The column used was a Porapak N (80/100 mesh, diameter 2·3 mmx2·0 m; GL-Sciences), and the carrier gas was N2 delivered at a flow rate of 30 ml min–1. This assay was performed three times. Strain 6H33bT was observed to reduce acetylene to ethylene at an oxygen concentration of 2–6 % (Fig. 1). The highest nitrogenase activity was detected at 4 % oxygen, with no activity at 0 %. This result indicated that the nitrogenase activity of 6H33bT was controlled against oxygen tension, as reported for P. stutzeri A15 (A1501) (Desnoues et al., 2003) and for some diazotrophs (Bergersen, 1991).
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by using the total DNA of strain 6H33bT, which was extracted using the method of Perego et al. (1988). The product (331 bp) was cloned with the pGEM-T vector system (Promega). To obtain the full-length nifH gene sequence, a Southern blot analysis and subsequent colony hybridization were performed with the cloned PCR fragment as a probe using a DIG DNA labelling kit (Roche Diagnostics) and a DIG nucleic acid detection kit (Roche Diagnostics). In the Southern blot analysis, total DNA of strain 6H33bT was digested with EcoRI, HindIII or PstI and blotted on a Hybond-N+ membrane (Amersham Biosciences). Hybridization signals were observed at about 9·0 kb, at more than 10 kb and at 3·2 kb, respectively, with these fragments (data not shown). The 3·2 kb PstI fragment was selected and cloned into pUC118 by colony hybridization, using a Hybond-C membrane (Amersham Biosciences). Sequencing of the cloned fragment was performed with a CEQ DTCS-quick start kit (Beckman Coulter) and a CEQ 2000 XL sequencing system (Beckman Coulter) using the M13 forward and reverse primers and primers generated from the internal sequence of the fragment. The sequence of the PstI fragment was 3196 bp in length (GenBank accession no. AB189453) and contained the complete nifH gene sequence (882 bp) and a partial nifD gene sequence (1428 bp). Similarity searches in BLASTN (Altschul et al., 1997) and FASTA (Pearson, 2000) indicated that the nifH and nifD gene sequences of 6H33bT showed the highest similarity to those of the nifH (GenBank accession no. X96609) and nifD (GenBank accession no. X95565) genes of P. stutzeri A15 (A1501) (96·6 and 93·2 % similarity, respectively). Moderate similarities were found to the nifH and nifD gene sequences of strains of other representative - and -proteobacteria (see Supplementary Fig. S1 in IJSEM Online).
To determine the phylogenetic position of strain 6H33bT, its 16S rRNA gene sequence was analysed. PCR amplification of a 16S rRNA gene fragment was conducted with primers Eubac27F and 1492R (DeLong, 1992) using Ex Taq (Takara Shuzo) and a PCR System 9700 (PE Applied Biosystems). After agarose-gel electrophoresis, the fragment was purified using an EZNA gel extraction kit (Omega). Sequencing was performed with the same system described above for the nifH and nifD gene sequences using the primers reported by Hiraishi (1992) and Hiraishi et al. (1994). The 16S rRNA gene sequence of strain 6H33bT was aligned at positions 32–1489 (Escherichia coli numbering system; Brosius et al., 1978) with those of representative strains of the genus Pseudomonas sensu stricto using the CLUSTAL X software package (Thompson et al., 1997). Distance values were calculated using the method described by Kimura (1980), which were then used in CLUSTAL X to construct a phylogenetic tree via the neighbour-joining method (Saitou & Nei, 1987) with bootstrap values (Felsenstein, 1985) based on 1000 replications. A maximum-likelihood analysis was performed in DNAML with bootstrap values based on 100 replications using the SEQBOOT and CONSENSE programs of the PHYLIP package, version 3.6a3 (Felsenstein, 2002). Visualization of these results was provided by TreeView software (Page, 1996). The 16S rRNA gene sequence of 6H33bT (1458 bp) showed closest similarity to that of Pseudomonas indica IMT37T (97·3 % similarity), and a relationship to strains of the genus Pseudomonas sensu stricto was tested. Phylogenetic analyses based on the 16S rRNA gene sequences, performed using the neighbour-joining (Fig. 2) and maximum-likelihood (data not shown) methods, indicated that strain 6H33bT belongs to the genus Pseudomonas sensu stricto. Although differences in branching were found between the two trees, that between 6H33bT and P. indica IMT37T was supported with high bootstrap values (above 96 %) in both. These observations suggest that 6H33bT and P. indica IMT37T are representative of different species.
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and the medium described by Luisetti et al. (1972). Nitrate reduction and denitrification were tested according to the methods of Lelliott et al. (1966) and Stanier et al. (1966), respectively. Nitrogen fixation was tested using the method described by us above. Utilization of various carbon sources was determined in triplicate using a Biolog GN2 MicroPlate, as recommended by the manufacturer. The G+C content of the DNA of strain 6H33bT and the reference strains was determined by the method of Tamaoka & Komagata (1984). Other physiological characteristics were tested as described by Palleroni (1984). Strain 6H33bT grew over a pH range of 6·1–9·8 and at 28–41 °C in LB medium. It reacted positively in oxidase and catalase tests and in a test for hydrolysis of Tween 80, as did the three reference strains. Other physiological characteristics of strain 6H33bT differed from those of the reference strains, such as the production of pigments, nitrate reduction, denitrification, resistance to NaCl and the utilization of some substrates (Table 1). Of the four strains tested, only 6H33bT was able to fix nitrogen. Although nitrogen fixation was reported in P. stutzeri A15 (A1501), the type strain of P. stutzeri indicated a negative reaction. The G+C content of the DNA of 6H33bT was 66·3 mol%, which was in good agreement with the values of the genus Pseudomonas sensu stricto (58–70 mol%; Palleroni, 1984).
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, between 6H33bT and its closest relative, P. indica DSM 14015T (1–5 % similarity; data not shown). Strain 6H33bT therefore represents a novel species of the genus Pseudomonas sensu stricto, for which the name Pseudomonas azotifigens is proposed. From the phylogenetic tree constructed based on the nifH gene sequences, the nifH genes of P. azotifigens 6H33bT and P. stutzeri A15 (A1501) appear to have evolved from a common ancestral gene (Supplementary Fig. S1). However, the nitrogenase genes were not found in other Pseudomonas species, and Southern blot analysis with the nifH fragment of strain 6H33bT as a probe revealed the absence of nifH-like genes in the genomes of its most closely related type strains, P. indica DSM 14015T and P. stutzeri NCIMB 11358T (data not shown). This was in good agreement with the results of the nitrogenase assay. These results strongly indicated that nitrogen fixation ability in Pseudomonas sensu stricto is a strain-specific characteristic, and that there may have been a complex evolutionary history of the nitrogenase genes in the genus. One possible explanation is that the nitrogenase genes may have been lost from the other Pseudomonas strains, especially the type strain of P. stutzeri, after the strains NCIMB 11358T and A15 (A1501) diverged. Alternatively, the nitrogenase genes of P. azotifigens 6H33bT and P. stutzeri A15 (A1501) may have been horizontally transferred from other diazotroph(s) after these strains were established. Isolation and characterization of novel nitrogen-fixers in the genus Pseudomonas sensu stricto, together with analysis of nitrogen fixation within recognized Pseudomonas species, may provide further data on the evolutionary history of the nitrogenase genes in this genus.
Description of Pseudomonas azotifigens sp. nov.
Pseudomonas azotifigens [a.zo.ti.fi'gens. French n. azote (from Gr. pref. a- and Gr. n. zoê) nitrogen; N.L. n. azotum -i nitrogen; L. part. figens (from L. v. figo) fixing; N.L. part. adj. azotifigens nitrogen fixing].
Cells are Gram-negative straight rods, 2–5 µm long and 0·5 µm wide, and motile. Colonies are translucent, wrinkled and with obscure edges when grown on LB agar. Growth in LB medium is observed at pH 6·1–9·8, 28–41 °C and in the presence of 2·5–5 % NaCl. Nitrogen fixation in NFMM is observed in the presence of 2–6 % oxygen in the gas phase. Catalase and oxidase are produced. No pigments are produced on King medium A or Luisetti medium. Nitrate reduction and denitrification are negative. Starch and Tween 80 are hydrolysed. The following substrates are oxidized: acetic acid, cis-aconitic acid, L-alaninamide, D-alanine, L-alanine, D-arabitol, L-asparagine, bromosuccinic acid, citric acid, formic acid, D-gluconic acid, 
-D-glucose, L-glutamic acid, glycogen, hydroxy-L-proline, -hydroxybutyric acid, -hydroxybutyric acid, itaconic acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, DL-lactic acid, L-leucine, D-mannitol, methyl pyruvate, monomethyl succinate, L-proline, propionic acid, D-psicose, L-pyroglutamic acid, D-saccharic acid, sebacic acid, succinamic acid, succinic acid and Tweens 40 and 80. The following substrates are oxidized weakly: L-alanylglycine, L-aspartic acid, D-cellobiose, D-fructose, -hydroxybutyric acid, p-hydroxyphenylacetic acid, D-serine, L-serine and L-threonine. The G+C content of the type strain is 66·3 mol%.
The type strain, 6H33bT (=ATCC BAA-1049T=JCM 12708T), was isolated from a compost pile in Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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