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Phylogenetic Analyses of the Nitrogen-Fixing Genus Derxia

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Phylogenetic Analyses of the Nitrogen-Fixing Genus Derxia J. Gen. Appl. Microbiol., 50, 129–135 (2004) Full Paper Phylogenetic analyses of the nitrogen-fixing genus Derxia Cheng-Hui Xie* and Akira Yokota Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–0032, Japan (Received August 27, 2003; Accepted May 21, 2004) Phylogenetic analyses of the 16S rRNA gene sequence indicate that the genus Derxia forms a distinct lineage in the b-Proteobacteria. On the NJ tree Derxia has a low bootstrap value (30.9%) with Alcaligeneceae, and on the ML tree it shows an independent cluster separated from other families. Moreover, there is below 93.4% 16S rDNA sequence similarity between genus Derxia and the genera of the b-Proteobacteria. These facts reveal that Derxia is not grouped with any known family of b-Proteobacteria and should be placed as a separate genus of b-Proteobacteria. -The data on high G؉C content (71 mol%), the cellular fatty acid composition, and the physiologi cal characteristics of facultative hydrogen autotrophy and nitrogen fixation are unique for Derxia. The nifH gene sequence was found in this genus and phylogenetically compared among nitrogen-fixing bacteria to indicate that Derxia is clustered with the diazotrophs of b-Proteobac- teria. Key Words——cellular fatty acid; Derxia; nifH; phylogeny; b-Proteobacteria; 16S rDNA Introduction thinobacterium, Alcaligenes, and a few other taxa of b- Proteobacteria; the genus was, however, placed in the The genus Derxia Jensen et al. 1960 consists of a family Beijerinckiaceae (a-Proteobacteria) in the latest single species, Derxia gummosa. Cells are Gram-neg- edition of “Bergey’s Manual of Systematic Bacteriol- ative, rod-shaped with rounded ends, motile by means ogy” without any comments regarding this placement of a short polar flagellum, and catalase-negative. Mo- (Garrity and Holt, 2000). The phylogenetic position of lecular nitrogen can be fixed under both aerobic condi- Derxia has not previously been studied, making the tions and decreased oxygen pressures, and can grow taxonomy of Derxia a source of confusion. In the pres- as a facultative hydrogen autotroph (Derxia Jensen et ent study, we investigated the phylogenetic position of al. 1960). Derxia is usually found in tropical soils. the species of the genus Derxia based on 16S rDNA, The genus Derxia has been considered to have a nifH gene and chemotaxonomic analyses. relationship with the diazotrophic genera, Azotobacter, Azomonas, Beijerinckia, and Pseudomonas, based on Materials and Methods morphological, physiological, and chemotaxonomic characteristics (Becking, 1984, 1991); based on the Bacterial strains. The two strains of D. gummosa method of rRNA cistron similarity, De Smedt et al. were obtained from the IAM Culture Collection (Japan) (1980) have also grouped the genus Derxia with Jan- and LMG Culture Collection (Belgium). The studied strains, the type strain of D. gummosa, IAM 13946T ϭ Tϭ T * Address reprint requests to: Dr. Cheng-Hui Xie, Institute of ( ATCC 15594 LMG 3977 ) and the reference ϭ Molecular and Cellular Biosciences, The University of Tokyo, strain, IAM 14990 ( LMG 3975) were isolated from 1–1–1 Hongo, Bunkyo-ku, Tokyo 113–0032, Japan. the soil in West Bengal, India, in 1960 by Jensen et al. E-mail: [email protected] (1960). Bacterial strains were grown in the medium 130 XIE and YOKOTA Vol. 50 IAM B-1 (peptone 5.0 g, beef extract 3.0 g, NaCl 3.0 g, cataway, NJ, USA). distilled water 1.0 L, pH 7.0) or in LMG Medium-10 DNA sequencing. Sequencing of the complete 16S (glucose 10.0 g, CaCl2 ·2H2O 0.1 g, MgSO4 ·7H2O rDNA gene was performed on the coding and comple- Ј 0.1 g, K2HPO4 0.9 g, KH2PO4 0.1 g, CaCO3 5g, mentary strands by using four primer pairs: 520F: 5 - Ј Ј FeSO4 ·7H2O 10 mg, Na2MoO4 ·2H2O 5 mg, distilled CAGCAGCCGCGGTAATAC-3 (520–537)/1100R: 5 - water 1.0 L, pH 7.3) at 25°C. GGGTTGCGCTCGTTTG-3Ј (1100–1114); 926F: 5Ј-A- Biochemical analyses. API 20NE and 50CHL (bio- AACTCAAAGGAATTGACGG-3Ј (926–945)/1510R: 5Ј- Mérieux, S. A., Marcy-l’Etoile, France) were used to GGCTACCTTGTTACGTA-3Ј (1510–1527). 8F: 5Ј-AG- determine the physiological and biochemical character- AGTTTGATCCTGGCTCAG-3Ј (8–27)/700R: 5Ј-TCTA- istics. The API strips were incubated for 2 days at 25°C. CGCATTTCACC-3Ј (700–714). Sequencing reactions Chemotaxonomic investigations. Respiratory qui- were performed using a BigDye Terminator Cycle Se- nones were extracted with chloroform/methanol (2 : 1, quencing Ready Reaction Kit (Applied Biosystems, v/v), and were purified by TLC on silica-gel F254 plates Foster City, CA, USA) according to the manufacturer’s (Merck, Darmstadt, Germany) with hexane/diethyl protocol. Sequences were obtained with the ABI ether (85 : 15, v/v) being used as the solvent. The PRISMTM 310 Genetic Analyzer (Applied Biosystems). ubiquinone fraction was extracted with acetone, dried Phylogenetic analyses. The DNA sequences of D. under a stream of nitrogen, and subsequently ana- gummosa were compared with the sequences ob- lyzed by high-performance liquid chromatography tained from the DNA database. The sequences were (Model LC-10A apparatus, Shimadzu, Kyoto, Japan). aligned with the CLUSTAL W software package Fatty acid methyl esters were prepared from cells (Thompson et al., 1994), and evolutionary distances grown on TSA (Trypticase soy agar, Becton Dickinson and Knuc values (Kimura, 1980) were generated. Align- and Co., Sparks, MD, USA) for 48 h. The fatty acid ment gaps and ambiguous bases were not taken into methylesters (FAMES) were obtained from the cells by consideration and comparison. Phylogenetic trees saponification, methylation, and extraction according were constructed using the neighbor-joining method to the manual for the MIDI System (Microbial ID, Inc., (Saitou and Nei, 1987) and the maximum likelihood Newark, MD, USA). Analysis by gas chromatography method in PHYLIP Package (Felsenstein, 1989). The was controlled by MIS software. Following the stan- topology of the phylogenetic tree was evaluated by the dard protocol of the MIDI/Hewlett Packard Microbial bootstrap resampling method of Felsenstein with 1,000 Identification System, fatty acid methyl ester extracts replicates. The similarity values were calculated using were analyzed in a Hewlett Packard (model HP PAUP 4.068 PPC (Swofford, 1998). 6890A) GC equipped with a flame-ionization detector, Nucleotide sequence accession numbers. The an automatic sampler, and a computer. EMBL/GenBank accession numbers for the 16S rDNA PCR and sequencing. Genomic DNA was pre- sequences in this study are: D. gummosa (IAM pared from bacterial cells suspended in TE buffer by 13946T) AB089482 and D. gummosa (IAM 14990) heating at 95°C for 5 min, followed by cooling and cen- AB089481. The accession number of the nifH se- trifugation to collect the lysate. An approximately quences are: D. gummosa (IAM 13946T) AB089483 1,500-bp fragment of the 16S rDNA was amplified and D. gummosa (IAM 14990) AB089485. from the extracted DNA by using eubacterial universal Ј primers specific to the 16S rDNA gene: 8F: 5 - Results and Discussion AGAGTTTGATCCTGGCTCAG-3Ј [8–27, the E. coli numbering system of Brosius et al. (1978)] and 1510R: The phylogenetic trees were constructed by the 5Ј-GGCTACCTTGTTACGTA-3Ј (1510–1527). The 360- comparison of the 16S rDNA sequence of D. gum- base fragment of the nifH gene was amplified from the mosa and related genera from a database using the extracted DNA with the primers following forward and neighbor-joining (NJ) algorithm and the maximum like- backward: TGCGAYCCSAARGCBGACTC and ATS- lihood (ML) method, which are shown in Figs. 1 and 2, GCCATCATYTCRCCGGA (YϭC/T; SϭG/C; RϭA/G; respectively. They revealed that the genus Derxia be- BϭC/G/T) (Poly et al., 2001). The amplified fragments longs to the b-Proteobacteria. On the NJ phylogenetic were purified by GFXTM PCR DNA and a Gel Band Pu- tree Derxia has a low bootstrap value (30.9%) with the rification Kit (Amersham Pharmacia Biotech, Inc., Pis- Alcaligeneceae group, which strongly supports that is 2004 Phylogenetic analyses of the nitrogen-fixing genus Derxia 131 Fig. 1. Neighbor-joining tree showing phylogenetic relationships among members of b-Proteobacteria and the genus Derxia based on 1,138 nucleotide positions of the 16S rDNA sequence after excluding the positions with gaps and ambiguous bases. Beijerinckia indica ATCC 9093T (M59060) was used as the outgroup. Only bootstrap values over 80% are shown. not grouped with any known family such as the Burk- b-Proteobacteria, and it is not at all related to other holderiaceae, Alcaligeneceae, Oxalobacteraceae, or free-living nitrogen-fixing bacteria such as Beijerinkia, Comamonadaceae. The ML phylogenetic analyses Azospirillum, Azomonas, or Pseudomonas. confirms that Derxia is a distinct lineage and not close We also analyzed the chemotaxonomic characteris- to any other taxa. Moreover, there is less than 93.4% tics of D. gummosa (Table 1). The major cellular fatty 16S rDNA sequence similarity between the genus acid content was 18 : 1 w7c, 16 : 1 w7c, and 16 : 0, while Derxia and the genera of b-Proteobacteria. So far, 3OH-12 : 0, 3OH-14 : 0, and 2OH-14 : 0 were the major Derxia shows no close to phylogenetic relationship to hydroxy fatty acids. The high GϩC content of DNA 132 XIE and YOKOTA Vol. 50 Fig. 2. Maximum likelihood tree showing phylogenetic relationships among members of b-Proteobacteria and the genus Derxia based on 1,138 nucleotide positions of the 16S rDNA sequence after excluding the positions with gaps and ambiguous bases. Only bootstrap values over 80% are shown. (71 mol%), the facultative hydrogen autotrophy, and ase, shows a high degree of conservation of structure, the catalase-negative characteristics of this genus are function, and amino acid sequences across wide phy- different from those of other bacteria of b-Proteobacte- logenetic ranges.
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